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[ "BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.", "The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.", "Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.", "High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .", "DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.", "To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.", "The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.", "Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.", "Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.", "The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .", "These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.", "Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.", "Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.", "First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .", "1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .", "Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.", "Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.", "The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.", "Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.", "Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.", "Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.", "Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .", "H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .", "The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .", "Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.", "We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.", "A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.", "Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.", "To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .", "In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .", "The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .", "As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.", "Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.", "H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.", "Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .", "Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .", "To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .", "Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.", "It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .", "Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .", "Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .", "Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.", "However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.", "Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.", "Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments." ]

[ "BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.", "The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.", "Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.", "High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .", "DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.", "To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.", "The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.", "Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.", "Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.", "The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .", "These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.", "Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.", "Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.", "First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .", "1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .", "Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.", "Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.", "The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.", "Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.", "Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.", "Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.", "Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .", "H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .", "The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .", "Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.", "We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.", "A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.", "Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.", "To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .", "In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .", "The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .", "As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.", "Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.", "H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.", "Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .", "Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .", "To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .", "Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.", "It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .", "Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .", "Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .", "Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.", "However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.", "Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.", "Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments." ]

[ "BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.", "The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.", "Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.", "High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .", "DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.", "To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.", "The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.", "Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.", "Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.", "The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .", "These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.", "Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.", "Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.", "First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .", "1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .", "Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.", "Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.", "The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.", "Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.", "Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.", "Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.", "Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .", "H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .", "The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .", "Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.", "We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.", "A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.", "Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.", "To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .", "In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .", "The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .", "As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.", "Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.", "H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.", "Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .", "Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .", "To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .", "Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.", "It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .", "Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .", "Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .", "Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.", "However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.", "Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.", "Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments." ]

[ "BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.", "The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.", "Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.", "High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .", "DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.", "To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.", "The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.", "Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.", "Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.", "The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .", "These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.", "Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.", "Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.", "First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .", "1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .", "Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.", "Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.", "The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.", "Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.", "Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.", "Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.", "Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .", "H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .", "The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .", "Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.", "We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.", "A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.", "Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.", "To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .", "In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .", "The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .", "As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.", "Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.", "H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.", "Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .", "Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .", "To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .", "Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.", "It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .", "Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .", "Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .", "Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.", "However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.", "Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.", "Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments." ]

[ "BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.", "The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.", "Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.", "High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .", "DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.", "To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.", "The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.", "Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.", "Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.", "The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .", "These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.", "Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.", "Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.", "First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .", "1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .", "Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.", "Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.", "The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.", "Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.", "Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.", "Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.", "Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .", "H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .", "The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .", "Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.", "We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.", "A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.", "Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.", "To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .", "In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .", "The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .", "As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.", "Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.", "H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.", "Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .", "Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .", "To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .", "Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.", "It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .", "Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .", "Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .", "Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.", "However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.", "Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.", "Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments." ]

[ "BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.", "The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.", "Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.", "High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .", "DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.", "To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.", "The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.", "Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.", "Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.", "The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .", "These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.", "Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.", "Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.", "First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .", "1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .", "Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.", "Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.", "The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.", "Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.", "Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.", "Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.", "Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .", "H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .", "The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .", "Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.", "We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.", "A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.", "Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.", "To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .", "In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .", "The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .", "As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.", "Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.", "H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.", "Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .", "Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .", "To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .", "Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.", "It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .", "Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .", "Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .", "Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.", "However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.", "Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.", "Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments." ]

[ "BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.", "The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.", "Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.", "High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .", "DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.", "To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.", "The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.", "Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.", "Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.", "The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .", "These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.", "Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.", "Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.", "First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .", "1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .", "Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.", "Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.", "The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.", "Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.", "Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.", "Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.", "Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .", "H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .", "The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .", "Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.", "We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.", "A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.", "Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.", "To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .", "In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .", "The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .", "As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.", "Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.", "H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.", "Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .", "Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .", "To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .", "Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.", "It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .", "Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .", "Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .", "Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.", "However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.", "Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.", "Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments." ]

[ "BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.", "The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.", "Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.", "High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .", "DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.", "To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.", "The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.", "Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.", "Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.", "The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .", "These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.", "Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.", "Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.", "First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .", "1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .", "Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.", "Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.", "The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.", "Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.", "Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.", "Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.", "Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .", "H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .", "The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .", "Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.", "We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.", "A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.", "Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.", "To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .", "In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .", "The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .", "As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.", "Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.", "H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.", "Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .", "Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .", "To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .", "Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.", "It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .", "Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .", "Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .", "Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.", "However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.", "Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.", "Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments." ]

[ "BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.", "The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.", "Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.", "High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .", "DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.", "To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.", "The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.", "Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.", "Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.", "The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .", "These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.", "Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.", "Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.", "First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .", "1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .", "Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.", "Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.", "The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.", "Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.", "Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.", "Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.", "Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .", "H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .", "The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .", "Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.", "We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.", "A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.", "Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.", "To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .", "In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .", "The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .", "As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.", "Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.", "H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.", "Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .", "Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .", "To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .", "Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.", "It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .", "Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .", "Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .", "Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.", "However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.", "Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.", "Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments." ]

[ "BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.", "The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.", "Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.", "High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .", "DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.", "To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.", "The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.", "Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.", "Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.", "The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .", "These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.", "Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.", "Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.", "First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .", "1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .", "Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.", "Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.", "The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.", "Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.", "Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.", "Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.", "Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .", "H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .", "The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .", "Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.", "We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.", "A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.", "Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.", "To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .", "In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .", "The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .", "As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.", "Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.", "H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.", "Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .", "Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .", "To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .", "Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.", "It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .", "Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .", "Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .", "Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.", "However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.", "Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.", "Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments." ]

[ "BACKGROUND: Mother-to-child transmission MTCT is the main cause of HIV-1 infection in children worldwide. Given that the C-type lectin receptor, dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node–specific ICAM-grabbing non-integrin L-SIGN , can interact with pathogens including HIV-1 and is expressed at the maternal-fetal interface, we hypothesized that it could influence MTCT of HIV-1. METHODS AND FINDINGS: To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of 197 HIV-infected mothers and their infants recruited in Harare, Zimbabwe. Infants harbouring two copies of DC-SIGNR H1 and/or H3 haplotypes H1-H1, H1-H3, H3-H3 had a 3.6-fold increased risk of in utero IU P = 0.013 HIV-1 infection and a 5.7-fold increased risk of intrapartum IP P = 0.025 HIV-1 infection after adjusting for a number of maternal factors. The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro.", "The implicated H1 and H3 haplotypes share two single nucleotide polymorphisms SNPs in promoter region p-198A and intron 2 int2-180A that were associated with increased risk of both IU P = 0.045 and P = 0.003, respectively and IP P = 0.025, for int2-180A HIV-1 infection. The promoter variant reduced transcriptional activity in vitro. In homozygous H1 infants bearing both the p-198A and int2-180A mutations, we observed a 4-fold decrease in the level of placental DC-SIGNR transcripts, disproportionately affecting the expression of membrane-bound isoforms compared to infant noncarriers P = 0.011 . CONCLUSION: These results suggest that DC-SIGNR plays a crucial role in MTCT of HIV-1 and that impaired placental DC-SIGNR expression increases risk of transmission. Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa.", "Text: Without specific interventions, the rate of HIV-1 mother-tochild transmission MTCT is approximately 15-45% . UNAIDS estimates that last year alone, more than 400,000 children were infected worldwide, mostly through MTCT and 90% of them lived in sub-Saharan Africa. In the most heavilyaffected countries, such as Zimbabwe, HIV-1 is responsible for one third of all deaths among children under the age of five. MTCT of HIV-1 can occur during pregnancy in utero, IU , delivery intrapartum, IP or breastfeeding postpartum, PP . High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy.", "High maternal viral load, low CD4 cells count, vaginal delivery, low gestational age have all been identified as independent factors associated with MTCT of HIV-1 . Although antiretrovirals can reduce MTCT to 2%, limited access to timely diagnostics and drugs in many developing world countries limits the potential impact of this strategy. A better understanding of the mechanisms acting at the maternal-fetal interface is crucial for the design of alternative interventions to antiretroviral therapy for transmission prevention. Dendritic cell-specific ICAM-grabbing non-integrin-related DC-SIGNR, also known as CD209L or liver/lymph node-specific ICAM-grabbing non-integrin L-SIGN can interact with a plethora of pathogens including HIV-1 and is expressed in placental capillary endothelial cells . DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms .", "DC-SIGNR is organized in three distinct domains, an N-terminal cytoplasmic tail, a repeat region containing seven repeat of 23 amino acids and a C-terminal domain implicated in pathogen binding. Alternative splicing of DC-SIGNR gene leads to the production of a highly diversify isoforms repertoire which includes membrane-bound and soluble isoforms . It has been proposed that interaction between DC-SIGNR and HIV-1 might enhance viral transfer to other susceptible cell types but DC-SIGNR can also internalize and mediate proteasome-dependant degradation of viruses that may differently affect the outcome of infection. Given the presence of DC-SIGNR at the maternal-fetal interface and its interaction with HIV-1, we hypothesized that it could influence MTCT of HIV-1. To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta.", "To investigate the potential role of DC-SIGNR in MTCT of HIV-1, we carried out a genetic association study of DC-SIGNR in a well-characterized cohort of HIV-infected mothers and their infants recruited in Zimbabwe, and identified specific DC-SIGNR variants associated with increased risks of HIV transmission. We further characterized the functional impact of these genetic variants on DC-SIGNR expression and show that they affect both the level and type of DC-SIGNR transcripts produced in the placenta. Samples consisted of stored DNA extracts obtained from 197 mother-child pairs co-enrolled immediately postpartum in the ZVITAMBO Vitamin A supplementation trial Harare, Zimbabwe and followed at 6 weeks, and 3-monthly intervals up to 24 months. The ZVITAMBO project was a randomized placebocontrolled clinical trial that enrolled 14,110 mother-child pairs, between November 1997 and January 2000, with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on MTCT of HIV-1. The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment.", "The samples used in the present study were from mother-child pairs randomly assigned to the placebo group of the ZVITAMBO project. Antiretroviral prophylaxis for HIV-1-positive antenatal women was not available in the Harare public-sector during ZVITAMBO patient recruitment. The samples were consecutively drawn from two groups: 97 HIV-1-positive mother/HIV-1-positive child pairs and 100 HIV-1-positive mother/HIV-negative child pairs. Mother's serological status was determined by ELISA and confirmed by Western Blot. Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age.", "Infants were considered to be infected if they were HIV-1 seropositive at 18 months or older and had two or more positive HIV-1-DNA polymerase chain reaction PCR results at earlier ages. 100 infants were considered to be uninfected as they were ELISA negative at 18 months or older and had two DNA PCR negative results from samples collected at a younger age. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP as determined by PCR analyses of blood samples collected at birth, 6 weeks, 3 and 6 months of age and according to the following definitions adapted from Bryson and colleagues . Briefly, infants who were DNA PCR positive at birth were infected IU. Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period.", "Infants with negative PCR results from sample obtained at birth but who become positive by 6 weeks of age were infected IP. Infants with negative PCR results at birth and 6 weeks of age but who subsequently became DNA PCR positive were considered to be infected during the PP period. In the analysis comparing the 3 different modes of MTCT, 12 HIV-1-infected infants were excluded because the PCR results were not available at 6 weeks of age. Full methods for recruitment, baseline characteristics collection, laboratory procedures have been described elsewhere . The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%.", "The nucleotide sequence variation of the entire promoter, coding and part of 39-UTR regions of DC-SIGNR gene in the study population was determined previously . Haplotype reconstruction was performed using Bayesian statistical method implemented in PHASE , version 2.1.1, using single nucleotide polymorphism SNP with a minimum allele frequency MAF of 2%. We applied the algorithm five times, using different randomly generated seeds, and consistent results were obtained across runs Figure 1 . Fifteen haplotype-tagged SNPs htSNPs were identified by the HaploBlockFinder software with a MAF $5%. These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels .", "These htSNPs were genotyped in the 197 infants by direct PCR sequencing analysis as we have described previously . The DC-SIGNR exon 4 repeat region genotype was determined by PCR amplification followed by migration in 1.5% agarose gels . DNA sequences in the promoter region were analysed with the TESS interface for putative transcription factors binding sites using the TRANSFAC database. Luciferase reporter assays using pGL2-Basic vector were performed in order to investigate the functional effect of mutations on DC-SIGNR promoter activity. Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing.", "Genomic DNA from subjects homozygous for the promoter variants and WT was amplified from nucleotide position 2715 to 21 and cloned between the BglII and HindIII multiple cloning sites in the pGL2-Basic vector which harbours a reporter firefly luciferase gene downstream Invitrogen Canada inc, Burlington, Canada . All recombinants clones were verified by DNA sequencing. The firefly luciferase test reporter vector was co-transfected at a ratio of 10:1 with the constitutive expressor of Renilla luciferase, phRL-CMV Promega, Madison, WI, USA . We cultured HeLa cells in 6 wells plates 2610 5 cells and transfected them the following day using lipofectamine Invitrogen according to the manufacturer. Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.", "Cells were lysed and luciferase assays were performed using 20 mg of protein extract according to the manufacturer Promega at 44 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments. We carried out lucierase assays in triplicate in three independent experiments. Results are expressed as mean6 standard error of the mean S.E.M . First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression.", "First-term placental tissues were obtained from abortions following voluntary interruption of pregnancy at CHUM Hôpital Saint-Luc Montreal, Canada . Tissues from 3 H1 associated with MTCT of HIV-1 and 3 H15 wild-type homozygous haplotypes were used to analyse possible differences in isoform expression. Total placental RNAs were extracted by MasterPure DNA and RNA Extraction Kit Epicentre Biotechnologies, Madison, WI, USA according to the manufacturer. Fragments corresponding to the DC-SIGNR coding region were reversed transcribed RT and then amplified by nested PCR with the following primers; RT primers RR, first PCR RF and RR and second PCR RcF and RcR according to Liu and colleagues . 1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen .", "1 mg of total RNA was reverse transcribed with Expand RT Roche Applied Science, Indianapolis, IN, USA according to the manufacturer and were PCR-amplified with DNA Platinum Taq Polymerase Invitrogen . Major PCR products from the second PCR reaction were gel extracted with the Qiagen Gel Extraction Kit Qiagen Canada inc, Mississauga, ON, Canada and cloned using the TOPO TA Cloning Kit for sequencing Invitrogen . For each placenta, 15 different clones were randomly selected and amplified with M13 primers and sequenced with ABI PRISM 3100 capillary automated sequencer Applied Biosystems, Foster City, CA, USA . Sequences were analysed and aligned with GeneBank reference sequence NM_014257 using Lasergene software DNA Stars, Madison, WI, USA . Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia .", "Quantitative expression of DC-SIGNR isoforms 1,5 mg of placental RNA was reverse transcribed using 2.5 mM of Oligo dT 20 and Expand RT in 20 ml volume according to the manufacturer Roche Applied Science . 15 ng of total cDNA in a final volume of 20 ml was used to perform quantitative real-time PCR using Universal Express SYBR GreenER qPCR Supermix Invitrogen on a Rotor Gene Realtime Rotary Analyser Corbett Life Science, Sydney, Australia . Samples from 2 subjects in each group were used because RNA quality of others was not suitable for a qRT-PCR analysis. Amplification of all DC-SIGNR isoforms was performed using an exon 5 specific primer pair Table S1 . Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA.", "Membrane-bound isoforms were amplified using primers specific for exon 3, corresponding to the common trans-membrane domain of DC-SIGNR. Primers were targeted to the exon-exon junction and RNA extracts were treated with DNase Fermantas International inc, Burlington, ON, Canada to avoid amplification of contaminant DNA. Standard curves 50-500 000 copies per reaction were generated using serial dilution of a full-length DC-SIGNR or commercial GAPDH Invitrogen plasmid DNA. All qPCR reactions had efficiencies ranging from 99% to 100%, even in the presence of 20 ng of non-specific nucleic acids, and therefore could be compared. The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts.", "The copy number of unknown samples was estimated by placing the measured PCR cycle number crossing threshold on the standard curve. To correct for differences in both RNA quality and quantity between samples, the expression levels of transcripts were normalised to the reference GAPDH gene transcripts. GAPDH primer sequences were kindly provided by A. Mes-Masson at the CHUM. The results are presented as target gene copy number per 10 5 copies of GAPDH. The ratio of membrane-bound isoforms was calculated as E3/E5. Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M.", "Soluble isoforms were calculated by subtracting membrane-bound from total isoforms. We carried out qPCR assays in triplicate in three independent experiments. Results are expressed as mean6S.E.M. Statistical analysis was performed using the GraphPad PRISM 5.0 for Windows GraphPad Software inc, San Diego, CA, USA . Differences in baseline characteristics and genotypic frequencies of haplotypes or htSNPs were compared between groups using the x 2 analysis or Fisher's exact test. Logistic regression analysis was used to estimate odds ratios OR for each genotype and baseline risk factors. Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method.", "Multiple logistic regression was used to define independent predictors identified as significant in the crude analysis. ORs and 95% confidence interval were calculated with the exact method. Comparisons of continuous variables between groups were assessed with the unpaired two-tailed Student's t test when variables were normally distributed and with the Mann-Whitney U test when otherwise. Differences were considered significant at P,0.05. Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected.", "Written informed consent was obtained from all mothers who participated in the study and the ZVITAMBO trial and the investigation reported in this paper were approved by The We carried out an association study of DC-SIGNR polymorphism in 197 infants born to untreated HIV-1-infected mothers recruited in Harare, Zimbabwe. Among them, 97 infants were HIV-1-infected and 100 infants remained uninfected. Of the 97 HIV-1-infected infants, 57 were infected IU, 11 were infected IP, and 17 were infected PP. Timing of infection was not determined for 12 HIV-1-infected infants. Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups.", "Baseline characteristics of mothers and infants are presented in Table 1 . Maternal age and CD4 cell count, child sex, mode of delivery, duration of membrane rupture and gestational age were similar among all groups. However, maternal viral load .29 000 copies/ml was associated with increased risk in both IU and PP with odds ratios OR of 3.64 95% CI = 1.82-7.31, P = 0.0002 and 4.45 95% CI = 1.50-13.2, P = 0.0045 for HIV-1 transmission, respectively. Fifteen haplotype-tagged SNPs htSNPs corresponding to the 15 major DC-SIGNR haplotypes Figure 1 described among Zimbabweans were genotyped in our study samples Tables S2 and S3 . H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 .", "H1 31% and H3 11% were the most frequent haplotypes observed Figure 1 . Being homozygous for the H1 haplotype was associated with increased risk of both IU OR: 4.42, P = 0.022 and PP OR: 7.31, P = 0.016 HIV-1 transmission Table 2 . Infants harbouring two copy combinations of H1 and/ or H3 haplotypes H1-H1, H1-H3 or H3-H3 had increased risk of IU OR: 3.42, P = 0.007 and IP OR: 5.71, P = 0.025 but not PP P = 0.098 HIV-1 infection compared to infant noncarriers Table 2 . The latter associations remained significant after adjustment was made for the maternal viral load for both IU OR: 3.57, 95% CI = 1.30-9.82, P = 0.013 and IP OR: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 .", "The H1 and H3 haplotypes share a cluster of mutations p-198A, int2-391C, int2-180A, ex4RPT, int5+7C Figure 1 . Of these, the p-198A and int2-180A variants were significantly associated with MTCT of HIV-1 Table S2 . In the unadjusted regression analysis, homozygous infants for the p-198A and int2-180A variants had increased risk of IU OR: 2.07 P = 0.045, OR: 3.78, P = 0.003, respectively and IP OR: 2.47, P = 0.17, O.R: 5.71, P = 0.025, respectively HIV-1 infection compared to heterozygote infants or noncarriers Table 3 . When adjustment was made for maternal factors, only the association with the int2-180A variant remained significant for IU OR: 3.83, 95% CI = 1.42-10.4, P = 0.008 and IP O.R: 5.71, 95% CI = 1.40-23.3, P = 0.025 HIV-1 transmission. Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires .", "Thus, infants homozygous for DC-SIGNR variant int2-180A contained in H1 and H3 haplotypes were 4-fold to 6-fold more likely to be infected by HIV-1 during pregnancy or at delivery, respectively. Alternative splicing of the DC-SIGNR gene in the placenta produces both membrane-bound and soluble isoform repertoires . The relative proportion of membrane bound and soluble DC-SIGNR could plausibly influence the susceptibility to HIV-1 infection . We therefore hypothesized that the DC-SIGNR mutations associated with MTCT of HIV-1 would have an impact on both the level of DC-SIGNR expression and in the isoform repertoire produced. We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples.", "We investigated DC-SIGNR transcript expression in first-term placentas obtained after elective abortion. We cloned DC-SIGNR from placental tissues by RT-PCR from 3 homozygous H1 samples containing both the DC-SIGNR p-198AA and int2-180AA variants associated with HIV-1 transmission and 3 homozygous wild-type WT p-198CC, int2-180GG samples. Fifteen clones per sample were randomly selected for sequencing. As expected, we found an extensive repertoire of DC-SIGNR transcripts in all samples with 9 to 16 different isoforms per individual. A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons.", "A total of 65 distinct transcripts were identified Figure S1 , of which 3 were full-length transcripts. 64 of the sequenced clones contained a total of 69 amino acid substitutions with 3 new C termini and 2 premature stop codons. However, the diversity was mostly attributable to the entire deletion of exon 2 or exon 3 or to variations in the length of the neck region exon 4 of DC-SIGNR. The deletion of exon 3 eliminates the trans-membrane domain of the protein and leads to the expression of soluble DC-SIGNR isoforms . Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype.", "Interestingly, the abundance of membrane-bound isoforms in placental tissues of the H1 homozygotes appears to be lower than that observed in samples from WT individuals Figure S1 . The deletion of exon 3 was confirmed by sequencing and we hypothesize that the skipping of exon 3, could be due to the presence of the int2-180A mutation observed in infants with the H1 haplotype. In fact, this intron mutation is located 180 bp downstream from exon 3 and potentially modifies splicing events Figure 2A . We confirmed that the variation in transcript proportions seen between the two groups was also reflected at the level of mRNA expression in the placenta. To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio.", "To quantify membrane-bound vs soluble isoforms in placental samples from homozygous H1 and WT infants, we amplified the exon 5 E5 sequence present in all DC-SIGNR isoforms total transcripts . We then amplified exon 3 E3 which is deleted in the soluble forms and then calculated the E3:E5 ratio. We found that placental tissues from homozygous H1 infants express a significantly lower proportion of membrane-bound DC-SIGNR 18% compared to that in WT individuals 36% P = 0.004 Figure 2B suggesting that exon 3 skipping happens more frequently in presence of the DC-SIGNR int2-180A variant associated with MTCT of HIV-1. The DC-SIGNR int2-180A variant is always transmitted with the promoter mutation p-198A Figure 1 . In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 .", "In the unadjusted regression analysis, the p-198A variant was significantly associated with IU but not with IP and PP HIV-1 transmission Table 3 . Computational transcription factor binding site analysis predicts Table 1 . Baseline characteristics of mother and infants risk factors for intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Figure 3A . The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 .", "The luciferase activity of the p-198A variant construct was significantly lower than that of the WT p-198C promoter construct p-198C/A ratio = 2, P = 0.006 Figure 3B suggesting that DC-SIGNR p-198A affects promoter activity. The other promoter mutants p-577C and p-323A observed in the Zimbabwean population did not affect DC-SIGNR transcription in this assay Figure S2 . To determine the net impact of the DC-SIGNR p-198A mutation on DC-SIGNR expression in the placenta, we quantitated the absolute number of total and membrane-bound DC-SIGNR transcripts in the H1 homozygote and wild-type placental samples as described earlier. The total number of DC-SIGNR transcripts was determined to be 6856213 DC-SIGNR copies6S.E.M per 10 5 GAPDH copies in the placental samples from homozygous H1 infants and was 4-fold lower compared to that found in placentas from WT individuals 27816638, P = 0.011 Figure 3C . As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C .", "As suggested earlier, the int2-180A mutation might induce exon 3 skipping leading to a lower production of membrane-bound DC-SIGNR. Although, the decrease in the total number of DC-SIGNR transcripts in H1 homozygous placental samples containing both the p-198AA and int2-180AA variants affected the proportion of membrane-bound and soluble isoforms, the effect of these mutations was more pronounced on the membrane-bound isoforms with an 8-fold decrease H1 = 117636.2 vs WT = 9906220.6, P = 0.003 compared to a 3-fold decrease in total soluble isoforms H1 = 5686181.9 vs WT = 19256495.3, P = 0.03 Figure 3C . Therefore, DC-SIGNR p-198A and int2-180A mutations associated with MTCT of HIV-1 significantly decreased the level of total placental DC-SIGNR transcripts, disproportionately affecting the membrane-bound isoform production. Table 3 . Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1.", "Associations between infant DC-SIGNR promoter p-198 and intron 2 int2 -180 variants and intrauterine IU , intrapartum IP and postpartum PP mother-to-child HIV-1 transmission. Our genetic results, supported by expression assay in placenta, suggest the involvement of DC-SIGNR in MTCT of HIV-1. Homozygosity for the haplotype H1 was associated with IU transmission in the unadjusted regression analysis. However, the association disappeared after adjustment was made for the maternal factors presumably because of the small number of H1 homozygote infants analysed in each groups. H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1.", "H1 and H3 were the most frequent haplotypes observed in the study population and they share a cluster of mutations Figure 1 . Grouping haplotypes H1 and H3 increased the power of the study and permitted the identification of specific DC-SIGNR mutations associated with MTCT of HIV-1. Indeed, two mutations shared by haplotypes H1 and H3 were associated with vertical transmission of HIV-1. The int2-180A was associated with a 4-fold increased risk of IU and 6fold increased risk of IP after adjustment for the maternal factors. Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant.", "Although the p-198A variant was associated with IU transmission, the association disappeared after adjustment was made for the maternal viral load. Nevertheless, we showed that this mutation reduces DC-SIGNR transcriptional activity in vitro and produces lower level of DC-SIGNR transcripts in placental tissues in combination with the int2-180A variant. Since int2-180A is always transmitted with p-198A on the MTCT associated combined haplotypes H1/H3, whereas p-198A is carried on other nonassociated haplotypes Figure 1 , we can speculate that the p-198A mutation alone may have a minor effect in vivo whereas in combination with the int2-180A variant, they both act to reduce the level of placental DC-SIGNR expression resulting in an increased risk of MTCT of HIV-1. The majority of IU transmission occurs during the last trimester of pregnancy reviewed in . Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein .", "Full-term placenta samples were not available for the current study and the expression assays were performed on first-term placental tissues. A previous study looking at DC-SIGNR placental isoforms repertoire in full-term placenta samples demonstrated similar diversity of DC-SIGNR transcripts as in the first-term placental tissues studied herein . However, since levels of DC-SIGNR expression have never been compared between the different terms of pregnancy, it is not known whether DC-SIGNR expression varies during the course of pregnancy. Nevertheless, it is reasonable to assume that the inter-individual differences in both DC-SIGNR isoform repertoire and transcript levels observed between the H1 and WT homozygous infants would be reflected throughout the pregnancy. To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo .", "To date, most studies have focused on the potential role of DC-SIGNR in trans infection of HIV-1 in vitro . However, the multiple mechanisms involved in trans infection and redundancy among C-type lectin functions make it difficult to determine the actual participation of DC-SIGNR in this mode of infection in vivo . The strong correlation we observed between MTCT of HIV-1 and DC-SIGNR genetic variants producing low levels of DC-SIGNR in the placenta suggested that mechanisms other than DC-SIGNR-mediated trans infection might operate during vertical transmission of HIV-1. For example, DC-SIGNR has also been shown to function as a HIV-1 antigen-capturing receptor . Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response .", "Chan and colleagues recently demonstrated that DC-SIGNR transfected CHO cells diminish SARS-CoV titers by enhanced capture and degradation of the virus in a proteasome-dependent manner . Since endothelial cells express MHC-I and II, degraded viral antigens could then be presented to immune cells to elicit an adaptive immune response . The HIV-1 coreceptor CCR5, but not CD4, is co-expressed with DC-SIGNR on placental and blood-brain barrier BBB endothelial cells . HIV-1 gp120 binding to CCR5 receptor on endothelial cells compromises BBB integrity and enhances monocytes adhesion and transmigration across the BBB . It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery.", "It is thus possible that reduced expression of DC-SIGNR, particularly the membranebound isoforms, on placental capillary endothelial cells might favour HIV-1 binding to CCR5 receptor, instead of DC-SIGNR receptor, facilitating the migration of maternal HIV-1-infected cells across the placental barrier resulting in IU transmission of HIV-1. The int2-180A variant contained in the H1 and H3 haplotypes was associated with IP transmission suggesting that DC-SIGNR also affect transmission of HIV-1 during delivery. Little is known about the mechanisms underlying transmission of HIV-1 during delivery. Passage through the birth canal could potentially expose infants through a mucosal portal entry presumably ophthalmic, skin, or gastrointestinal , whereas placental insult during delivery physical or inflammatory may enhance transplacental passage of maternal HIV-1-infected cells into foetal circulation . Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery .", "Such process called microtransfusion has been proposed in regards to the results obtain in a Malawian cohort. Kweik and colleagues found a significant association between levels of maternal DNA in umbilical cord blood and IP transmission of HIV-1 suggesting that passage of maternal infected cells through the placenta is likely to occur during delivery . Thus, in a similar fashion as suggested earlier for IU transmission, the relatively lower level of DC-SIGNR in the placenta of homozygous infants harbouring the int2-180A variant could promote HIV-1 binding to CCR5 receptor on endothelial cells affecting the placental barrier integrity and facilitating the passage of maternal infected cells in foetal circulation during delivery. Beside DC-SIGNR, other HIV-1 receptors are known to influence MTCT of HIV-1 reviewed in . Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans .", "Genetic variants in CCR5 have been shown to influence vertical transmission of HIV-1. CCR5 promoter variants resulting in higher expression of the receptor were associated with increased risk of MTCT of HIV-1 among sub-Saharan Africans . The 32-pb deletion polymorphism in CCR5 has be shown to protect from vertical transmission of HIV-1 , but this variant is virtually absent among African populations . High copy numbers of CCL3L1, a potent HIV-1 suppressive ligand for CCR5, are associated with higher chemokine production and lower risk of MTCT of HIV-1 among South African infants . Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 .", "Mannose-binding lectin MBL is an innate immune receptor synthesised in the liver and secreted in the bloodstream in response to inflammation signal. MBL promotes pathogen elimination by opsonization and phagocytosis, and reduced expression of MBL resulting from polymorphism in coding and non-coding regions has been associated with an increased risk of MTCT of HIV-1 . In this study, we demonstrate for the first time, the potential functional impact of DC-SIGNR mutations on its expression in the placenta and in vertical transmission of HIV-1. We believe that the presence of DC-SIGNR at the placental endothelial cell surface may protect infants from HIV-1 infection by capturing virus and promoting its degradation/presentation. However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation.", "However, in placenta containing low levels of DC-SIGNR, HIV-1 would preferentially binds CCR5 on endothelial cells resulting in a loss of placental barrier integrity and enhanced passage of maternal HIV-1-infected cells in foetal circulation leading to MTCT of HIV-1. This mechanism may also apply to other vertically-transmitted pathogens known to interact with DC-SIGNR such as HIV-2, hepatitis C and dengue viruses and warrant further investigation. Associations between child DC-SIGNR exon 4 repeated region genotypes and mother-to-child HIV-1 transmission.CI, Confidence interval; N, number; NA; not applicable; OR, odds ratio a P-value as determined by the Chi-square test. b Comparison between genotype and all others. Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced.", "Found at: .1371/journal.pone.0007211.s003 Figure S1 DC-SIGNR transcripts repertoire in placenta. Major RT-PCR products from RNA extract from 3 homozygous H1 and 3 homozygous WT placenta samples were purified, cloned and sequenced. Sequenced were analysed according to NCBI reference sequence NM_014257. CT; cytoplasmic tail, TM; trans-membrane domain; WT; wild-type Found at: .1371/journal.pone.0007211.s004 Figure S2 Effect of DC-SIGNR promoter variant on transcriptional activity in luciferase reporter assay in vitro in transfected HeLa cells. Relative luciferase expression from pGL2-Basic, parental vector without promoter. Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate.", "Expression DC-SIGNR promoter constructs, spanning p-577C variant or p-323A variant were calculated relatively to this value. Data are presented in mean values6S.E.M of three independent experiments performed in triplicate. One-way ANOVA test followed by the Dunnett test for multiple comparison was used to compare the relative luciferase expression of the p-557C and p-323A variant reporters against the wild-type WT construct not significant . 0 mg, 0,5 mg or 1 mg CMV-Tat vector was transfected with LTR-Luc as a positive control in these experiments." ]

[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]

[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]

[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]

[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]

[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]

[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]

[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]

[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]

[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]

[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]

[ "Recently, one of the interferon-induced transmembrane IFITM family proteins, IFITM3, has become an important target for the activity against influenza A H1N1 virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane TM1 domain and in the cytoplasmic CP loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression.", "IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 FKBP11 to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid 17-ODYA , and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP , revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11.", "Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.", "The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane IFITM protein family also known as the Fragilis family in mice is a part of the dispanin family and is composed of double-transmembrane α-helices connected by a cytoplasmic CP loop and extracellular EC amino-and carboxyl-terminal polypeptide sequences Figure 1-A . The IFITM proteins are evolutionarily conserved in vertebrates . Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present.", "Recent genomic research has revealed that there are 5 IFITM members in humans IFITM1, 2, 3, 5 and 10 and 7 members in mice IFITM1, 2, 3, 5, 6, 7, and 10 . These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation IFITM1-3 , cell-to-cell adhesion IFITM1 , antiviral activity IFITM1-3 , and bone formation IFITM5 , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A H1N1 virus infection and internalization . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein . The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice.", "The S-palmitoylation is a post-translational modification on proteins by C 16 saturated-fatty acids palmitic acids covalently attached to certain cysteine residues via a thioester linkage Figure 1-B . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 top and IFITM3 bottom . The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively.", "The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage.", "The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C The amino acid sequence identity similarity among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. .1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity.", "The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane TM1 domain, and Cys105 in the CP loop Figure 1-A . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization . A resent study has revealed that murine IFITM1 has four cysteine residues Cys49, Cys50, Cys83, and Cys103 for the S-palmitoylation, which is required for the antiviral activity and the protein stability . The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein .", "The other IFITM family members also possess these cysteines Figure 1-A , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like BRIL protein . Among the IFITM family proteins, IFITM5 is unique. i Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C .", "Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells , while the other IFITM proteins are expressed ubiquitously ii . Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins ~ 65% similarity , while IFITM1-3 proteins share ~ 85% similarity with each other Figure 1 -C . In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding Figure 1 -A . iii Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones .", "Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies . The ifitm5-gene knockout mice have smaller bones . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation . iv Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 FKBP11 to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator FPRP complex . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity.", "When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 Bst2 , interferon inducible protein 1 Irgm , interferoninduced protein with tetratricopeptide repeats 3 Ifit3 , b.microglobulin B2m , and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid 17-ODYA , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid 2BP . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less .", "The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A Cys-less . The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11.", "The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan .", "The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 IFITM5-WT and FLAG-fused FKBP11 FKBP11-FLAG were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector Takara Bio, Shiga, Japan . The details of the recombinant DNA constructs were the same as described previously . The genes of IFITM5 mutants IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A Cys-less were prepared using a QuikChange site-directed mutagenesis kit Stratagene, La Jolla, CA . The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector.", "The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b Novagen, Madison, WI expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO .", "The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively Sigma-Aldrich, St. Louis, MO . Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank RCB 1126 . The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system.", "When necessary, 2-bromopalmitic acid 2BP; Wako, Osaka, Japan and 17-octadecynoic acid 17-ODYA; Sigma-Aldrich were dissolved in 99.5% dimethyl sulfoxide DMSO; Wako and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21 DE3 cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside IPTG , cells were harvested by centrifugation 6,400 × g for 10 min at 4°C . The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C .", "The cells were suspended in 50 mM Tris-HCl buffer pH 8 and disrupted by a French press Ohtake, Tokyo, Japan 100 MPa × 4 times . The crude membrane fraction was collected by ultracentrifugation 178,000 × g for 90 min at 4°C . The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside DDM Dojindo Lab, Kumamoto, Japan in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin Qiagen, Hilden, Germany . The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole.", "The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl pH 8 , 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column GE Healthcare UK, Ltd., Amersham Place, England . The experimental details are described in previous reports . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot.", "Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit BioChain Institute Inc., Newark, CA . Then, the cell lysate was incubated with anti-FLAG M2 agarose gel Sigma-Aldrich at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide Sigma-Aldrich dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE 15% ePAGEL; ATTO, Tokyo, Japan and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence TSYPREDPRAPSSRC , and anti-FLAG monoclonal antibody Sigma-Aldrich were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG H+L Zymed Laboratories, San Francisco, CA and goat anti-mouse IgG H+L Sigma-Aldrich antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins.", "The proteins were detected by chemiluminescent reaction MercK-Millipore, Billerica, MA . The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5.-carboxytetramethylrhodamine TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ with reference to previous studies and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA 565 nm was detected using a 575nm long-path filter Typhoon FLA 9000; GE Healthcare . The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation.", "The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium α-MEM; Sigma-Aldrich containing 10% v/v fetal bovine serum FBS; Nichirei Biosciences Inc., Tokyo, Japan . On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used.", "Mineralized nodules were stained with Alizarin Red S Sigma-Aldrich . The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation Figure 2-A . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells.", "For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker see lane 1 in the absence of 2BP. However, in the presence of 2BP see lane 4 , the band appeared at a lower position than that in the absence of 2BP lane 1 . These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine .", "These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine . Following hydroxylamine treatment see lane 2 , the band appeared at the same position as in the presence of 2BP lane 4 . In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | does not occur. Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results .", "Hence, the band was also observed at the same lower position see lane 3 . In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA Figure 2-C , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells.", "Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu I -catalyzed 3+2 azidealkyne cycloaddition method 10, 29, 30, 32 . An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT see lane 2 in Figure 2 -D showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling lanes 1 and 5 . In addition, after the hydroxylamine treatment see lane 6 , the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above lane 2 of Figure 2-B . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively.", "Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop Figure 1-A . All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less .", "All of these cysteines are highly conserved among the mammalian IFITM family proteins Figure 3-A . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A Cys-less . The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation.", "In the C52A/C53A and Cys-less mutants see lanes 2 and 4 , the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine lanes 3 and 7 , respectively, but not in the Cys-less mutant lanes 4 and 8 . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B .", "Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel lower panel of Figure 2 -D and Figure 3-B . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain.", "Previous studies have revealed that IFITM5 interacts with FKBP11 . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times.", "Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes KEGG database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3.", "Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated lanes 1 and 3 . The C52A/C53A lane 2 and Cys-less lane 4 mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. .1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG.", ".1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes upper panel . Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11.", "The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP see lane 1 , whereas IFITM5 was not S-palmitoylated in the presence of 2BP see lane 2 . After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP see lane 3 , indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared see lane 4 , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured.", "Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants C52A/ C53A, C86A, and Cys-less and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel.", "Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes upper panels because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant lanes 1 and 3 but not for the -C52A/C53A and Cys-less mutants lanes 2 and 4 . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11.", "Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants see Figures 2-D, 3 -B and the lower panel of Figure 4 -C . Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 also in Figure 4-A , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site s , Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B .", "Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP Figure 5-A and -B . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 .", "In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP Figure 5-A, -C, and -D , the mineralization was started 15 days after the initiation of the cell differentiation Day 0 . On the other hand, in the presence of 2BP Figure 5-B and -D , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP Figure 5-D . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21.", "In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP panel b , whereas in the absence of 2BP the nodules formed a large cluster panels a and c . Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells .", "In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity Figure 1-C , two cysteines in the TM1 domain Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2 and one cysteine in the CP loop Cys105 for IFITM3, Cys104 for IFITM2 are all S-palmitoylated in cells . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain Cys52 or Cys53 and one cysteine in the CP loop Cys86 are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins Figures 1-A and 3-A , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane .", "The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization the roles are summarized in Figure 6 -A and discussed in detail below . A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 lower panels , the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively .", "Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 FKBP11-FLAG in the osteoblast cells in the absence and the presence of 2BP denoted as \"-\" and \"+\", respectively . Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG.", "B Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus.", "A The functional mechanism of IFITM3 is summarized from previous studies. i IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, ii which induces clustering and correct positioning in the membrane, iii resulting in the antiviral activity against influenza virus. B The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression.", "The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells iii . The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in i - iii are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 .", "Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid s attached to the cysteine s summarized in Figure 6 -B, discussed in detail later . Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction.", "Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 BCAP31 , and the hydroxysteroid 17-beta dehydrogenase 7 HSD17b7 . These three proteins bind to the IFITM5 without the S-palmitoylation low molecular-mass form; see Figure 3 -b in ref . and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient .", "and Figure 1 -B in ref . . In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex.", "The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity.", "If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells Figure 5 . It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules panel b of Figure 5 -E ; we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP.", "While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii .", "In the case of IFITM3, as shown in Figure 6 -A, the following are observed. i The three cysteines are all S-palmitoylated ii . The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane iii . The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5.", "When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. i Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated ii . The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11.", "The interaction is presumed to be mediated by the palmitic acid s attached to the cysteine s facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes.", "The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 iii . The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states i ~ iii illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected.", "The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation.", "In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address i whether the S-palmitoylation on IFITM5 is constitutive or regulated, or ii when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway." ]

[ "Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors.", "Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction.", "The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s .", "Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al.", "Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS.", "In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR.", "Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor.", "NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected .", "Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.", "For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\".", "The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment." ]

[ "Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors.", "Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction.", "The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s .", "Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al.", "Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS.", "In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR.", "Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor.", "NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected .", "Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.", "For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\".", "The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment." ]

[ "Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors.", "Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction.", "The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s .", "Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al.", "Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS.", "In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR.", "Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor.", "NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected .", "Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.", "For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\".", "The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment." ]

[ "Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors.", "Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction.", "The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s .", "Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al.", "Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS.", "In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR.", "Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor.", "NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected .", "Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.", "For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\".", "The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment." ]

[ "Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors.", "Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction.", "The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s .", "Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al.", "Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS.", "In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR.", "Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor.", "NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected .", "Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.", "For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\".", "The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment." ]

[ "Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors.", "Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction.", "The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s .", "Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al.", "Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS.", "In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR.", "Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor.", "NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected .", "Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.", "For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\".", "The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment." ]

[ "Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus HBV and its satellite hepatitis D virus HDV still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma HCC cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors.", "Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction.", "The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender . HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s .", "Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 amino acids 1-47 necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor s . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al.", "Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor s . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS.", "In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control homologous peptide without specific binding , they identified one cellular protein, NTCP sodium taurocholate cotransporting peptide by LC/MS/MS. The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR.", "Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor.", "NTCP is a transmembrane protein, usually located in the lateral surface canalicular of hepatocytes, which mediates bile acid transport . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected .", "Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one hundreds or thousands fold viral amplification . For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro.", "For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors maybe carboxypeptidase D and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\".", "The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says \"a thousand-mile journey starts from one incremental step\". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "BACKGROUND: This paper reports findings from the prevalence survey conducted in Shandong China in 2010, a province with a population of 94 million. This study aimed to estimate TB prevalence of the province in 2010 in comparison with the 2000 survey; and to compare yields of TB cases from different case finding approaches. METHODS: A population based, cross-sectional survey was conducted using multi-stage random cluster sampling. 54,279 adults participated in the survey with a response rate of 96%. Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture.", "Doctors interviewed and classified participants as suspected TB cases if they presented with persistent cough, abnormal chest X-ray CXRAY , or both. Three sputum specimens of all suspected cases were collected and sent for smear microscopy and culture. RESULTS: Adjusted prevalence rate of bacteriologically confirmed cases was 34 per 100,000 for adults in Shandong in 2010. Compared to the 2000 survey, TB prevalence has declined by 80%. 53% of bacteriologically confirmed cases did not present persistent cough. The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s.", "The yield of bacteriologically confirmed cases was 47% by symptom screening and 95% by CXRAY. Over 50% of TB cases were among over 65’s. CONCLUSIONS: The prevalence rate of bacteriologically confirmed cases was significantly reduced compared with 2000. The survey raised challenges to identify TB cases without clear symptoms. Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 .", "Text: China, with an estimated prevalence of all TB cases of 108 per 100,000 in 2010, has the second highest TB burden in the world, accounting for 13% of all cases worldwide . The World Health organization WHO estimated that China had reached the targets of 85% treatment success by 1993 and 70% case detection rate by 2005 . National TB prevalence surveys were conducted in China in 1979 China in , 1990 China in , 2000 , and 2010 . Survey results provide more accurate estimates for TB prevalence rates than the WHO estimates and can be used to assess the likelihood of China achieving global targets for TB prevalence. Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 .", "Shandong province has a population of 94 million. It is a relatively developed province with a per capita GDP 1.6 times of the national average in 2010 . The prevalence rate of TB in Shandong was lower compared with the average rate of China in 2000 . Population representative samples were drawn in Shandong in the surveys of 2000 and 2010 using similar methods. The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months.", "The study aimed to estimate the TB prevalence in Shandong based on the 2010 survey, and compare results of the two cross sectional surveys. The target population of the TB prevalence survey was residents of 15 years old or above who had lived in the selected clusters for more than 6 months. A population based, cross-sectional survey was conducted using multistage random cluster sampling method. The survey employed the same sampling methods as the China national survey in 2010, which was similar to the sampling methods used in 2000 . The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 .", "The design of the surveys was in accordance with WHO recommendations . The design effect factor due to cluster sampling was estimated at 1.28 . A sample size of 52500 adults ≧15 years old , in 35 clusters, was calculated based on detecting a change of 20% in prevalence rate of TB smear positive cases compared with the rate of the 2000 survey 95 per 100,000 , with a probability greater than 95% and 95% power, accounting for 90% response rate of participants . A stratified multi stage random sampling was used to select the 35 clusters within 17 prefectures in Shandong province. The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older .", "The number of clusters was randomly allocated in proportion to the provincial population at the prefectural, county/district and township levels. A cluster was defined as a community a village in the rural area or a resident community in an urban area with a population of 1250 to 1750 adults i.e., those of 15 years or older . If the community contained less than 1250 adult residents, the neighboring community to the north was annexed. If the community or combined communities containing more than 1750 adults, we randomly selected households and then included all adults in the household for the survey until the total number of selected adults reached 1750. Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses.", "Military barracks and prisons located in the cluster were excluded . The survey was conducted from March to June 2010 by survey teams consisting of clinicians, public health doctors, radiologists, laboratory technicians and nurses. Local media was used to promote awareness of the survey. Community workers conducted a house-to-house census to update the database of residents, inform survey participants and obtain informed consent. The study did not involve children under 15 years old. Written informed consent was obtained from all participants of 16 years old or above. While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital.", "While from those of 15 years old, written informed consents were obtained from their parents or guardians. All documents were properly stored in the Shandong Chest Hospital. Ethical approvals for the study and consent procedures were obtained from the Institutional Review Board IRB of Shandong Chest Hospital NIH register numberIRB00006010 . Those who agreed to participate in the survey were invited to the county TB dispensary, where they completed a consultation with a trained clinical TB doctor regarding any symptoms suggestive of TB, such as persistent cough lasting two weeks or longer , haemoptysis, weight loss and fever. All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases.", "All participants had a chest X-ray CXRAY taken that then were reviewed by a panel of radiologists. Those with symptoms or CXRAY films suggestive of TB were classified as suspected TB cases. All suspected cases were asked to produce three sputum samples, one at the time of consultation, another at night and the third in the early morning of the following day. Identified suspects completed an additional questionnaire regarding their social-economic situation, smoking status, and the presence of TB related symptoms in the preceding six months cough, fever, weight loss, chest pain and haemoptysis . Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation.", "Sputum smears were conducted in local TB dispensaries. All sputum samples were cultured using the Löwenstein-Jensen medium in the provincial laboratory within 24 hours using cold chain transportation. Samples were excluded from TB when non-tuberculosis bacilli were identified from the culture. All sputum smear and culture were conducted strictly according the national TB laboratory external quality control measure, which is in consistent with the WHO TB prevalence survey guideline . TB classification was made according to the China national TB guideline . A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases.", "A positive smear had at least one acid fast bacillus identified during examination of at least 100 fields. Participants with positive sputum smear specimens were classified as sputum positive cases. Those with positive smear or culture sputum specimens were classified as sputum bacteriologically confirmed cases. Those being culture negative with abnormal CXRAY suggestive of TB and having been ruled out from other diseases by clinicians and radiologists were classified as CXRAY suggestive bacteriologically negative cases. Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system.", "Due to resource limitations the recommendation of broad-spectrum antimicrobial agents to confirm the diagnosis of negative TB cases was not applied in this survey . Newly diagnosed cases were distinguished from previously diagnosed cases through checks during the interviews and against the TB registration system. Initial diagnosis was made by a group of local clinicians and radiologists. Subsequently, samples and CXRAY films of all suspected and confirmed cases were re-assessed by a group of senior clinicians and radiologists at provincial and national levels. CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases.", "CXRAY films of 100% of those scored as abnormal and 10% random sampling of those scored as normal were transferred for independent reading. The provincial laboratory team randomly examined one slide from the three samples of sputum positive cases, all three samples of CXRAY suggestive TB cases, and randomly selected 10% of the non-TB cases. Prevalence estimates of sputum positive, bacteriologically confirmed and all TB cases were calculated. In all analyses, population weightings were employed to adjust for the stratified multi-stage sampling design effect . The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups .", "The survey results in 2010 and 2000 were standardized against the age structures of China's census population in 2010. The 2000 TB prevalence survey included all age groups . The WHO recommended method was used to enable comparison between the two surveys that prevalence rates of child TB were assumed to reduce to the same extent as adult TB from 2000 to 2010 . Subgroup analysis in gender, age groups and urban/rural residence were conducted. Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases.", "Case identification rate was calculated as the number of cases identified by a screening method over all suspected cases found by the method. Yields of the symptom consultation and CXRAY were calculated as a proportion of the total number of bacteriologically confirmed cases. The survey selected 17 urban clusters and 18 rural clusters. It covered a total population of 89,093, of which 56,671 were eligible for the survey Figure 1 . The response rate ranged from 95% to 97% in different clusters. 54,279 participants attended clinical consultation and were examined by CXRAY. Among them, 47% were males. The average age was 46 years with 14% of 65 years and older.", "Among them, 47% were males. The average age was 46 years with 14% of 65 years and older. A total of 572 suspected TB cases were found. Of these, 264 46% were identified based on CXRAY abnormalities, 228 40% were based on persistent cough, 80 14% were based on both. The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program.", "The survey diagnosed 172 new cases, including 19 new bacteriologically confirmed cases including 11 sputum and culture positive cases, and 8 sputum negative but culture positive cases and 153 CXRAY suggestive bacteriologically negative cases. The survey also identified 11 existing cases registered on the national TB program. In addition, the survey found four cases with culture positive non-tuberculosis bacilli, and excluded them from TB patients. All participants of the survey were first screened by symptoms and CXRAY. Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% .", "Those who had symptoms of consistent cough or haemoptysis, or CXRAY abnormalities were then screened by smear and culture. Case identification rates of new bacteriologically confirmed cases from the suspected cases were significantly higher with CXRAY as a primary tool Figure 1 , 3.8%, P = 0.012 and further increased by both symptom screen of persistent cough and CXRAY 10%, P < 0.001 compared with symptom screen alone 0.4% . The same pattern of case identification rate was observed in the sputum positive cases 7.5%, 1.9% and 0% respectively . The proportion reporting persistent cough was not significantly higher among bacteriologically confirmed cases compared with other suspects P = 0.565 . The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB.", "The symptom consultation alone identified 308 suspects, including 6 1.9% sputum smear positive TB and 9 2.9% bacteriologically confirmed TB. Among the 344 suspects with CXRAY abnormalities, 11 3.2% had sputum positive TB and 18 5.2% had bacteriologically confirmed TB. The yield of bacteriologically confirmed cases was 47.4% by screening consultation and 94.7% by CXRAY. In the population of over 65 years old, symptom consultation and the CXRAY identified 174 and 182 suspected cases respectively, yielding5 2.9% and 9 4.9% of bacteriologically confirmed cases. Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 .", "Yields of bacteriologically confirmed cases were 55.6% by symptom consultation and 100% by CXRAY among over 65's. Of the 512 suspected cases that completed the additional questionnaire, 42% were farmers and 31% were current smokers Table 1 . Per capita household income of bacteriologically confirmed cases was less than 50% of that of the non-TB cases P < 0.05 . Though smoking rate was higher among TB cases compared with non-TB cases, no significant differences were found P > 0.05 . Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 .", "Of the ten bacteriologically confirmed cases not presenting with persistent cough at the prevalence survey, one coughed for two days, one had chest pain, and the other eight had no symptoms of TB in the last six months. The crude prevalence rate in Shandong in 2010 of sputum positive cases was 22.1 95% CI: 9.6-34.6 , bacteriologically confirmed cases was 36.8 95% CI: 17.8-55.8 , and all cases were 337.1 95% CI: 254.1-420.0 per 100,000 in adult population Table 2 . The adjusted prevalence rates of the whole population in Shandong were17.8 95% CI: 8.3-17.5 , 27.8 95% CI: 14.8-28.0 and 239.4 95% CI: 179.9-298.9 per 100,000 in 2010. A remarkable decline of 82.0%, 80.2% and 31.4% was observed in TB prevalence rates of sputum positive, bacteriologically confirmed, and all cases, respectively, compared to the adjusted rates in 2000 . Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively.", "Large declines were observed in males between 40 and 65 years old, and in females over 60 years old Figure 2 . The adjusted prevalence rates in the adult population were 21.4 95% CI: 10.0-32.8 , 33.5 95% CI: 17.8-49.2 and 285.8 95% CI: 254.2-356.4 for sputum positive cases, bacteriologically confirmed cases and all cases, respectively. Significant differences regarding adjusted TB prevalence rates were observed between males and females, over 65's and 15 to 64 years old, in rural and urban areas Table 2 , P < 0.001 . The male to female ratios were 5.5 in sputum positive cases and 2.8 in bacteriologically confirmed cases, while the ratios climbed to 6.0 and 4.1, respectively, among those over 65 years. The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases.", "The majority of TB patients, 54.5% of sputum positive cases and 47.3% of bacteriologically confirmed cases, were from people 65 years or older. The ratio between over 65's and 15 to 64 years old was 8.4 in sputum positive cases and 5.9 in bacteriologically confirmed cases. The ratio between rural and urban areas was 2.7 in sputum positive cases and 4.8 in bacteriologically confirmed cases. The most striking finding was that a large proportion of TB patients did not present consistent cough. Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY.", "Passive case finding is the routine practice in developing countries where sputum microscopy is performed to identify TB cases among people with persistent cough. A large proportion of TB cases may be missed using this method as 53% of bacteriologically confirmed cases and 45% sputum positive cases in this study had no persistent cough but were identified through abnormal CXRAY. Nearly half of bacteriologically confirmed cases reported no symptoms in the last six months. This finding, although initially surprising, is consistent with reports from Vietnam 47% of bacteriologically confirmed cases not presenting persistent cough , Myanmar 38% and Ethiopia 48% . CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput .", "CXRAY was sensitive in detecting TB cases, as yields of bacteriologically confirmed cases were much higher by CXRAY compared with by symptom screening, as reported in Vietnam and some high HIV prevalence settings . CXRAY, though expensive at the initial installment, may improve TB case finding due to its short turnover time and high throughput . Our findings suggest that the strategy of case finding using CXRAY followed by sputum or culture as the primary and secondary screening tests could be more effective, especially among the population of over 65 year olds, as the yields were higher in over 65's compared with the general Table 2 Prevalence rates of sputum positive TB cases, bacteriologically confirmed TB cases and all cases in Shandong, China, 2010 No population. Although using CXRAY to examine everyone is not feasible, it can be used in routine elder physical examinations. The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before.", "The China public health package now covers free CXRAY for elders, as well annual employee body examinations provided free CXRAY. In this survey, only one sputum positive patient had been detected and treated by the national program, though specific clinical consultation was conducted to identify any patients who have been diagnosed and treated for TB before. This may reflect the difference between the active case finding approach in the survey and the passive casing finding approach in practice. Nevertheless, it indicated that a large proportion of bacteriologically confirmed TB cases are missed by the national TB program. Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 .", "Another notable change is the sharp decline of the proportion of sputum positive cases, which accounted for 30.5% of all cases in the 2000 survey but was reduced to 6.6% in the 2010 survey. The proportion of notified sputum cases out of all TB cases in Shandong also declined from 80.9% in 2005 to 64.6% in 2010 . The prevalence rate of bacteriologically confirmed cases has reduced by 80% in the last decade in Shandong, compared with a national decline of 45% from 216/ 100,000 in 2000 to 119/ 100,000 in 2010 . The rapid decline of TB prevalence rate of bacteriologically confirmed cases in the recent decade may be attributed to China's strengthened public health system following the outbreak of severe acute respiratory syndrome in 2003 . Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries .", "Another reason may be due to improved reporting of TB cases in the online communicable disease reporting system, and the improved collaboration between public hospitals and TB dispensaries . Other factors such as social economic development may also have played an important role in the reduction of TB prevalence, as found in a study of TB notification rates trends in 134 countries . The adjusted prevalence rate of bacteriologically confirmed cases in Shandong was lower than the WHO estimates for China in 2010 . But the national prevalence rates of bacteriologically confirmed cases, 119/100,000 in 2010 , was higher than the WHO estimate, 108/ 100,000, even the survey did not collect negative and extra-pulmonary TB cases. Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding.", "Vietnam reported similar findings in its 2006 survey . One reason is that prevalence surveys results are based on active case finding while WHO estimates are based on notification rates from passive case finding. A re-evaluation of the reported TB prevalence in China is needed based on the recent survey. CXRAY suggestive bacteriologically negative cases may be smear or culture negative TB cases if they had any TB symptoms, while some may be caused by suboptimal smear or culture. As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough.", "As reported in China's previous surveys , including these cases as TB cases may result in an over-estimate of all pulmonary cases . The survey revealed that over half of the TB patients were 65 years and older in Shandong, while the over 65's were more likely to present with abnormal CXRAY and persistent cough. Similar trends have been documented in other developed cities such as Hong Kong and Singapore . These high rates may reflect the higher TB rates in the past and decline in immunity in the over 65's. How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels.", "How to treat elders with TB and other complications such as diabetes remains an ongoing challenge in China and similar settings. The survey results can be generalized to the Shandong population of 94 million or similar international settings with middle income and middle TB prevalence levels. The patterns of the TB epidemic found in Shandong, i.e., the proportion of patients with symptoms, ratios between urban and rural areas, men and women, were similar to those found in the national survey . However, the prevalence rates cannot be extrapolated to western provinces in China with a higher TB prevalence. For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population .", "For logistical reasons, the eligible population did not include adults staying in the sampled clusters less than 6 months, which was the same practice in the 2000 survey. However, shortterm migrants may have a potentially higher prevalence of TB than the general population . This may result in a lower estimate of the true prevalence rate. The survey did not collect social-economic indicators, smoking status and HIV status of all participants, so comparisons between TB cases and all non-TB patients are not available. However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB.", "However, the HIV prevalence in Shandong China is below 0.01%, and would not significantly alter the TB prevalence rate. In addition, the survey did not evaluate child TB and extra pulmonary TB. Discussions of using CXRAY as a screening tool was on the technical aspect, but not on costing side as we did not conduct any cost effectiveness analysis or the social willingness to pay for such a strategy in similar settings. This study has shown that the prevalence of bacteriologically confirmed TB in Shandong has reduced substantially over the last decade. Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention.", "Importantly, the majority of these cases did not present with persistent cough and the proportion of sputum positive cases has declined sharply. Further studies are recommended to assess the feasibility of adopting CXRAY in the existing health care services to detect TB cases and the cost effectiveness of such intervention. The authors declare that they have no competing interests." ]

[ "RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.", "Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.", "Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.", "Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.", "It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .", "Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .", "Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.", "Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.", "Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.", "Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.", "Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.", "In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.", "A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.", "For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.", "Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.", "Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.", "The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.", "In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.", "For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.", "The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .", "The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.", "Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.", "The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .", "Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.", ". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .", "To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.", "Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.", "Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .", "An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .", "Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.", "This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .", "Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.", "Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.", "Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.", "Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.", "The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.", "Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.", "Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .", "Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .", "ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.", "Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.", "RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.", "Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .", "PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .", "These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .", "COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.", "Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.", "nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.", "Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.", "The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.", "However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .", "The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .", "It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.", "In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .", "MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.", "This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .", "There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.", "Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.", "Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.", "A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .", "Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.", "Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.", "In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .", "Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .", "Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.", "Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .", "The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.", "MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.", "Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .", "Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.", "It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.", "They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.", "This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.", "Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.", "Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.", "A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.", "Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients." ]

[ "RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.", "Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.", "Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.", "Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.", "It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .", "Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .", "Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.", "Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.", "Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.", "Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.", "Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.", "In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.", "A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.", "For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.", "Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.", "Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.", "The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.", "In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.", "For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.", "The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .", "The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.", "Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.", "The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .", "Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.", ". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .", "To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.", "Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.", "Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .", "An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .", "Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.", "This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .", "Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.", "Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.", "Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.", "Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.", "The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.", "Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.", "Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .", "Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .", "ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.", "Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.", "RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.", "Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .", "PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .", "These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .", "COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.", "Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.", "nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.", "Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.", "The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.", "However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .", "The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .", "It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.", "In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .", "MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.", "This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .", "There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.", "Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.", "Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.", "A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .", "Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.", "Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.", "In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .", "Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .", "Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.", "Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .", "The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.", "MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.", "Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .", "Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.", "It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.", "They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.", "This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.", "Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.", "Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.", "A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.", "Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients." ]

[ "RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.", "Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.", "Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.", "Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.", "It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .", "Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .", "Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.", "Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.", "Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.", "Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.", "Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.", "In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.", "A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.", "For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.", "Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.", "Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.", "The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.", "In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.", "For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.", "The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .", "The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.", "Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.", "The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .", "Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.", ". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .", "To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.", "Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.", "Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .", "An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .", "Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.", "This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .", "Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.", "Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.", "Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.", "Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.", "The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.", "Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.", "Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .", "Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .", "ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.", "Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.", "RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.", "Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .", "PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .", "These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .", "COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.", "Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.", "nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.", "Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.", "The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.", "However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .", "The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .", "It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.", "In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .", "MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.", "This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .", "There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.", "Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.", "Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.", "A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .", "Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.", "Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.", "In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .", "Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .", "Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.", "Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .", "The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.", "MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.", "Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .", "Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.", "It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.", "They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.", "This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.", "Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.", "Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.", "A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.", "Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients." ]

[ "RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.", "Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.", "Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.", "Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.", "It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .", "Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .", "Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.", "Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.", "Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.", "Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.", "Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.", "In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.", "A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.", "For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.", "Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.", "Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.", "The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.", "In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.", "For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.", "The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .", "The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.", "Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.", "The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .", "Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.", ". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .", "To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.", "Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.", "Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .", "An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .", "Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.", "This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .", "Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.", "Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.", "Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.", "Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.", "The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.", "Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.", "Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .", "Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .", "ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.", "Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.", "RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.", "Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .", "PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .", "These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .", "COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.", "Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.", "nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.", "Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.", "The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.", "However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .", "The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .", "It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.", "In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .", "MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.", "This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .", "There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.", "Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.", "Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.", "A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .", "Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.", "Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.", "In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .", "Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .", "Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.", "Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .", "The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.", "MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.", "Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .", "Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.", "It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.", "They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.", "This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.", "Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.", "Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.", "A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.", "Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients." ]

[ "RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.", "Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.", "Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.", "Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.", "It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .", "Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .", "Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.", "Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.", "Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.", "Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.", "Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.", "In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.", "A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.", "For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.", "Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.", "Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.", "The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.", "In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.", "For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.", "The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .", "The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.", "Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.", "The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .", "Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.", ". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .", "To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.", "Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.", "Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .", "An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .", "Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.", "This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .", "Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.", "Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.", "Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.", "Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.", "The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.", "Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.", "Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .", "Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .", "ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.", "Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.", "RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.", "Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .", "PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .", "These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .", "COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.", "Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.", "nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.", "Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.", "The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.", "However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .", "The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .", "It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.", "In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .", "MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.", "This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .", "There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.", "Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.", "Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.", "A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .", "Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.", "Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.", "In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .", "Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .", "Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.", "Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .", "The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.", "MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.", "Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .", "Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.", "It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.", "They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.", "This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.", "Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.", "Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.", "A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.", "Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients." ]

[ "RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.", "Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.", "Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.", "Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.", "It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .", "Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .", "Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.", "Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.", "Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.", "Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.", "Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.", "In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.", "A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.", "For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.", "Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.", "Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.", "The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.", "In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.", "For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.", "The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .", "The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.", "Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.", "The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .", "Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.", ". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .", "To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.", "Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.", "Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .", "An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .", "Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.", "This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .", "Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.", "Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.", "Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.", "Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.", "The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.", "Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.", "Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .", "Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .", "ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.", "Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.", "RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.", "Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .", "PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .", "These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .", "COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.", "Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.", "nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.", "Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.", "The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.", "However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .", "The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .", "It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.", "In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .", "MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.", "This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .", "There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.", "Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.", "Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.", "A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .", "Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.", "Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.", "In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .", "Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .", "Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.", "Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .", "The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.", "MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.", "Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .", "Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.", "It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.", "They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.", "This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.", "Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.", "Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.", "A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.", "Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients." ]

[ "RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.", "Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.", "Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.", "Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.", "It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .", "Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .", "Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.", "Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.", "Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.", "Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.", "Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.", "In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.", "A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.", "For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.", "Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.", "Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.", "The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.", "In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.", "For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.", "The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .", "The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.", "Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.", "The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .", "Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.", ". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .", "To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.", "Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.", "Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .", "An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .", "Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.", "This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .", "Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.", "Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.", "Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.", "Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.", "The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.", "Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.", "Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .", "Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .", "ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.", "Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.", "RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.", "Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .", "PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .", "These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .", "COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.", "Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.", "nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.", "Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.", "The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.", "However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .", "The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .", "It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.", "In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .", "MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.", "This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .", "There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.", "Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.", "Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.", "A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .", "Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.", "Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.", "In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .", "Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .", "Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.", "Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .", "The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.", "MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.", "Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .", "Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.", "It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.", "They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.", "This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.", "Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.", "Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.", "A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.", "Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients." ]

[ "RNA interference RNAi is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA mRNA degradation. Two types of small RNA molecules, i.e. small interfering RNAs siRNAs and microRNAs miRNAs , are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route.", "Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases. Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique.", "Text: traditional surgery, Bivas-Benita et al. reported that no mortality occurred as a result of the use of the endotracheal technique. Endotracheal applications are currently being used by many practitioners in the pulmonary field ; this is useful for studying pulmonary drug delivery in mice. However, the approach is more complex in humans because an artificial path for the delivery of drugs into the lungs is used. Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings.", "Therefore, the method is being used in animal models to test and evaluate its reliability for possible clinical applications. Intratracheal route: under anesthesia, the trachea is exposed surgically, and a tube or needle is inserted through an incision made between the tracheal rings. Complications, such as vascular injury and air leakage, are possible due to the tracheotomy. b Endotracheal route: siRNAs are sprayed directly from the mouth into the lungs using a MicroSprayer ® aerolizer Penn-Century, Philadelphia, PA, USA and a laryngoscope. It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies.", "It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details .", "Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus RSV infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products see Section 3.1.1. for details . Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application .", "Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history.", "Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.", "Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination. For most MDIs, the propellant is one or more gases called chlorofluorocarbons CFCs . Although CFCs in drugs are safe for patients to inhale, they are harmful to the environment. Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder.", "Therefore, further development of inhalable siRNAs may not be the best way forward. DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.", "Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated . In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable.", "In the future, the technique is desirable to estimate the in vivo study on siRNA therapy for inhalation. In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.", "A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.", "For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.", "Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.", "Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural \"remodeling\" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions.", "The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells.", "In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression.", "For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes.", "The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane. Despite their small size, the negative charge and chemical degradability of siRNA molecules prevent them from readily crossing biological membranes. Therefore, efficient siRNA delivery approaches need to overcome this limitation by facilitating cellular uptake. One of the main functions of a delivery vector is to facilitate the cellular uptake of siRNAs . The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis .", "The electrostatic complexation of siRNA molecules with cationic lipids and polymers helps to mask their net negative charge. The positively charged siRNA carrier complex interacts with anionic proteoglycans on the cell membrance, forms an endocytic vesicle, and enters the cells by endocytosis . After cellular internalization, the siRNA carrier complex in endocytic vesicles is transported along microtubules to lysosomes that are co-localized with the microtubule-organizing center. To avoid lysosomal degradation, siRNAs must escape from the endosome into the cytoplasm, where they can associate with the RNAi machinery. Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors.", "Endosomal escape is a major barrier for efficient siRNA delivery . The endosomal entrapment and lysosomal degradation of siRNA and carriers contribute to the low transfection efficiency and is a major difficulty for delivery vectors. An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells.", "The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth .", "Recently, Guo et al. showed that lentivirus-mediated siRNA was used to specifically knock down the expression of nuclear protein 1 NUPR1 in vivo, which resulted in inhibited tumor growth . However, viral-based delivery has several disadvantages. The immune response to viruses not only impedes gene delivery but also has the potential to cause severe complications . Recent well-documented cases, such as the death of Jesse Gelsinger due to complications related with an adenoviral delivery vector, highlight this problem . In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery.", ". Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size . After siRNAs are successfully taken into the target cells by endocytosis, the main barriers for delivering siRNAs to its site of action are the endosomal entrapment and lysosomal degradation of siRNA and carriers. To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity .", "To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results. Lipid-based delivery vectors are successfully used to deliver siRNA in vitro and in vivo . Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance.", "Cationic lipids are composed of positively charged head, a linker and hydrophobic. In general, lipid-based complexes are easy to formulate and good transfection efficacy is achieved due to interaction with negative charged cell membrance. Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching.", "Cationic polymer polyethylenimine PEI is widely used for siRNA delivery . PEI is considered as the gold standard for in vitro gene delivery and its transfection efficiency depends on the molecular weight and degree of branching. On the other hand, lipid-based vectors can also induce toxicity and non-specific activation of inflammatory cytokine and interferon responses . Although polymer-based vectors elicit a relatively less strong immune response than lipid-based vectors, effective siRNA delivery to a local area in lung diseases requires more attention to the development of non-toxic delivery vectors. An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response .", "An important point for siRNA-mediated inhibition of gene expression is whether the observed effects are specific rather than due to off-target effects and free from potential interferon responses . Interestingly, some studies have shown that it was possible to administer \"naked siRNAs\" to mice and down-regulate an endogenous or exogenous target without inducing an interferon response . The term \"naked siRNAs\" refers to the delivery of siRNAs without any delivery vectors. Naked siRNAs are degraded by serum endonucleases and are usually removed by glomerular filtration, resulting in a short plasma half-life of < 10 min. Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs .", "Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs . Indeed, in one clinical trial, the delivery of naked siRNAs for the treatment of RSV has been used . This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally.", "This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial .", "Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors. Nevertheless, the advantage of naked siRNAs over delivery vectors in the treatment of lung diseases is controversial . Further in vivo investigations about both naked siRNAs and non-viral vectors are required. Lung disease is a major cause of death, and diminished quality of life is responsible for the suffering of many patients. Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure.", "Various lung diseases make life extremely difficult for the patients, and severe cases of these lung diseases can result in death. The high death rates associated with lung cancer are partially due to the fact that it is unfortunately difficult to cure. Above all, COPD is the fourth-leading cause of death in most industrialized countries and is predicted to become third by 2020 . Therefore, decisive action is needed to stem the rising health and economic burden this represents. Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases.", "Chronic lung diseases, such as COPD and asthma, are disorders of the airways largely related to the presence of persistent inflammation. The approval of inhaled corticosteroids pioneered a new generation of therapy in treating chronic inflammatory diseases. This was the first time that an anti-inflammatory product was available to reduce the characteristic lung inflammation in airways and the associated obstruction. Corticosteroids are still an important therapeutic intervention. However, they are used with limitations in COPD and moderate to severe asthma. Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia.", "Likewise, the treatment of various refractory lung diseases also depends on systemic corticosteroid therapy. Many of these patients also suffered various side effects from systemic corticosteroid use, such as weight gain and uncontrolled hyperglycemia. Treatment of lung disease using cell-specific targeting as well as RNAi techniques represents a novel strategy and could possibly provide new opportunities in nanomedicine. Pulmonary applications of siRNA in in vivo conditions are frequently studied and often result in clinical trials . The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized.", "The findings of recent clinical studies of pulmonary RNAi therapeutics are discussed. Since the discovery of RNAi, the therapeutic potential of siRNAs has been rapidly recognized. In 2004, the first human clinical trial of RNAi-based therapy was initiated for the treatment of age-related macular degeneration with a siRNA targeting VEGF-receptor 1 delivered intravitreally . Many studies have been conducted over the past few years that involve the delivery of siRNAs to the lungs for the treatment of various lung diseases. Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection.", "Delivery to the lungs will be most important to moving siRNA technology into the clinic. A number of siRNA-based therapies are being evaluated in clinical trials for the treatment of different conditions, including lung diseases such as asthma and RSV infection. Table 1 is a summary of clinical trials of siRNA-based therapeutics . SiRNA shows potential for the treatment of various pulmonary viral infections, and it has been reported that siRNA-based therapeutics can also be used in the treatment of influenza , parainfluenza virus , severe acute respiratory syndrome SARS , and RSV . Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children.", "Above all, RSV is the most promising therapeutic target of siRNAs. RSV is a common cause of serious respiratory infections in infants and children. It also produces significant morbidity and mortality in adult immunocompromised or elderly populations . An RSV vaccine is not available, and the only approved antiviral therapy for RSV is undesirable for pediatric patients due to its potential teratogenicity and limited effectiveness. Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection .", "Thus, a safe and efficacious RSV therapy has long been awaited for both pediatric and adult patients. RNAi-based therapy has shown promising effects in murine models of RSV infection . The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection .", "ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated . Additionally, ALN-RSV01 has been evaluated in a randomized, double-blind, placebo-controlled phase II trial in lung transplant patients with RSV respiratory tract infection . The administration of ALN-RSV01 to RSV infected lung transplant patients was safe and well tolerated and associated with a statistically significant improvement in symptoms. Based on these results, a larger multinational, randomized, double-blind Phase IIb trial of ALN-RSV01 has been initiated in lung transplant patients to confirm and extend these findings. Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality.", "Cancer is a major target of RNAi-based therapy, as oncogenes, mutated tumor suppressor genes, and several other genes contributing to tumor progression are potentially important targets for gene silencing by RNAi. Lung cancer is one of the most frequent tumors worldwide with regard to incidence rates and mortality. Patients with lung cancer are commonly diagnosed at an advanced stage of the disease and have limited therapeutic options. Although the knowledge regarding the genetic and molecular basis of lung cancer has regularly increased, the median survival rates of individuals with advanced lung cancer are still poor. RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression.", "RNAi-based therapy is an attractive strategy for the development of more effective anticancer therapies with reduced treatment-related toxicity. The major advantage of RNAi therapeutics in cancer might be the simultaneous targeting of multiple genes belonging to different cellular pathways that are involved in tumor progression. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 , Wilms tumor 1 WT1 , overexpressed genes such as the insulin-like growth factor receptor 1 IGF-1R , NUPR1 and EZH2 . Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer.", "Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment. Atu027, a siRNA-lipoplex targeted against protein kinase N3 PKN3 , prevented lung metastasis in a phase I trial of various cancer models . PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration .", "PKN3 is a downstream effector of the phosphoinositide 3-kinase PI3K signaling pathway , which regulates diverse cellular responses, including development, growth, and survival . Recently, PKN3 has also been considered as a suitable therapeutic target for modulating tumor angiogenesis because loss of function analysis with Atu027 in cultured primary endothelial cells showed an essential role of PKN3 for endothelial tube formation and migration . Atu027 can be considered as a potential siRNA for preventing lung metastasis and might be suitable for preventing hematogenous metastasis combined with conventional cancer therapy. Inflammatory lung disease, also called COPD, includes a wide range of lung ailments. These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components .", "These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible. Systemic and local airway inflammation has been implicated in the pathogenesis of COPD . COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening .", "COPD is mainly associated with tobacco smoking, and recent studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence. Smoking might cause cells to senesce due to DNA damage through increased cell turnover, which in turn leads to accelerated telomere shortening . Lately, a lot of studies have investigated the role of cellular senescence in the development and progression of COPD . Although several medication classes, including inhaled corticosteroids, are used for COPD treatment, none of these medications have been shown to significantly improve long-term lung function during the progression of the disease. Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood.", "Current interventions that have been shown to improve mortality in COPD are cessation of smoking and delivery of supplemental oxygen when hypoxemia is present. Many people are developing COPD, and the cause of this condition is complicated and not thoroughly understood. One key factor is genetic susceptibility. Some studies have shown a large genetic contribution to the variability in pulmonary function and COPD . Polymorphisms in multiple genes have been reported to be associated with COPD , such as transcription factor e.g. nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g.", "nuclear factor-kappa B NFκB , extracellular matrix e.g., matrix metalloproteinase-12 MMP-12 , cytokines e.g. tumor necrosis factor TNF -α , chemokines e.g. interleukins IL -8, IL-8 receptor and chemokine receptor CCR 1 , and apoptosis e.g., caspase-3 and vascular endothelial growth factor VEGF . Many of these have been identified as possible targets for therapeutic intervention using molecule inhibitors or antagonists. Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD.", "Although several new treatments that target the inflammatory process are now in clinical development, such as TNF-α inhibitors and I-kappaB kinase complex 2 IKK2 inhibitors , clinical trials with siRNAs have never been performed in COPD. The delay of drug development for COPD might be due to the relatively recent emergence of research addressing the molecular basis of COPD. Furthermore, more research is needed to understand the essential molecular mechanisms about the pathogenesis of COPD and to develop monitoring techniques to support the development of RNAi therapies. Currently, no available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma. The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction.", "The RNAi-based approach for the key molecules also has potential implications for the treatment of COPD. Asthma is also a chronic inflammatory disease of the airways characterized by variable and recurring symptoms and reversible airflow obstruction. The World Health Organization estimates that 300 million people are currently affected and that, by the year 2025, another 100 million will be affected by the disease . Inhaled corticosteroids are very effective in mild asthma because they improve symptoms and decrease exacerbations. However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects.", "However, in moderate and severe asthma, inhaled corticosteroids have important therapeutic limitations. Although corticosteroids remain an important therapeutic intervention for inflammatory lung diseases, their use is not always completely effective and is associated with side effects. Due to such limitations, it is clear that there is a need for new types of medications that can treat and improve the prognosis of moderate to severe asthma. Many target genes have been identified that participate in the pathogenesis of asthma. The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported .", "The most promising targets include genes coding for cytokines IL-4, IL5, and IL-13 , cytokine and chemokine receptors IL-4 receptor and CCR3 , and tyrosine kinases spleen tyrosine kinase Syk and LCK/YES-related novel tyrosine kinase Lyn , as well as for transcription factors signal transducers and activators of transcription 1 STAT1 , STAT6, GATA3, and NFκB that are involved in asthma . The genes that have been assessed as siRNA targets for the treatment of asthma in preclinical models are reported . Currently, in a clinical trial for asthma, Excellair TM ZaBeCor, Bala Cynwyd, PA, USA , a siRNA that targets Syk, is being used. The kinase is involved in signaling from a B cell receptor and is a key regulator of downstream signaling cascades that ultimately lead to the activation of several pro-inflammatory transcription factors. It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells .", "It has been reported that antisense oligonucleotides administered by aerosol were potent to decrease Syk expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation . Moreover, inhibition of inflammatory mediators was shown in a study using siRNA targeting Syk in airway epithelial cells . Following the successful results of the company's Phase I clinical trial, a Phase II trial for its asthma drug candidate Excellair TM has already been initiated. Some of the current treatments for asthma and other inflammatory conditions, such as TNF-α inhibitors or leukotriene inhibitors, inhibit only one of the mediators of inflammation. In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases.", "In contrast, siRNA targeting Syk seeks to inhibit an initial signaling step of inflammation and, thereby, prevent the release of multiple inflammatory mediators. Overall, recent progress of siRNAs to the lungs has also improved the therapeutic feasibility of RNAi for inflammatory lung diseases. The rapid progress will put siRNA-based therapeutics on a fast track to the clinic. MiRNAs are small endogenous noncoding RNAs that regulate gene expression by repressing translation or promoting the degradation of their target mRNA. MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs .", "MiRNAs regulate gene expression by binding to the 3′ untranslated region UTR of their target mRNAs and mediating mRNA degradation or translational inhibition. In the human genome, transcripts of approximately 60% of all mRNAs are estimated to be targeted by miRNAs . According to their function, miRNAs play an important role in cellular processes as development, proliferation, and apoptosis of pulmonary pathologies . A growing number of miRNAs have been shown to be involved in different lung diseases. This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications.", "This evidence makes miRNAs a promising technology for current and future therapeutic development. We discuss the role of some miRNAs in various lung diseases as well as the possible future of these discoveries in clinical applications. Table 2 shows the summary of miRNAs in therapeutic development. At this point, a miRNA-based therapy has already entered a phase II clinical trial. There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 .", "There is evidence that upregulation or downregulation of miRNAs is critical for lung homeostasis and, thus, may contribute to the development of pathological pulmonary conditions. Many studies have focused on the role of miRNAs in inflammatory lung diseases, such as COPD , pulmonary fibrosis , and asthma Table 3 . The pathogenesis of COPD is attributed to not only chronic inflammation in the airways but also systemic inflammation . Cigarette smoking is the main risk factor for the development of COPD. Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components.", "Smoking has been shown to cause biological change in the gene expression of the lungs , and there are some reports about smoking-related miRNAs . However, there are few reports that focus on the miRNAs related to the pathogenesis of this disease with systemic inflammatory components. Recent study on pulmonary fibroblasts of COPD patients presents less expression of miR-146a after stimulation with proinflammatory cytokines when compared with non-COPD subjects with similar smoking histories . The downregulation of miR-146a resulted in a prolonged mRNA half-life of cyclooxygenase-2, thus increasing prostaglandin E2 in fibroblasts from COPD subjects. Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD.", "Moreover, Ezzie et al. researched the difference of miRNA profiles expressed in the lungs of smokers with and without COPD. They concluded that miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD . Yet, COPD is a complex, multi-component, and heterogeneous disorder with a number of different pathological processes and subgroups with their own characteristics and natural history . A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited.", "A better understanding of the complexity of the disease and potential clinical relevance of the identified miRNAs is needed. Pulmonary fibrosis can be caused by an identifiable irritation to the lungs, but, in many cases, the cause is unknown, and the therapeutic possibilities are limited. Cigarette smoking is one of the most recognized risk factors for the development of pulmonary fibrosis. This disorder is mainly accompanied by increased expression of the key fibrotic mediator transforming growth factor β TGF-β and other cytokines produced at the lesion of active fibrosis . Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening .", "Recently, it was reported that miRNAs may play an important regulatory role in the pulmonary fibrotic change in the lungs. The downregulation of let-7d in idiopathic pulmonary fibrosis IPF resulted in increased collagen deposition and alveolar septal thickening . In addition, Liu et al. reported that the oncogenic miR-21 was found to be upregulated in IPF patients and in the murine lungs with bleomycin-induced fibrosis . Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes.", "Although these miRNAs may be potential therapeutic targets because their expression is related to the regulation of TGF-β, the factor is necessary but not sufficient for pathologic fibrosis of the lungs. Pulmonary fibrosis is also a complicated illness that can have many different causes. Focus on the role of miRNAs in asthma has recently increased. Asthma is an inflammatory disease of the airway that is characterized by an abnormal response of T helper-2 Th2 -type CD4+T lymphocytes against inhaled allergens . In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response.", "In a different asthmatic mouse model, there was an observed increase in the expression of miR-21 in the lungs . This report might contribute to the understanding of the inflammatory mechanism in the airway through the inhibition of IL-12, favoring the Th2 lymphocyte response. A toll-like receptor 4 TLR4 -induced Th2 lymphocyte induces high expression of miR-126, and selective blockade of miR-126 suppressed the asthmatic phenotype . In addition, airway remodeling is a characteristic feature of asthma and has important functional implications. Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling .", "Rodriguez et al. have shown that miR-155 is related to the development of inflammatory infiltration into the lung and airway remodeling . Thus, some studies present a functional connection between miRNA expression and asthma pathogenesis and suggest that targeting miRNAs in the airways may lead to anti-inflammatory treatments for allergic asthma. Despite the evidence from experimental models, the expression profiling of miRNAs in airway biopsies from patients with mild asthma before and after treatment with inhaled corticosteroids and in healthy volunteers revealed no differences in miRNA expression . Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer .", "Further investigations about the role of miRNAs related to asthma pathogenesis are required. Although the basic evidence of miRNA biology is still providing new insights, applications of miRNA-based therapy for inflammatory lung diseases are less advanced than those for lung cancer . One reason for this could be that the disease heterogeneity is caused by the effects of many environmental air pollutants, including smoke and volatile organic compounds. The presence of several risk factors makes the understanding of the pathogenesis of inflammatory lung diseases complicated. Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs.", "Understanding the role that miRNAs play in the modulation of gene expression, leading to sustain the pathogenesis of lung diseases, is important for the development of new therapies that focus on the prevention of disease progression and symptom relief. Given the significant roles that miRNAs play in multiple pathways of lung carcinogenesis, increasing efforts are dedicated to the research and development of miRNA-based therapies, including restoring functions of tumor suppressive miRNAs or inhibiting oncogenic miRNAs. The development of miRNA-based therapies for lung cancer is growing prosperously with the help of new RNAi technologies. Compared to siRNA-based therapies, which are already in clinical trials, miRNAs are less toxic and have the potential to target multiple genes. The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects .", "The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer . One strategy is miRNA replacement therapy, which involves the re-introduction of a tumor suppressor miRNA mimic to restore a loss of the function. MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA.", "MiRNA mimics are synthetic RNA duplexes designed to mimic the endogenous functions of miRNAs with chemical modifications for stability and cellular uptake. The concept of miRNA replacement therapy is most exemplified by the let-7 miRNA. let-7 is a tumor-suppressor miRNA in non-small-cell lung cancer that inversely correlates with the expression of the RAS oncoprotein, a key cancer gene . Intranasal administration of let-7 mimic into mouse models of lung cancer significantly reduced tumor growth, suggesting that miRNA replacement therapy is indeed promising . Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth.", "Another miRNA that shows the value of miRNA replacement is provided by miR-34a . Local and/or systemic delivery of a synthetic miR-34a mimic led to accumulation of miR-34a in the tumor tissue and inhibition of lung tumor growth. Lately, Ling et al. also showed that tumor suppressor miR-22 exhibited anti-lung cancer activity through post-transcriptional regulation of ErbB3 . Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells .", "Thus, therapeutic miRNA mimics have a powerful potential by attacking multiple genes relevant to several diseases. However, it is necessary to pay attention to the potential toxicity in normal tissues under conditions in which the therapeutic delivery of miRNA mimics will lead to an accumulation of exogenous miRNAs in normal cells . Although the assumptions are well founded, there is still insufficient evidence for toxicity caused by miRNA mimics. Indeed, several in vivo studies failed to reveal side effects caused by the miRNA mimics and suggested that delivery of miRNA mimics to normal tissues was well tolerated . It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs.", "It will be important to research miRNA mimic-induced effects in normal cells and to carefully assess toxicity before using them in clinical practice. The second strategy is directed toward a gain of function and aims to inhibit oncomiRs by using anti-miRNAs. Chemical modifications, such as 2'-O-methyl-group and locked nucleic acid LNA , would increase oligo stability against nucleases . Antisense oligonucleotides contained in these modifications are termed antagomirs or \"LNA-antimiRs\" . They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics.", "They are oligonucleotides with sequences complementary to the endogenous miRNA and inhibit the specific miRNA function. An LNA-antimiR against miR-122 has been shown to effectively silence miR-122 in non-human primates , and the findings support the potential of these compounds as a new class of therapeutics. Moreover, it has also been reported that anti-miR-150 delivered into lung tumor xenografts in mice led to inhibited tumor growth . Relative to studies on miRNA mimics, studies with antisense oligonucleotides have shown effective evidence with naked oligonucleotides. This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach.", "This illustrates the potential of chemical modifications of oligonucleotides to improve their stability, resistance to RNase, and pharmacologic properties. Therefore, inhibition of miRNA function by chemically modified antimiR oligonucleotides has become an important and widely used approach. Recent data from the first phase II study in patients with chronic HCV infection treated with the LNA-modified antimiR-122 showed that this compound was well tolerated and provided continuing viral suppression. An increasing number of studies have examined the therapeutic potential of miRNAs. Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy.", "Recently, the evidence of roles for miRNAs in determining drug resistance has emerged . Cytotoxic and molecular target drugs have been widely used in the treatment of advanced lung cancer; unfortunately, many cases are still refractory to chemotherapy. In this situation, combining miRNA mimics or antimiR with chemotherapy may potentiate the efficacy of the cancer treatment in the future. In addition, miRNAs related with cancer stem cells may significantly broaden the field of miRNA-based therapy and suggest that miRNAs can be potential tools to kill cancer cells associated with therapy resistance, recurrence, and metastasis . Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases.", "Hence, the main challenge is the successful delivery and chemical modifications of the therapeutic miRNAs to the target tissue without harming normal tissues. RNAi-based approaches provide a promising therapeutic modality for the treatment of various lung diseases. One of the greatest challenges in RNAi-based therapy continues to be the delivery method of the therapeutic siRNAs and miRNAs to the target cells. Pulmonary delivery applications are very attractive, since they tend to be non-invasive, are locally restricted, and can be administered by the patient. A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases.", "A realistic therapeutic intervention, such as aerosolization, can enhance drug delivery to the site of action and decrease systemic exposure of the patient to the therapy, thereby reducing off-target effects. The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors. Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development.", "Increased evidence has indicated that miRNAs fulfill causative roles in a variety of lung diseases and have prompted investigations into their potential as therapeutic targets. Further understanding of the detailed mechanisms of RNAi-based therapy and investigations of more effective delivery methods are required for future development. These novel approaches could open new avenues for various lung diseases and improve the clinical outcome of the patients." ]

[ "BACKGROUND: The epidemiology of local viral etiologies is essential for the management of viral respiratory tract infections. Limited data are available in China to describe the epidemiology of viral respiratory infections, especially in small–medium cities and rural areas. OBJECTIVES: To determine the viral etiology and seasonality of acute respiratory infections in hospitalized children, a 3-year study was conducted in Shenzhen, China. METHODS: Nasopharyngeal aspirates from eligible children were collected. Influenza and other respiratory viruses were tested by molecular assays simultaneously. Data were analyzed to describe the frequency and seasonality.", "METHODS: Nasopharyngeal aspirates from eligible children were collected. Influenza and other respiratory viruses were tested by molecular assays simultaneously. Data were analyzed to describe the frequency and seasonality. RESULTS: Of the 2025 children enrolled in the study, 971 48·0% were positive for at least one viral pathogen, in which 890 91·7% were <4 years of age. The three most prevalent viruses were influenza A IAV; 35·8% , respiratory syncytial virus RSV; 30·5% and human rhinovirus HRV; 21·5% . Co-infections were found in 302 cases 31·1% , and dual viral infection was dominant. RSV, HRV and IAV were the most frequent viral agents involved in co-infection.", "Co-infections were found in 302 cases 31·1% , and dual viral infection was dominant. RSV, HRV and IAV were the most frequent viral agents involved in co-infection. On the whole, the obvious seasonal peaks mainly from March to May were observed with peak strength varying from 1 year to another. CONCLUSIONS: This study provides a basic profile of the epidemiology of acute respiratory viral infection in hospitalized children in Shenzhen. The spectrum of viruses in the study site is similar to that in other places, but the seasonality is closely related to geographic position, different from that in big cities in northern China and neighboring Hong Kong. Text: Acute respiratory tract infections ARTIs are a persistent and pervasive public health problem in both developed and developing countries.", "The spectrum of viruses in the study site is similar to that in other places, but the seasonality is closely related to geographic position, different from that in big cities in northern China and neighboring Hong Kong. Text: Acute respiratory tract infections ARTIs are a persistent and pervasive public health problem in both developed and developing countries. They cause a great burden of disease worldwide. Especially in developing countries including China, ARTIs, mainly pneumonia, are the leading cause of death among children under the age of 5 years. 1,2 A great variety of pathogens can cause ARTIs, and viruses have been considered as the predominant pathogens in this children population. 3, 4 The most frequently reported viruses include respiratory syncytial virus RSV , influenza viruses A and B IAV, IBV , parainfluenza viruses PIVs , human rhinovirus HRV and adenovirus ADV , which are responsible for most episodes of ARTIs in children.", "1,2 A great variety of pathogens can cause ARTIs, and viruses have been considered as the predominant pathogens in this children population. 3, 4 The most frequently reported viruses include respiratory syncytial virus RSV , influenza viruses A and B IAV, IBV , parainfluenza viruses PIVs , human rhinovirus HRV and adenovirus ADV , which are responsible for most episodes of ARTIs in children. 1 In the past decade, several new viruses associated with ARTIs such as human metapneumovirus HMPV , novel strains of coronaviruses SARS-CoV, HCoV-NL63 and HKUI , human bocavirus BOV , WU polyomavirus WUPoyV and KI polyomavirus KIPoyV have been discovered in human respiratory tract specimens. Among them, some have been identified to be causative pathogens of ARTIs. 1, 4, 5 Currently, there are no approved vaccines or medications available for most of the respiratory viruses. 1 A better understanding of the epidemiology of viral respiratory tract infections in children plays a key role for the prevention, control and treatment of ARTIs.", "1, 4, 5 Currently, there are no approved vaccines or medications available for most of the respiratory viruses. 1 A better understanding of the epidemiology of viral respiratory tract infections in children plays a key role for the prevention, control and treatment of ARTIs. Studies showed that many viral respiratory infections exhibited predictable seasonal variations. However, the epidemiological profiles of viral respiratory infections from different climate zones or different countries in the same climate zone may be varied. China is a large country crossing three climate zones, and great differences in climate are found from region to region. A better understanding of the epidemiology of ARTIs in different regions could be helpful to develop effective surveillance, prevention and treatment strategies.", "China is a large country crossing three climate zones, and great differences in climate are found from region to region. A better understanding of the epidemiology of ARTIs in different regions could be helpful to develop effective surveillance, prevention and treatment strategies. Although some studies on the epidemiology of ARTIs have recently been reported in big cities such as Beijing, Shanghai and Hong Kong, the epidemic characteristics of viruses in ARTIs are still not well established all around China, especially in other cities and rural areas. Shenzhen is the largest migratory city of China with high population density and population mobility. It is located in southern China at 22°27 0 -22°52 0 N and 113°46 0 -114°37 0 E, immediately north of Hong Kong, with a typical subtropical monsoon climate. The annual average temperature and relative humidity of Shenzhen are about 23°C 12-33°C and 77%, respectively.", "It is located in southern China at 22°27 0 -22°52 0 N and 113°46 0 -114°37 0 E, immediately north of Hong Kong, with a typical subtropical monsoon climate. The annual average temperature and relative humidity of Shenzhen are about 23°C 12-33°C and 77%, respectively. The purpose of this study is to investigate the prevalence, seasonality and clinical characteristics of acute viral respiratory infections in hospitalized children in Shenzhen and to provide insights into etiologies of ARTIs in local infants and children. A consecutive 3-year prospective study from July 2007 to June 2010 was conducted in Shenzhen, a coastal city neighboring Hong Kong. Four hospitals including a children's hospital were chosen for the study. Selected patients with ARTIs admitted to the pediatric wards were enrolled.", "Four hospitals including a children's hospital were chosen for the study. Selected patients with ARTIs admitted to the pediatric wards were enrolled. The inclusion criteria were as follows: less than 14 years old, acute fever T ≥ 38°C , with any one of respiratory symptoms such as sore throat, cough, wheezing and dyspnoea/ tachypnoea , normal or low leukocyte count, the onset of illness within 3 days before hospitalization. The diagnosis of pneumonia was based on the guideline of the management of childhood community acquired pneumonia CAP issued by the Chinese Medical Association in 2006. 17 In the guideline, the clinical symptoms and signs for the diagnosis of childhood CAP include fever, cough, tachypnoea defined according to different age , difficulty breathing and/or lower chest wall indrawing. X-ray evaluation has been carried out when necessary.", "17 In the guideline, the clinical symptoms and signs for the diagnosis of childhood CAP include fever, cough, tachypnoea defined according to different age , difficulty breathing and/or lower chest wall indrawing. X-ray evaluation has been carried out when necessary. The study protocol was approved by the medical ethical committees of the hospitals. Written informed consent was obtained from the parents or legal guardians of the children. Nasopharyngeal aspirates NPA were obtained by trained personnel following standard operating procedures within 24 hour after admission. The specimens were transported immediately to the laboratory by sterile viral transport media, then divided into aliquots and immediately frozen at À80°C until further processing.", "Nasopharyngeal aspirates NPA were obtained by trained personnel following standard operating procedures within 24 hour after admission. The specimens were transported immediately to the laboratory by sterile viral transport media, then divided into aliquots and immediately frozen at À80°C until further processing. Total viral nucleic acids DNA and RNA were extracted from 200 ll of NPA specimen using the AxyPrep Body Fluid DNA/RNA Miniprep Kit Axygen, Union City, CA, USA , according to the manufacturer's instructions. Purified DNA and RNA were stored at À80°C in aliquots for further PCR analysis. For each specimen, assays for ten common and newly identified viruses were performed. Briefly, WUPoyV and BOV were tested using monoplex PCRs described previously.", "For each specimen, assays for ten common and newly identified viruses were performed. Briefly, WUPoyV and BOV were tested using monoplex PCRs described previously. 18, 19 Other viruses were tested using the Luminex platform and multiplex xTAG TM respiratory viral panel assay RVP Assay according to the manufacturer's instructions. 20 All multiple infection samples were retested. If there was discordance between two tests, the sample was confirmed by monoplex PCR. Statistical Package for the Social Sciences SPSS for Windows version 11.0 SPSS Inc. Chicago, IL, USA was used. For comparison of categorical data, chi-square or Fisher's exact test was used.", "Statistical Package for the Social Sciences SPSS for Windows version 11.0 SPSS Inc. Chicago, IL, USA was used. For comparison of categorical data, chi-square or Fisher's exact test was used. All tests were two-tailed, and a P value below 0Á05 was considered statistically significant. A total of 2025 specimens were obtained from 2025 eligible patients ranging from 15 days to 14 years old with a median age of 12 months, in which 89Á6% of patients were < 4 years old. There were 964 47Á6% females and 1061 52Á4% males included. Of all hospitalized children enrolled in this study, 84Á0% involved lower respiratory infection and 16Á0% had upper respiratory tract infection Table 1 .", "There were 964 47Á6% females and 1061 52Á4% males included. Of all hospitalized children enrolled in this study, 84Á0% involved lower respiratory infection and 16Á0% had upper respiratory tract infection Table 1 . Among 971 positive cases, 572 58Á9% were diagnosed as pneumonia. About 971 of the 2025 cases 48Á0% were positive for at least one viral pathogen. Among them, IAV, RSV, HRV and PIVs were detected in 348 35Á8% , 296 30Á5% , 209 21Á5% and 169 17Á4% cases, respectively. Single infection was observed in 669 68Á9% cases, and multiple infection was found in 302 31Á1% .", "Among them, IAV, RSV, HRV and PIVs were detected in 348 35Á8% , 296 30Á5% , 209 21Á5% and 169 17Á4% cases, respectively. Single infection was observed in 669 68Á9% cases, and multiple infection was found in 302 31Á1% . Our results also showed that RSV, IAV and HRV were the main pathogens in single viral infection cases Table 1 . The monthly positive rates varied from 32Á5% to 75Á0% with a mean of 45Á1% Figure 1 . In the year 2009, when influenza A H1N1 was pandemic worldwide, the positive rate started to increase in March and the highest positive rate 75Á0% was observed in May. Among the 971 positive cases, a total of 1335 viral pathogens were detected.", "In the year 2009, when influenza A H1N1 was pandemic worldwide, the positive rate started to increase in March and the highest positive rate 75Á0% was observed in May. Among the 971 positive cases, a total of 1335 viral pathogens were detected. The most frequently detected pathogen was IAV 26Á1%, 348/1335 , followed by RSV 22Á2%, 296/1335 , HRV 15Á7%, 209/1335 , PIV1 and PIV3 12Á7%, 169/1335 About 302 co-infection cases were identified, accounting for 14Á9% of all 2025 hospitalized children. During the H1N1 outbreak from March to August 2009, co-infection cases and co-infection rate increased significantly Figure 2 . 143 of 302 47Á4% co-infection cases were detected during that time. Among them, 121 cases were involved in IAV infection, including 90 dual infection cases.", "143 of 302 47Á4% co-infection cases were detected during that time. Among them, 121 cases were involved in IAV infection, including 90 dual infection cases. Of all co-infection cases, 247 81Á8% , 49 16Á2% and 5 1Á7% were infected with two, three and four potential viral pathogens, respectively. One multiple infection with five viruses was detected in a RSV IAV HRV BOV PIV3 ADV HMPV WUPoyV PIV1 IBV Total A Total cases 163 172 85 55 51 49 37 26 24 7 669 Bronchitis 12 19 7 7 5 2 2 3 5 0 62 Bronchiolitis 45 19 20 7 13 9 8 2 5 0 128 Pneumonia 93 97 55 28 24 28 25 18 14 5 387 URTI 13 37 3 13 9 10 2 3 0 2 *Case number and percentage in all enrolled children. **Incidence rate in this age group significantly higher than the other age groups. ***No significant difference between these three age groups.", "**Incidence rate in this age group significantly higher than the other age groups. ***No significant difference between these three age groups. †No significant differences between these two age groups, but significantly lower than the other age groups. 6-month-old infant. IAV, RSV and HRV were the three most frequently found viruses in co-infection and detected in 176, 133 and 124 cases with co-infection rates of 50Á6%, 44Á9% and 59Á3%, respectively Table 2 . Various multiple infection patterns were observed in the study. A total of 152 50Á3% co-infection cases involved at least two viruses of RSV, HRV and IAV.", "Various multiple infection patterns were observed in the study. A total of 152 50Á3% co-infection cases involved at least two viruses of RSV, HRV and IAV. Co-infection rate of each individual virus detected varied significantly. The lowest and highest co-infection rates were observed in WUPoyV 33Á3% and IBV 66Á7% , respectively. 91Á4% 276/302 of co-infection cases were tested in the age group of 4 years old or younger Table 2 , but among all age groups, no statistical difference in co-infection rate was found v 2 = 1Á83, P = 0Á8721 . Gender-specific difference in co-infection rate was not observed v 2 = 2Á17, P = 0Á1404 .", "91Á4% 276/302 of co-infection cases were tested in the age group of 4 years old or younger Table 2 , but among all age groups, no statistical difference in co-infection rate was found v 2 = 1Á83, P = 0Á8721 . Gender-specific difference in co-infection rate was not observed v 2 = 2Á17, P = 0Á1404 . There was no significant difference in co-infection rates between PICU and non-PICU cases. Similarly, no significant difference in clinical symptoms was observed between co-infection and single cases data not shown . In general, respiratory viruses were detected more often in the period of March to May than in other months 55Á4% and 40Á6%, respectively, v 2 = 28Á06, P = 0Á0000 , and obvious seasonal peaks were observed during those months with peak strength varying from 1 year to another. A weaker seasonal peak could also be distinguished in some winter months in different years Figure 1 .", "In general, respiratory viruses were detected more often in the period of March to May than in other months 55Á4% and 40Á6%, respectively, v 2 = 28Á06, P = 0Á0000 , and obvious seasonal peaks were observed during those months with peak strength varying from 1 year to another. A weaker seasonal peak could also be distinguished in some winter months in different years Figure 1 . The seasonality profile of each individual virus detected was diversified. A seasonal distribution of IAV can be observed from late spring to summer mainly March to May and sometimes in fall October, November or December . A wide seasonal peak of IAV infection was detected from March to August 2009 Figure 3A . Although RSV was tested almost a whole year, two yearly peaks were identified.", "A wide seasonal peak of IAV infection was detected from March to August 2009 Figure 3A . Although RSV was tested almost a whole year, two yearly peaks were identified. One was found in November and/or December and the other stronger one was found in March to May of the year. The peak duration in 2009 was longer than those in other years. The seasonal trends of HRV and PIVs were similar to that of RSV, but the peaks of these three viruses fluctuated and shifted mildly Figure 3B . Although IBV and ADV had a low detection rate in the study, similar seasonality was observed and their infection peaks were mainly in midwinter.", "The seasonal trends of HRV and PIVs were similar to that of RSV, but the peaks of these three viruses fluctuated and shifted mildly Figure 3B . Although IBV and ADV had a low detection rate in the study, similar seasonality was observed and their infection peaks were mainly in midwinter. Peaks in spring and summer were also observed in some years Figure 3C . Our investigation did not find regular seasonality in BOV infections. A sudden increase in BOV infection was recorded in April and May 2010. Although the positive rate of HMPV infection was only 4Á8%, regular seasonality was observed from March to May of each year.", "A sudden increase in BOV infection was recorded in April and May 2010. Although the positive rate of HMPV infection was only 4Á8%, regular seasonality was observed from March to May of each year. Of 39 patients with WUPoyV infection, 36 were detected after July 2008. Our data implied that peak months of WUPoyV infection were from March to May Figure 3D . The positive rates of viral infections in male and female were 52Á5% and 47Á5%, respectively. No significant gender difference was revealed v 2 = 0Á012, P = 0Á9118 . The distribution of viral agents and infection patterns in different age groups are shown in Table 2 .", "No significant gender difference was revealed v 2 = 0Á012, P = 0Á9118 . The distribution of viral agents and infection patterns in different age groups are shown in Table 2 . Of all 971 positive children, 890 91Á7% were 4 years old or younger. The positive rate in this age group was significantly higher than that in children more than 4 years old v 2 = 8Á26, P = 0Á0041 . Children under 6 months were the most susceptible to respiratory viral pathogens with a positive rate of 14Á8% Table 2 . Very few long-term prospective studies were performed for viral etiologies of ARTIs among hospitalized children.", "Children under 6 months were the most susceptible to respiratory viral pathogens with a positive rate of 14Á8% Table 2 . Very few long-term prospective studies were performed for viral etiologies of ARTIs among hospitalized children. In this present study, the infection frequency, seasonality, co-infection pattern and clinical features of viral respiratory infections were investigated based on prospective analysis of three consecutive year's data from hospitalized children with ARTIs. Our results provided a distinctive epidemiological profile of viral respiratory infections in hospitalized children with ARTIs in the study areas, which was different from those in the big cities in northern China such as Beijing and Shanghai and also different from that in adjacent Hong Kong. Overall, 48Á0% of our cases were positive for respiratory virus infections, which resembled the latest study in the same city. 21 A similar incidence rate has been obtained in neighboring regions 13, 22 and other cities such as Rome 23 and Milan, 24 but it was different from other studies.", "Overall, 48Á0% of our cases were positive for respiratory virus infections, which resembled the latest study in the same city. 21 A similar incidence rate has been obtained in neighboring regions 13, 22 and other cities such as Rome 23 and Milan, 24 but it was different from other studies. In China, the overall positive rate reported varied from 27Á3 to 74Á8% depending on different areas and detection methods. 15, 16, The rate of respiratory viral infections varied worldwide, and many factors such as geographic distribu-tion, study design and detection protocols could lead to these variations. 1, 7, 8, 32 In our study, leukocyte count was used as an indicator of inclusion criteria and it probably affected the positive rate. Viruses not considered in the study, for example coronaviruses, would underestimate the positive rate.", "1, 7, 8, 32 In our study, leukocyte count was used as an indicator of inclusion criteria and it probably affected the positive rate. Viruses not considered in the study, for example coronaviruses, would underestimate the positive rate. Most studies showed that RSV or HRV was the most prevalent viruses in children with viral respiratory tract infection. 1 In this study, IAV was the most frequently detected respiratory virus, followed by RSV and HRV. IAV H1N1 outbreak in 2009 could explain this shift. Data showed that about 60% of IAV infections were detected during the outbreak period. Studies showed that the H1N1 outbreak could change viral distribution patterns.", "Data showed that about 60% of IAV infections were detected during the outbreak period. Studies showed that the H1N1 outbreak could change viral distribution patterns. 24, 29, 33 Regardless of the IAV H1N1 outbreak, RSV and HRV were the two most common viral pathogens in ARTIs, which was consistent with most previous studies. 1, 10, 15, 16, 22, Our study further confirmed the importance of RSV and HRV in children with ARTIs, especially in children < 4 years of age. 10, 14, 23 Our results also showed that 12Á7% of viral pathogens detected were PIV1 and PIV3, which implied that PIVs played an important role in children with ARTIs. Similar findings were obtained in the studies conducted in Shanghai, 14, 34 Changsha, 26 Harbin, 30 Hong Kong 13 and Rome.", "10, 14, 23 Our results also showed that 12Á7% of viral pathogens detected were PIV1 and PIV3, which implied that PIVs played an important role in children with ARTIs. Similar findings were obtained in the studies conducted in Shanghai, 14, 34 Changsha, 26 Harbin, 30 Hong Kong 13 and Rome. 23 The prevalence of PIV3 was twofold higher than that of PIV1, particularly in infants, which was similar with other reports, 25, 26, 30, 35 implying that infants could be more vulnerable to infection with PIV3 than PIV1. HMPV has been proven to be one of the main viral pathogens responsible for ARTIs in children. 5 The positive rate found in the study was consistent with previously published results. 10, 36, 37 In China, the infection rate of HMPV varied from 3Á2 to 10Á6%.", "5 The positive rate found in the study was consistent with previously published results. 10, 36, 37 In China, the infection rate of HMPV varied from 3Á2 to 10Á6%. 22, 26, 28, 29, 31 The seasonality of HMPV in this study was mainly from March to May, similar to that in Hong Kong, 36 but different from other places. 5, 37 In our study, 4Á9% of cases were positive for BOV, which coincided with 5Á0% in Hong Kong 38 and higher than Guangzhou 39 and eastern Guangdong. 22 Our result suggested that BOV might be present throughout the year with no seasonal distribution. However, seasonal distribution was noted from September to February in Hong Kong 38 and May and June in Guangzhou.", "22 Our result suggested that BOV might be present throughout the year with no seasonal distribution. However, seasonal distribution was noted from September to February in Hong Kong 38 and May and June in Guangzhou. 39 The use of multiple PCR made it possible to simultaneously detect a broad spectrum of viruses with excellent sensitivity, at the same time, with increased viral detection rate and co-infection rate for ARTIs. 12,40 Among our positive cases, co-infection rate was 31Á1%, which was similar to 27Á9% reported by Do et al. 10 Co-infection rate reported elsewhere varied widely from 25Á4 to 47Á9%. 40 The relatively lower co-infection rates ranging from 0Á24 to 26Á9% were reported in the studies conducted in various cities of China.", "10 Co-infection rate reported elsewhere varied widely from 25Á4 to 47Á9%. 40 The relatively lower co-infection rates ranging from 0Á24 to 26Á9% were reported in the studies conducted in various cities of China. 22, In most of these studies, immunofluorescence kits were used to test a lower number of respiratory viruses. It was worth to note that in the study by Peng et al. 32 in Wuhan, China, 69Á5% of co-infection rate was reported with immunofluorescence kit. These variations might be attributed to geographic differences, diagnostic methods for viral agents and study design.", "32 in Wuhan, China, 69Á5% of co-infection rate was reported with immunofluorescence kit. These variations might be attributed to geographic differences, diagnostic methods for viral agents and study design. 12, 32, 34, 41, 42 Pathogens in those negative patients need to be further investigated as only ten common and newly identified viruses were included in our study, which might underestimate positive rate or coinfection rate. It was notable that the correlation between co-infection rate and positive rate was not observed. Of multiple infections, dual infection was predominant in this study whether or not considering the IAV H1N1 outbreak in 2009, which was consistent with previous studies. 28, 32, 42, 43 Similar with the studies conducted in the cities of Guangzhou and Wuhan, China, 28, 29 our study showed that IAV, RSV and HRV were the main viruses involved in multiple infections.", "Of multiple infections, dual infection was predominant in this study whether or not considering the IAV H1N1 outbreak in 2009, which was consistent with previous studies. 28, 32, 42, 43 Similar with the studies conducted in the cities of Guangzhou and Wuhan, China, 28, 29 our study showed that IAV, RSV and HRV were the main viruses involved in multiple infections. High co-infection rate between these three viruses could be explained from the overlap of their seasonal distributions. A variety of predominant multiple infection patterns between respiratory viruses were observed in different studies. 12, 32, 42, 43 For example, it was shown in Martin et al. 's study 43 that ADV and coronaviruses were the most common co-infection pattern.", "12, 32, 42, 43 For example, it was shown in Martin et al. 's study 43 that ADV and coronaviruses were the most common co-infection pattern. Our study showed that RSV and HRV were the two most viruses involved in multiple infection, followed by IAV and PIVs, regardless of IAV infection in the H1N1 outbreak period. It was difficult to explain the variations of coinfection patterns based only on seasonal distribution. A recent study suggested that co-infection patterns were not random and certain pathogens had higher frequency of coinfection. 41 As molecular assays only detect nucleic acid and positive result does not mean the presence of the pathogen, when studying co-infection patterns of respiratory viruses, the ability to differentiate the real causative pathogens needs to be solved first.", "A recent study suggested that co-infection patterns were not random and certain pathogens had higher frequency of coinfection. 41 As molecular assays only detect nucleic acid and positive result does not mean the presence of the pathogen, when studying co-infection patterns of respiratory viruses, the ability to differentiate the real causative pathogens needs to be solved first. Viral load detection could provide some clues for solving this issue. 43, 44 Although high co-infection rates have been reported in various studies, the associations among multiple infections, hospitalization rate and severity of ARTIs were still not clear with inconsistent results in different studies. 42, 43, 45 Our data suggested that multiple infection had less association with the severity of disease, consistent with Peng et al. 's study.", "42, 43, 45 Our data suggested that multiple infection had less association with the severity of disease, consistent with Peng et al. 's study. 32 The relationship between co-infection rates and age group was also investigated in our study, and little correlation was observed. Several previous studies observed that co-infection rates were more frequent in a certain age group, but results were varied. 32, 43 In contrast to temperate region, where most viruses had winter-spring seasonality, the respiratory viral infections in tropical and subtropical regions appeared mainly to be spring-summer seasonality. 9 In this study, due to the high detection rate and similar seasonality of RSV, HRV, IAV, PIV and HMPV, an overall spring-summer seasonality of viral respiratory infections in children was concluded.", "32, 43 In contrast to temperate region, where most viruses had winter-spring seasonality, the respiratory viral infections in tropical and subtropical regions appeared mainly to be spring-summer seasonality. 9 In this study, due to the high detection rate and similar seasonality of RSV, HRV, IAV, PIV and HMPV, an overall spring-summer seasonality of viral respiratory infections in children was concluded. Studies conducted in Hong Kong showed that a clear seasonal peak was from April to September, 36, 46 with a longer duration than our study. The overall seasonality in this study was also different from the studies conducted in northern or central cities of China, in which the seasonality of most viruses presented in autumn-winter and/or winter-spring. 15, 30 The winter-spring seasonality was also observed in Guangzhou, a city about 150 kilometers north of Shenzhen. 28 Different seasonal onset and duration were observed in various studies conducted in sub- tropical regions.", "15, 30 The winter-spring seasonality was also observed in Guangzhou, a city about 150 kilometers north of Shenzhen. 28 Different seasonal onset and duration were observed in various studies conducted in sub- tropical regions. In these studies, ambient temperature, humidity and rainfall were widely used to explain these differences in seasonality, but inconsistent results were observed. 9, 46, 47 Although most studies demonstrated that the seasonality of viral respiratory infections was correlated with increased rainfall, effects of climate factors such as humidity and temperature on the seasonality were complex and interactive. 9, 46, 48 The study areas have four indistinct seasons, and the coldest month usually emerges in January average 12°C . During the period from March to May, the weather featured warm ambient temperature average 18-25°C , high relative humidity average 85%-90% and increasing rainfall.", "9, 46, 48 The study areas have four indistinct seasons, and the coldest month usually emerges in January average 12°C . During the period from March to May, the weather featured warm ambient temperature average 18-25°C , high relative humidity average 85%-90% and increasing rainfall. These meteorological conditions were perhaps conducive to viral survival. 9, 48 In addition, intensive temperature fluctuations during seasonal alternation could increase the susceptibility to infections. 49 As reported in other studies in temperate, tropical and subtropical regions, viral infection rates in children population showed an inverse correlation with age, with younger individuals experiencing higher viral infection rates. 3, 4, 6, 9, 24 Our results suggested that children younger than 4 years of age, particularly <6 months, were at higher risk of hospitalization for ARTIs, compared with older children.", "49 As reported in other studies in temperate, tropical and subtropical regions, viral infection rates in children population showed an inverse correlation with age, with younger individuals experiencing higher viral infection rates. 3, 4, 6, 9, 24 Our results suggested that children younger than 4 years of age, particularly <6 months, were at higher risk of hospitalization for ARTIs, compared with older children. This was particularly substantiated in RSV infection. Our presumption was supported by other studies. 14,25-28 Of course, this speculation needed to be validated by the population-based study. The findings reported elsewhere suggested that more males than females were affected by ARTIs, which were not observed in our study.", "14,25-28 Of course, this speculation needed to be validated by the population-based study. The findings reported elsewhere suggested that more males than females were affected by ARTIs, which were not observed in our study. Notably, our study occurred over a span of 3 years, which included the IAV H1N1 outbreak in 2009. The impact of the outbreak on the results should be considered. Data showed that the detection rate of IAV increased significantly and co-infection rate during outbreak months was much higher than average co-infection rate. Unfortunately, we did not type these influenza strains based on the original study design. It was most likely that these strains contributed to the relatively high proportion of IAV.", "Unfortunately, we did not type these influenza strains based on the original study design. It was most likely that these strains contributed to the relatively high proportion of IAV. Relatively higher single and multiple infections of RSV, HRV and PIVs were also observed during the outbreak of IAV. Increased susceptible population and awareness, intensive testing and altered patient and physician behavior could lead to these increases. These factors could partly explain the relatively high proportion of pneumonia cases in the study. Furthermore, studies showed that the outbreak of IAV H1N1 could increase the risk of other viral infections such as RSV and HRV. 24, 33 Other limitations also existed in this study.", "Furthermore, studies showed that the outbreak of IAV H1N1 could increase the risk of other viral infections such as RSV and HRV. 24, 33 Other limitations also existed in this study. First, molecular methods allowed the detection of only viral nucleic acid even without virus replication, which complicates the interpretation of positive detection results. Second, the subtype identification of some common respiratory viruses such as IAV and HRV was not performed in our study, particularly during the IAV H1N1 outbreak in 2009. In summary, despite those aforementioned limitations, this three consecutive years' surveillance would provide a basic profile of the spectrum, seasonality, age and gender distribution, co-infection patterns as well as clinical association of viral respiratory infections in hospitalized children in the study sites. It could help the prediction, prevention and control of ARTIs in children." ]

[ "BACKGROUND: The epidemiology of local viral etiologies is essential for the management of viral respiratory tract infections. Limited data are available in China to describe the epidemiology of viral respiratory infections, especially in small–medium cities and rural areas. OBJECTIVES: To determine the viral etiology and seasonality of acute respiratory infections in hospitalized children, a 3-year study was conducted in Shenzhen, China. METHODS: Nasopharyngeal aspirates from eligible children were collected. Influenza and other respiratory viruses were tested by molecular assays simultaneously. Data were analyzed to describe the frequency and seasonality.", "METHODS: Nasopharyngeal aspirates from eligible children were collected. Influenza and other respiratory viruses were tested by molecular assays simultaneously. Data were analyzed to describe the frequency and seasonality. RESULTS: Of the 2025 children enrolled in the study, 971 48·0% were positive for at least one viral pathogen, in which 890 91·7% were <4 years of age. The three most prevalent viruses were influenza A IAV; 35·8% , respiratory syncytial virus RSV; 30·5% and human rhinovirus HRV; 21·5% . Co-infections were found in 302 cases 31·1% , and dual viral infection was dominant. RSV, HRV and IAV were the most frequent viral agents involved in co-infection.", "Co-infections were found in 302 cases 31·1% , and dual viral infection was dominant. RSV, HRV and IAV were the most frequent viral agents involved in co-infection. On the whole, the obvious seasonal peaks mainly from March to May were observed with peak strength varying from 1 year to another. CONCLUSIONS: This study provides a basic profile of the epidemiology of acute respiratory viral infection in hospitalized children in Shenzhen. The spectrum of viruses in the study site is similar to that in other places, but the seasonality is closely related to geographic position, different from that in big cities in northern China and neighboring Hong Kong. Text: Acute respiratory tract infections ARTIs are a persistent and pervasive public health problem in both developed and developing countries.", "The spectrum of viruses in the study site is similar to that in other places, but the seasonality is closely related to geographic position, different from that in big cities in northern China and neighboring Hong Kong. Text: Acute respiratory tract infections ARTIs are a persistent and pervasive public health problem in both developed and developing countries. They cause a great burden of disease worldwide. Especially in developing countries including China, ARTIs, mainly pneumonia, are the leading cause of death among children under the age of 5 years. 1,2 A great variety of pathogens can cause ARTIs, and viruses have been considered as the predominant pathogens in this children population. 3, 4 The most frequently reported viruses include respiratory syncytial virus RSV , influenza viruses A and B IAV, IBV , parainfluenza viruses PIVs , human rhinovirus HRV and adenovirus ADV , which are responsible for most episodes of ARTIs in children.", "1,2 A great variety of pathogens can cause ARTIs, and viruses have been considered as the predominant pathogens in this children population. 3, 4 The most frequently reported viruses include respiratory syncytial virus RSV , influenza viruses A and B IAV, IBV , parainfluenza viruses PIVs , human rhinovirus HRV and adenovirus ADV , which are responsible for most episodes of ARTIs in children. 1 In the past decade, several new viruses associated with ARTIs such as human metapneumovirus HMPV , novel strains of coronaviruses SARS-CoV, HCoV-NL63 and HKUI , human bocavirus BOV , WU polyomavirus WUPoyV and KI polyomavirus KIPoyV have been discovered in human respiratory tract specimens. Among them, some have been identified to be causative pathogens of ARTIs. 1, 4, 5 Currently, there are no approved vaccines or medications available for most of the respiratory viruses. 1 A better understanding of the epidemiology of viral respiratory tract infections in children plays a key role for the prevention, control and treatment of ARTIs.", "1, 4, 5 Currently, there are no approved vaccines or medications available for most of the respiratory viruses. 1 A better understanding of the epidemiology of viral respiratory tract infections in children plays a key role for the prevention, control and treatment of ARTIs. Studies showed that many viral respiratory infections exhibited predictable seasonal variations. However, the epidemiological profiles of viral respiratory infections from different climate zones or different countries in the same climate zone may be varied. China is a large country crossing three climate zones, and great differences in climate are found from region to region. A better understanding of the epidemiology of ARTIs in different regions could be helpful to develop effective surveillance, prevention and treatment strategies.", "China is a large country crossing three climate zones, and great differences in climate are found from region to region. A better understanding of the epidemiology of ARTIs in different regions could be helpful to develop effective surveillance, prevention and treatment strategies. Although some studies on the epidemiology of ARTIs have recently been reported in big cities such as Beijing, Shanghai and Hong Kong, the epidemic characteristics of viruses in ARTIs are still not well established all around China, especially in other cities and rural areas. Shenzhen is the largest migratory city of China with high population density and population mobility. It is located in southern China at 22°27 0 -22°52 0 N and 113°46 0 -114°37 0 E, immediately north of Hong Kong, with a typical subtropical monsoon climate. The annual average temperature and relative humidity of Shenzhen are about 23°C 12-33°C and 77%, respectively.", "It is located in southern China at 22°27 0 -22°52 0 N and 113°46 0 -114°37 0 E, immediately north of Hong Kong, with a typical subtropical monsoon climate. The annual average temperature and relative humidity of Shenzhen are about 23°C 12-33°C and 77%, respectively. The purpose of this study is to investigate the prevalence, seasonality and clinical characteristics of acute viral respiratory infections in hospitalized children in Shenzhen and to provide insights into etiologies of ARTIs in local infants and children. A consecutive 3-year prospective study from July 2007 to June 2010 was conducted in Shenzhen, a coastal city neighboring Hong Kong. Four hospitals including a children's hospital were chosen for the study. Selected patients with ARTIs admitted to the pediatric wards were enrolled.", "Four hospitals including a children's hospital were chosen for the study. Selected patients with ARTIs admitted to the pediatric wards were enrolled. The inclusion criteria were as follows: less than 14 years old, acute fever T ≥ 38°C , with any one of respiratory symptoms such as sore throat, cough, wheezing and dyspnoea/ tachypnoea , normal or low leukocyte count, the onset of illness within 3 days before hospitalization. The diagnosis of pneumonia was based on the guideline of the management of childhood community acquired pneumonia CAP issued by the Chinese Medical Association in 2006. 17 In the guideline, the clinical symptoms and signs for the diagnosis of childhood CAP include fever, cough, tachypnoea defined according to different age , difficulty breathing and/or lower chest wall indrawing. X-ray evaluation has been carried out when necessary.", "17 In the guideline, the clinical symptoms and signs for the diagnosis of childhood CAP include fever, cough, tachypnoea defined according to different age , difficulty breathing and/or lower chest wall indrawing. X-ray evaluation has been carried out when necessary. The study protocol was approved by the medical ethical committees of the hospitals. Written informed consent was obtained from the parents or legal guardians of the children. Nasopharyngeal aspirates NPA were obtained by trained personnel following standard operating procedures within 24 hour after admission. The specimens were transported immediately to the laboratory by sterile viral transport media, then divided into aliquots and immediately frozen at À80°C until further processing.", "Nasopharyngeal aspirates NPA were obtained by trained personnel following standard operating procedures within 24 hour after admission. The specimens were transported immediately to the laboratory by sterile viral transport media, then divided into aliquots and immediately frozen at À80°C until further processing. Total viral nucleic acids DNA and RNA were extracted from 200 ll of NPA specimen using the AxyPrep Body Fluid DNA/RNA Miniprep Kit Axygen, Union City, CA, USA , according to the manufacturer's instructions. Purified DNA and RNA were stored at À80°C in aliquots for further PCR analysis. For each specimen, assays for ten common and newly identified viruses were performed. Briefly, WUPoyV and BOV were tested using monoplex PCRs described previously.", "For each specimen, assays for ten common and newly identified viruses were performed. Briefly, WUPoyV and BOV were tested using monoplex PCRs described previously. 18, 19 Other viruses were tested using the Luminex platform and multiplex xTAG TM respiratory viral panel assay RVP Assay according to the manufacturer's instructions. 20 All multiple infection samples were retested. If there was discordance between two tests, the sample was confirmed by monoplex PCR. Statistical Package for the Social Sciences SPSS for Windows version 11.0 SPSS Inc. Chicago, IL, USA was used. For comparison of categorical data, chi-square or Fisher's exact test was used.", "Statistical Package for the Social Sciences SPSS for Windows version 11.0 SPSS Inc. Chicago, IL, USA was used. For comparison of categorical data, chi-square or Fisher's exact test was used. All tests were two-tailed, and a P value below 0Á05 was considered statistically significant. A total of 2025 specimens were obtained from 2025 eligible patients ranging from 15 days to 14 years old with a median age of 12 months, in which 89Á6% of patients were < 4 years old. There were 964 47Á6% females and 1061 52Á4% males included. Of all hospitalized children enrolled in this study, 84Á0% involved lower respiratory infection and 16Á0% had upper respiratory tract infection Table 1 .", "There were 964 47Á6% females and 1061 52Á4% males included. Of all hospitalized children enrolled in this study, 84Á0% involved lower respiratory infection and 16Á0% had upper respiratory tract infection Table 1 . Among 971 positive cases, 572 58Á9% were diagnosed as pneumonia. About 971 of the 2025 cases 48Á0% were positive for at least one viral pathogen. Among them, IAV, RSV, HRV and PIVs were detected in 348 35Á8% , 296 30Á5% , 209 21Á5% and 169 17Á4% cases, respectively. Single infection was observed in 669 68Á9% cases, and multiple infection was found in 302 31Á1% .", "Among them, IAV, RSV, HRV and PIVs were detected in 348 35Á8% , 296 30Á5% , 209 21Á5% and 169 17Á4% cases, respectively. Single infection was observed in 669 68Á9% cases, and multiple infection was found in 302 31Á1% . Our results also showed that RSV, IAV and HRV were the main pathogens in single viral infection cases Table 1 . The monthly positive rates varied from 32Á5% to 75Á0% with a mean of 45Á1% Figure 1 . In the year 2009, when influenza A H1N1 was pandemic worldwide, the positive rate started to increase in March and the highest positive rate 75Á0% was observed in May. Among the 971 positive cases, a total of 1335 viral pathogens were detected.", "In the year 2009, when influenza A H1N1 was pandemic worldwide, the positive rate started to increase in March and the highest positive rate 75Á0% was observed in May. Among the 971 positive cases, a total of 1335 viral pathogens were detected. The most frequently detected pathogen was IAV 26Á1%, 348/1335 , followed by RSV 22Á2%, 296/1335 , HRV 15Á7%, 209/1335 , PIV1 and PIV3 12Á7%, 169/1335 About 302 co-infection cases were identified, accounting for 14Á9% of all 2025 hospitalized children. During the H1N1 outbreak from March to August 2009, co-infection cases and co-infection rate increased significantly Figure 2 . 143 of 302 47Á4% co-infection cases were detected during that time. Among them, 121 cases were involved in IAV infection, including 90 dual infection cases.", "143 of 302 47Á4% co-infection cases were detected during that time. Among them, 121 cases were involved in IAV infection, including 90 dual infection cases. Of all co-infection cases, 247 81Á8% , 49 16Á2% and 5 1Á7% were infected with two, three and four potential viral pathogens, respectively. One multiple infection with five viruses was detected in a RSV IAV HRV BOV PIV3 ADV HMPV WUPoyV PIV1 IBV Total A Total cases 163 172 85 55 51 49 37 26 24 7 669 Bronchitis 12 19 7 7 5 2 2 3 5 0 62 Bronchiolitis 45 19 20 7 13 9 8 2 5 0 128 Pneumonia 93 97 55 28 24 28 25 18 14 5 387 URTI 13 37 3 13 9 10 2 3 0 2 *Case number and percentage in all enrolled children. **Incidence rate in this age group significantly higher than the other age groups. ***No significant difference between these three age groups.", "**Incidence rate in this age group significantly higher than the other age groups. ***No significant difference between these three age groups. †No significant differences between these two age groups, but significantly lower than the other age groups. 6-month-old infant. IAV, RSV and HRV were the three most frequently found viruses in co-infection and detected in 176, 133 and 124 cases with co-infection rates of 50Á6%, 44Á9% and 59Á3%, respectively Table 2 . Various multiple infection patterns were observed in the study. A total of 152 50Á3% co-infection cases involved at least two viruses of RSV, HRV and IAV.", "Various multiple infection patterns were observed in the study. A total of 152 50Á3% co-infection cases involved at least two viruses of RSV, HRV and IAV. Co-infection rate of each individual virus detected varied significantly. The lowest and highest co-infection rates were observed in WUPoyV 33Á3% and IBV 66Á7% , respectively. 91Á4% 276/302 of co-infection cases were tested in the age group of 4 years old or younger Table 2 , but among all age groups, no statistical difference in co-infection rate was found v 2 = 1Á83, P = 0Á8721 . Gender-specific difference in co-infection rate was not observed v 2 = 2Á17, P = 0Á1404 .", "91Á4% 276/302 of co-infection cases were tested in the age group of 4 years old or younger Table 2 , but among all age groups, no statistical difference in co-infection rate was found v 2 = 1Á83, P = 0Á8721 . Gender-specific difference in co-infection rate was not observed v 2 = 2Á17, P = 0Á1404 . There was no significant difference in co-infection rates between PICU and non-PICU cases. Similarly, no significant difference in clinical symptoms was observed between co-infection and single cases data not shown . In general, respiratory viruses were detected more often in the period of March to May than in other months 55Á4% and 40Á6%, respectively, v 2 = 28Á06, P = 0Á0000 , and obvious seasonal peaks were observed during those months with peak strength varying from 1 year to another. A weaker seasonal peak could also be distinguished in some winter months in different years Figure 1 .", "In general, respiratory viruses were detected more often in the period of March to May than in other months 55Á4% and 40Á6%, respectively, v 2 = 28Á06, P = 0Á0000 , and obvious seasonal peaks were observed during those months with peak strength varying from 1 year to another. A weaker seasonal peak could also be distinguished in some winter months in different years Figure 1 . The seasonality profile of each individual virus detected was diversified. A seasonal distribution of IAV can be observed from late spring to summer mainly March to May and sometimes in fall October, November or December . A wide seasonal peak of IAV infection was detected from March to August 2009 Figure 3A . Although RSV was tested almost a whole year, two yearly peaks were identified.", "A wide seasonal peak of IAV infection was detected from March to August 2009 Figure 3A . Although RSV was tested almost a whole year, two yearly peaks were identified. One was found in November and/or December and the other stronger one was found in March to May of the year. The peak duration in 2009 was longer than those in other years. The seasonal trends of HRV and PIVs were similar to that of RSV, but the peaks of these three viruses fluctuated and shifted mildly Figure 3B . Although IBV and ADV had a low detection rate in the study, similar seasonality was observed and their infection peaks were mainly in midwinter.", "The seasonal trends of HRV and PIVs were similar to that of RSV, but the peaks of these three viruses fluctuated and shifted mildly Figure 3B . Although IBV and ADV had a low detection rate in the study, similar seasonality was observed and their infection peaks were mainly in midwinter. Peaks in spring and summer were also observed in some years Figure 3C . Our investigation did not find regular seasonality in BOV infections. A sudden increase in BOV infection was recorded in April and May 2010. Although the positive rate of HMPV infection was only 4Á8%, regular seasonality was observed from March to May of each year.", "A sudden increase in BOV infection was recorded in April and May 2010. Although the positive rate of HMPV infection was only 4Á8%, regular seasonality was observed from March to May of each year. Of 39 patients with WUPoyV infection, 36 were detected after July 2008. Our data implied that peak months of WUPoyV infection were from March to May Figure 3D . The positive rates of viral infections in male and female were 52Á5% and 47Á5%, respectively. No significant gender difference was revealed v 2 = 0Á012, P = 0Á9118 . The distribution of viral agents and infection patterns in different age groups are shown in Table 2 .", "No significant gender difference was revealed v 2 = 0Á012, P = 0Á9118 . The distribution of viral agents and infection patterns in different age groups are shown in Table 2 . Of all 971 positive children, 890 91Á7% were 4 years old or younger. The positive rate in this age group was significantly higher than that in children more than 4 years old v 2 = 8Á26, P = 0Á0041 . Children under 6 months were the most susceptible to respiratory viral pathogens with a positive rate of 14Á8% Table 2 . Very few long-term prospective studies were performed for viral etiologies of ARTIs among hospitalized children.", "Children under 6 months were the most susceptible to respiratory viral pathogens with a positive rate of 14Á8% Table 2 . Very few long-term prospective studies were performed for viral etiologies of ARTIs among hospitalized children. In this present study, the infection frequency, seasonality, co-infection pattern and clinical features of viral respiratory infections were investigated based on prospective analysis of three consecutive year's data from hospitalized children with ARTIs. Our results provided a distinctive epidemiological profile of viral respiratory infections in hospitalized children with ARTIs in the study areas, which was different from those in the big cities in northern China such as Beijing and Shanghai and also different from that in adjacent Hong Kong. Overall, 48Á0% of our cases were positive for respiratory virus infections, which resembled the latest study in the same city. 21 A similar incidence rate has been obtained in neighboring regions 13, 22 and other cities such as Rome 23 and Milan, 24 but it was different from other studies.", "Overall, 48Á0% of our cases were positive for respiratory virus infections, which resembled the latest study in the same city. 21 A similar incidence rate has been obtained in neighboring regions 13, 22 and other cities such as Rome 23 and Milan, 24 but it was different from other studies. In China, the overall positive rate reported varied from 27Á3 to 74Á8% depending on different areas and detection methods. 15, 16, The rate of respiratory viral infections varied worldwide, and many factors such as geographic distribu-tion, study design and detection protocols could lead to these variations. 1, 7, 8, 32 In our study, leukocyte count was used as an indicator of inclusion criteria and it probably affected the positive rate. Viruses not considered in the study, for example coronaviruses, would underestimate the positive rate.", "1, 7, 8, 32 In our study, leukocyte count was used as an indicator of inclusion criteria and it probably affected the positive rate. Viruses not considered in the study, for example coronaviruses, would underestimate the positive rate. Most studies showed that RSV or HRV was the most prevalent viruses in children with viral respiratory tract infection. 1 In this study, IAV was the most frequently detected respiratory virus, followed by RSV and HRV. IAV H1N1 outbreak in 2009 could explain this shift. Data showed that about 60% of IAV infections were detected during the outbreak period. Studies showed that the H1N1 outbreak could change viral distribution patterns.", "Data showed that about 60% of IAV infections were detected during the outbreak period. Studies showed that the H1N1 outbreak could change viral distribution patterns. 24, 29, 33 Regardless of the IAV H1N1 outbreak, RSV and HRV were the two most common viral pathogens in ARTIs, which was consistent with most previous studies. 1, 10, 15, 16, 22, Our study further confirmed the importance of RSV and HRV in children with ARTIs, especially in children < 4 years of age. 10, 14, 23 Our results also showed that 12Á7% of viral pathogens detected were PIV1 and PIV3, which implied that PIVs played an important role in children with ARTIs. Similar findings were obtained in the studies conducted in Shanghai, 14, 34 Changsha, 26 Harbin, 30 Hong Kong 13 and Rome.", "10, 14, 23 Our results also showed that 12Á7% of viral pathogens detected were PIV1 and PIV3, which implied that PIVs played an important role in children with ARTIs. Similar findings were obtained in the studies conducted in Shanghai, 14, 34 Changsha, 26 Harbin, 30 Hong Kong 13 and Rome. 23 The prevalence of PIV3 was twofold higher than that of PIV1, particularly in infants, which was similar with other reports, 25, 26, 30, 35 implying that infants could be more vulnerable to infection with PIV3 than PIV1. HMPV has been proven to be one of the main viral pathogens responsible for ARTIs in children. 5 The positive rate found in the study was consistent with previously published results. 10, 36, 37 In China, the infection rate of HMPV varied from 3Á2 to 10Á6%.", "5 The positive rate found in the study was consistent with previously published results. 10, 36, 37 In China, the infection rate of HMPV varied from 3Á2 to 10Á6%. 22, 26, 28, 29, 31 The seasonality of HMPV in this study was mainly from March to May, similar to that in Hong Kong, 36 but different from other places. 5, 37 In our study, 4Á9% of cases were positive for BOV, which coincided with 5Á0% in Hong Kong 38 and higher than Guangzhou 39 and eastern Guangdong. 22 Our result suggested that BOV might be present throughout the year with no seasonal distribution. However, seasonal distribution was noted from September to February in Hong Kong 38 and May and June in Guangzhou.", "22 Our result suggested that BOV might be present throughout the year with no seasonal distribution. However, seasonal distribution was noted from September to February in Hong Kong 38 and May and June in Guangzhou. 39 The use of multiple PCR made it possible to simultaneously detect a broad spectrum of viruses with excellent sensitivity, at the same time, with increased viral detection rate and co-infection rate for ARTIs. 12,40 Among our positive cases, co-infection rate was 31Á1%, which was similar to 27Á9% reported by Do et al. 10 Co-infection rate reported elsewhere varied widely from 25Á4 to 47Á9%. 40 The relatively lower co-infection rates ranging from 0Á24 to 26Á9% were reported in the studies conducted in various cities of China.", "10 Co-infection rate reported elsewhere varied widely from 25Á4 to 47Á9%. 40 The relatively lower co-infection rates ranging from 0Á24 to 26Á9% were reported in the studies conducted in various cities of China. 22, In most of these studies, immunofluorescence kits were used to test a lower number of respiratory viruses. It was worth to note that in the study by Peng et al. 32 in Wuhan, China, 69Á5% of co-infection rate was reported with immunofluorescence kit. These variations might be attributed to geographic differences, diagnostic methods for viral agents and study design.", "32 in Wuhan, China, 69Á5% of co-infection rate was reported with immunofluorescence kit. These variations might be attributed to geographic differences, diagnostic methods for viral agents and study design. 12, 32, 34, 41, 42 Pathogens in those negative patients need to be further investigated as only ten common and newly identified viruses were included in our study, which might underestimate positive rate or coinfection rate. It was notable that the correlation between co-infection rate and positive rate was not observed. Of multiple infections, dual infection was predominant in this study whether or not considering the IAV H1N1 outbreak in 2009, which was consistent with previous studies. 28, 32, 42, 43 Similar with the studies conducted in the cities of Guangzhou and Wuhan, China, 28, 29 our study showed that IAV, RSV and HRV were the main viruses involved in multiple infections.", "Of multiple infections, dual infection was predominant in this study whether or not considering the IAV H1N1 outbreak in 2009, which was consistent with previous studies. 28, 32, 42, 43 Similar with the studies conducted in the cities of Guangzhou and Wuhan, China, 28, 29 our study showed that IAV, RSV and HRV were the main viruses involved in multiple infections. High co-infection rate between these three viruses could be explained from the overlap of their seasonal distributions. A variety of predominant multiple infection patterns between respiratory viruses were observed in different studies. 12, 32, 42, 43 For example, it was shown in Martin et al. 's study 43 that ADV and coronaviruses were the most common co-infection pattern.", "12, 32, 42, 43 For example, it was shown in Martin et al. 's study 43 that ADV and coronaviruses were the most common co-infection pattern. Our study showed that RSV and HRV were the two most viruses involved in multiple infection, followed by IAV and PIVs, regardless of IAV infection in the H1N1 outbreak period. It was difficult to explain the variations of coinfection patterns based only on seasonal distribution. A recent study suggested that co-infection patterns were not random and certain pathogens had higher frequency of coinfection. 41 As molecular assays only detect nucleic acid and positive result does not mean the presence of the pathogen, when studying co-infection patterns of respiratory viruses, the ability to differentiate the real causative pathogens needs to be solved first.", "A recent study suggested that co-infection patterns were not random and certain pathogens had higher frequency of coinfection. 41 As molecular assays only detect nucleic acid and positive result does not mean the presence of the pathogen, when studying co-infection patterns of respiratory viruses, the ability to differentiate the real causative pathogens needs to be solved first. Viral load detection could provide some clues for solving this issue. 43, 44 Although high co-infection rates have been reported in various studies, the associations among multiple infections, hospitalization rate and severity of ARTIs were still not clear with inconsistent results in different studies. 42, 43, 45 Our data suggested that multiple infection had less association with the severity of disease, consistent with Peng et al. 's study.", "42, 43, 45 Our data suggested that multiple infection had less association with the severity of disease, consistent with Peng et al. 's study. 32 The relationship between co-infection rates and age group was also investigated in our study, and little correlation was observed. Several previous studies observed that co-infection rates were more frequent in a certain age group, but results were varied. 32, 43 In contrast to temperate region, where most viruses had winter-spring seasonality, the respiratory viral infections in tropical and subtropical regions appeared mainly to be spring-summer seasonality. 9 In this study, due to the high detection rate and similar seasonality of RSV, HRV, IAV, PIV and HMPV, an overall spring-summer seasonality of viral respiratory infections in children was concluded.", "32, 43 In contrast to temperate region, where most viruses had winter-spring seasonality, the respiratory viral infections in tropical and subtropical regions appeared mainly to be spring-summer seasonality. 9 In this study, due to the high detection rate and similar seasonality of RSV, HRV, IAV, PIV and HMPV, an overall spring-summer seasonality of viral respiratory infections in children was concluded. Studies conducted in Hong Kong showed that a clear seasonal peak was from April to September, 36, 46 with a longer duration than our study. The overall seasonality in this study was also different from the studies conducted in northern or central cities of China, in which the seasonality of most viruses presented in autumn-winter and/or winter-spring. 15, 30 The winter-spring seasonality was also observed in Guangzhou, a city about 150 kilometers north of Shenzhen. 28 Different seasonal onset and duration were observed in various studies conducted in sub- tropical regions.", "15, 30 The winter-spring seasonality was also observed in Guangzhou, a city about 150 kilometers north of Shenzhen. 28 Different seasonal onset and duration were observed in various studies conducted in sub- tropical regions. In these studies, ambient temperature, humidity and rainfall were widely used to explain these differences in seasonality, but inconsistent results were observed. 9, 46, 47 Although most studies demonstrated that the seasonality of viral respiratory infections was correlated with increased rainfall, effects of climate factors such as humidity and temperature on the seasonality were complex and interactive. 9, 46, 48 The study areas have four indistinct seasons, and the coldest month usually emerges in January average 12°C . During the period from March to May, the weather featured warm ambient temperature average 18-25°C , high relative humidity average 85%-90% and increasing rainfall.", "9, 46, 48 The study areas have four indistinct seasons, and the coldest month usually emerges in January average 12°C . During the period from March to May, the weather featured warm ambient temperature average 18-25°C , high relative humidity average 85%-90% and increasing rainfall. These meteorological conditions were perhaps conducive to viral survival. 9, 48 In addition, intensive temperature fluctuations during seasonal alternation could increase the susceptibility to infections. 49 As reported in other studies in temperate, tropical and subtropical regions, viral infection rates in children population showed an inverse correlation with age, with younger individuals experiencing higher viral infection rates. 3, 4, 6, 9, 24 Our results suggested that children younger than 4 years of age, particularly <6 months, were at higher risk of hospitalization for ARTIs, compared with older children.", "49 As reported in other studies in temperate, tropical and subtropical regions, viral infection rates in children population showed an inverse correlation with age, with younger individuals experiencing higher viral infection rates. 3, 4, 6, 9, 24 Our results suggested that children younger than 4 years of age, particularly <6 months, were at higher risk of hospitalization for ARTIs, compared with older children. This was particularly substantiated in RSV infection. Our presumption was supported by other studies. 14,25-28 Of course, this speculation needed to be validated by the population-based study. The findings reported elsewhere suggested that more males than females were affected by ARTIs, which were not observed in our study.", "14,25-28 Of course, this speculation needed to be validated by the population-based study. The findings reported elsewhere suggested that more males than females were affected by ARTIs, which were not observed in our study. Notably, our study occurred over a span of 3 years, which included the IAV H1N1 outbreak in 2009. The impact of the outbreak on the results should be considered. Data showed that the detection rate of IAV increased significantly and co-infection rate during outbreak months was much higher than average co-infection rate. Unfortunately, we did not type these influenza strains based on the original study design. It was most likely that these strains contributed to the relatively high proportion of IAV.", "Unfortunately, we did not type these influenza strains based on the original study design. It was most likely that these strains contributed to the relatively high proportion of IAV. Relatively higher single and multiple infections of RSV, HRV and PIVs were also observed during the outbreak of IAV. Increased susceptible population and awareness, intensive testing and altered patient and physician behavior could lead to these increases. These factors could partly explain the relatively high proportion of pneumonia cases in the study. Furthermore, studies showed that the outbreak of IAV H1N1 could increase the risk of other viral infections such as RSV and HRV. 24, 33 Other limitations also existed in this study.", "Furthermore, studies showed that the outbreak of IAV H1N1 could increase the risk of other viral infections such as RSV and HRV. 24, 33 Other limitations also existed in this study. First, molecular methods allowed the detection of only viral nucleic acid even without virus replication, which complicates the interpretation of positive detection results. Second, the subtype identification of some common respiratory viruses such as IAV and HRV was not performed in our study, particularly during the IAV H1N1 outbreak in 2009. In summary, despite those aforementioned limitations, this three consecutive years' surveillance would provide a basic profile of the spectrum, seasonality, age and gender distribution, co-infection patterns as well as clinical association of viral respiratory infections in hospitalized children in the study sites. It could help the prediction, prevention and control of ARTIs in children." ]

[ "There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein GFP -expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas <30 in.", "However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas <30 in. during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient.", "We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient. IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks.", "Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients. Text: D espite early reports . . . . .", "Text: D espite early reports . . . . . , the premise that hand-wash sink traps can act as reservoirs of bacteria that cause nosocomial infections has been frequently overlooked. There has recently been an alarming increase in sink-related outbreaks worldwide, with many reports establishing an observational link . . . . . . . . . A sink often operates as an open conduit to wastewater in a patient care area that is often in the same room as the patient. Health care establishments often invest in desperate interventions to deal with nosocomial outbreaks. The preferred method for addressing most of the environmentrelated transmission is to employ enhanced cleaning using chemical and physical agents . .", "The preferred method for addressing most of the environmentrelated transmission is to employ enhanced cleaning using chemical and physical agents . . Unfortunately, routine approaches are inefficient in completely eliminating drug-resistant Gammaproteobacteria in an inaccessible microbiologically active area such as a sink trap 6, . . . . . . The wet, humid, and relatively protected environment in a sink trap favors the formation of rich stable microbial communities . . These communities will be exposed to liquids and waste that are discarded in a sink and may include antimicrobials, discarded beverages, soap, presumably pathogenic bacteria from health care workers' hands, and other items.", ". These communities will be exposed to liquids and waste that are discarded in a sink and may include antimicrobials, discarded beverages, soap, presumably pathogenic bacteria from health care workers' hands, and other items. In short, sink traps could serve as a breeding ground for opportunistic and highly antimicrobial-resistant bacteria that cannot be easily cleaned or removed . . . . . . . There are many reports of a genetic association between pathogens found in sink traps and those found in patients . . However, surprisingly little work has been done to understand the microscale transmission dynamics.", ". However, surprisingly little work has been done to understand the microscale transmission dynamics. It was previously demonstrated using a suspension of fluorescent particles Glo Germ; Glo Germ Co., Moab, UT that material injected into the P-trap gets dispersed around a hand-washing sink . . This result however has not been replicated hitherto in the follow-up studies. Dispersion has never been investigated with living organisms.", ". This result however has not been replicated hitherto in the follow-up studies. Dispersion has never been investigated with living organisms. Ultimately, many details remain unaddressed surrounding the spread of Enterobacteriaceae in sink-trap wastewater systems: i can organisms grow retrograde from the P-trap water to the sink strainer, ii can organisms spread from one sink to another along the internal surfaces of pipes with shared drainage systems, and iii which portion of a colonized drain pipe results in dispersion into the sink bowl during a hand-washing event? We aim to better understand the dispersion dynamics of Gammaproteobacteria living in the wastewater of a sink strainer and P-trap into an area where patients and health care workers could be exposed. To study this dynamic, we used a surrogate organism that could be easily tracked while remaining in the Enterobacteriaceae family, where some of the most concerning threats in antimicrobial resistance are developing .", "We aim to better understand the dispersion dynamics of Gammaproteobacteria living in the wastewater of a sink strainer and P-trap into an area where patients and health care workers could be exposed. To study this dynamic, we used a surrogate organism that could be easily tracked while remaining in the Enterobacteriaceae family, where some of the most concerning threats in antimicrobial resistance are developing . . Growth and colonization of GFP-expressing E. coli in the P-trap. In the first 14 days following the installation of the P-trap with established green fluorescent protein GFP -expressing Escherichia coli and just water running from the faucet, GFPexpressing E. coli was not detected in the tailpipe beyond 1.5 in. above the liquid level in the P-trap.", "In the first 14 days following the installation of the P-trap with established green fluorescent protein GFP -expressing Escherichia coli and just water running from the faucet, GFPexpressing E. coli was not detected in the tailpipe beyond 1.5 in. above the liquid level in the P-trap. GFP-expressing E. coli, however, was found to be viable in the P-trap without any nutrients added. A nutrient regimen was then instituted to understand the influence of nutrients on mobility and upward growth. The addition of tryptic soy broth TSB promoted GFP-expressing E. coli growth as early as day 1, with growth observed in the tailpipe 2 in. above the liquid surface in the P-trap Table 1 .", "The addition of tryptic soy broth TSB promoted GFP-expressing E. coli growth as early as day 1, with growth observed in the tailpipe 2 in. above the liquid surface in the P-trap Table 1 . On day 7, the strainer ϳ8 in. above the liquid in the P-trap was found to be colonized with GFP-expressing E. coli. This translates to an average growth rate of 1 in./day along the length of the tailpipe with the addition of nutrients and without faucet operation. GFP-expressing E. coli was not detected in the faucet water. Sink-to-sink transmission of bacteria.", "GFP-expressing E. coli was not detected in the faucet water. Sink-to-sink transmission of bacteria. In these experiments, a flanking sink sink 5 was the only P-trap inoculated with GFP-expressing E. coli and therefore was the sole source for transmission to the connected sinks. Starting with a lower inoculum concentration 10 3 CFU/ml in sink 5, on day 7, GFP-expressing E. coli was detected in the sink 2 and sink 3 P-traps Fig. 1a . With inoculum concentrations of 10 6 CFU/ml and Ͼ10 10 CFU/ml in sink 5, all of the sink P-traps in the sink gallery with the exception of sink 1 were found to be colonized with GFP-expressing E. coli after 7 days Fig.", "1a . With inoculum concentrations of 10 6 CFU/ml and Ͼ10 10 CFU/ml in sink 5, all of the sink P-traps in the sink gallery with the exception of sink 1 were found to be colonized with GFP-expressing E. coli after 7 days Fig. 1b and c . Faucet water and aerators tested negative for GFP-expressing E. coli. Irrespective of the starting inoculum concentration, on day 7 the highest level of colonization was recorded in the sink 3 P-trap. After day 7, when the nutrient regimen described previously was followed for an additional 7 days in each of the sinks in the sink gallery with an inoculum concentration of Ͼ10 10 CFU/ml, GFP-expressing E. coli was detected in the strainers of sinks 2 and 3 on day 14.", "Irrespective of the starting inoculum concentration, on day 7 the highest level of colonization was recorded in the sink 3 P-trap. After day 7, when the nutrient regimen described previously was followed for an additional 7 days in each of the sinks in the sink gallery with an inoculum concentration of Ͼ10 10 CFU/ml, GFP-expressing E. coli was detected in the strainers of sinks 2 and 3 on day 14. This finding validated the upward growth and growth rate in the tailpipe when nutrients were added. Nonfluorescent colonies were occasionally observed in the P-trap water samples collected from the sinks, which were subsequently identified to be Pseudomonas sp. or Stenotrophomonas maltophilia, and fluorescent colonies were confirmed to be E. coli. Dispersion of microspheres from sinks.", "or Stenotrophomonas maltophilia, and fluorescent colonies were confirmed to be E. coli. Dispersion of microspheres from sinks. In the first dispersion experiment, when fluorescent microspheres were inoculated into the offset drain tailpiece only 4 in. below the strainer, no microspheres were detected on the polyester sheets placed on the counter space. However, when the sink bowl was coated with the microspheres, polyester sheets overlaid on the counter space captured the dispersed microspheres caused by the faucet operation. Dispersion was observed on almost all zones of the sink counter space Fig. 2 .", "Dispersion was observed on almost all zones of the sink counter space Fig. 2 . Relatively higher levels of dispersion were observed along the major and minor axes of the elliptical sink bowl zones 2, 5, 6, 9, 11, and 12 . Anterior corners of the sink counter space zones 4 and 7 , which were most distant from the impact of water in the sink bowl, received the lowest dispersion. Dispersion of GFP-expressing E. coli from sinks. Initially the P-trap alone was inoculated with GFP-expressing E. coli and carefully installed, keeping the tailpipe and strainer free of GFP-expressing E. coli before operating the faucets.", "Dispersion of GFP-expressing E. coli from sinks. Initially the P-trap alone was inoculated with GFP-expressing E. coli and carefully installed, keeping the tailpipe and strainer free of GFP-expressing E. coli before operating the faucets. No fluorescent CFU were observed on the plates placed on the counter or attached to the bowl surface after faucet operation. Similarly, no fluorescent CFU were detected when GFPexpressing E. coli was inoculated into the offset drain tailpiece only 4 in. below the strainer. Interestingly, when there was conspicuous water backup over the strainer as a result of a higher water flow rate from the faucet than the drainage rate from the P-trap, dispersal was detected on the plates attached to the bowl surface.", "below the strainer. Interestingly, when there was conspicuous water backup over the strainer as a result of a higher water flow rate from the faucet than the drainage rate from the P-trap, dispersal was detected on the plates attached to the bowl surface. The dispersion pattern recorded when the sink bowl was coated with GFPexpressing E. coli was comparable to the pattern recorded when the sink bowl was coated with fluorescent microspheres Fig. 2 . Dispersion was significantly higher along the axes zones 6, 9, 11, and 12 and lower at the corners of the sink counter space zones 4, 7, and 10 . In contrast, dispersion of GFP-expressing E. coli caused by the faucet operation was much more extensive when the strainer was allowed to be colonized with GFPexpressing E. coli prior to the dispersion experiment.", "Dispersion was significantly higher along the axes zones 6, 9, 11, and 12 and lower at the corners of the sink counter space zones 4, 7, and 10 . In contrast, dispersion of GFP-expressing E. coli caused by the faucet operation was much more extensive when the strainer was allowed to be colonized with GFPexpressing E. coli prior to the dispersion experiment. In addition to the sink counter space, we measured dispersion to the sink bowl, faucet, faucet handles, splatter shields, and the extended counter surface. Dispersion of GFP-expressing E. coli was highest on the plates attached to the sink bowl Fig. 3b . Further, dispersion was greater along the Ϫ\" and \"ϩ\" denote the absence and presence of GFP-expressing E. coli, respectively.", "3b . Further, dispersion was greater along the Ϫ\" and \"ϩ\" denote the absence and presence of GFP-expressing E. coli, respectively. minor axis of the sink bowl Fig. 3b , zones B3, B4, and B10 than along the major axis of the sink bowl, associated with a shorter distance from the strike point of the faucet water to the bowl along this axis. The next highest CFU count from the dispersal was recorded on the counter area near the faucets Fig. 3a , zones 12 and 11 . A similar pattern of higher dispersion near the faucets and lower dispersion at the corners of the counter space Fig.", "3a , zones 12 and 11 . A similar pattern of higher dispersion near the faucets and lower dispersion at the corners of the counter space Fig. 3a , zones 4, 7, and 10 was also observed using microspheres. Dispersion was also recorded in other zones of the counter space, on the Plexiglas splatter shields, faucets, faucet handles, and extended surface Fig. 3c . There were no GFP-expressing E. coli CFU recorded on plates placed beyond 30 in. from the strainer, demarcating the range of dispersion under these experimental conditions. Table 2 gives a summary of the total distribution loads recorded using fluorescent microspheres and GFP-expressing E. coli across each experiment.", "from the strainer, demarcating the range of dispersion under these experimental conditions. Table 2 gives a summary of the total distribution loads recorded using fluorescent microspheres and GFP-expressing E. coli across each experiment. The loads of dispersion on the sink counter were comparable when the sink bowl was coated with microspheres or GFP-expressing E. coli before the faucet operation. Although the dispersion load on the sink counter was lower when the sink strainer was colonized, it is interesting to note that the sink bowl received the highest dispersion. To mimic dispersion in a hospital setting, we first investigated whether GFPexpressing E. coli would establish consistent colonization in a sink trap as many other Gammaproteobacteria implicated in nosocomial outbreaks have done . .", "To mimic dispersion in a hospital setting, we first investigated whether GFPexpressing E. coli would establish consistent colonization in a sink trap as many other Gammaproteobacteria implicated in nosocomial outbreaks have done . . Many recent reports demonstrate that P-traps become colonized with highly consequential Gammaproteobacteria, which then results in nosocomial transmission . . The retained water in a sink P-trap is present to provide a water barrier to prevent off-gassing of sewer smell, but it may inadvertently provide favorable conditions for pathogenic and opportunistic antibiotic-resistant microorganisms to survive and develop resilient biofilms . . However, the mechanism of dispersal of the bacteria in the P-trap to patients or the surrounding health care area had not been fully elucidated.", ". However, the mechanism of dispersal of the bacteria in the P-trap to patients or the surrounding health care area had not been fully elucidated. We began with the hypothesis that the bacteria originate from the P-trap via droplet creation when the water from the faucet hits the P-trap water, thus contaminating the sink bowl and the surrounding area. The finding supporting this theory had been previously reported using Glo Germ particles . . However, in the present study with careful attention to avoid strainer and tail piece contamination, the dispersal directly from the sink P-trap with either microspheres or GFP-expressing E. coli could not be reproduced as previously reported . .", "However, in the present study with careful attention to avoid strainer and tail piece contamination, the dispersal directly from the sink P-trap with either microspheres or GFP-expressing E. coli could not be reproduced as previously reported . . Rather this work demonstrates a different, more staged mode of transmission from a P-trap reservoir to the sink and surrounding environment. GFP-expressing E. coli in the P-trap alone was sustained for 14 days but did not grow or mobilize up the tailpipe to the strainer with just intermittent water exposure. However, when nutrients were subsequently added to the system, the organisms rapidly grew up the tailpipe to the strainer at approximately an inch per day. In a real-world setting, motility of bacteria inside the tailpipe is restricted to relatively sporadic and brief wetting events in which swimming is an opportunity to colonize new surfaces.", "However, when nutrients were subsequently added to the system, the organisms rapidly grew up the tailpipe to the strainer at approximately an inch per day. In a real-world setting, motility of bacteria inside the tailpipe is restricted to relatively sporadic and brief wetting events in which swimming is an opportunity to colonize new surfaces. It is assumed that once established, the biofilm promotes the upward growth of GFP-expressing E. coli in the tailpipe at an accelerated rate. The nutrient regimen that promoted colonization in our model reflects our and others' observations of items commonly disposed of in hospital sinks intravenous fluids, feeding supplements, and leftover beverages . . Transmission of bacteria between sinks via a common pipe was a key finding in this study as this highlights the concept that premise plumbing may be a more continuous system with shared microbiology than a single isolated sink.", ". Transmission of bacteria between sinks via a common pipe was a key finding in this study as this highlights the concept that premise plumbing may be a more continuous system with shared microbiology than a single isolated sink. The sink gallery used in this study provided a unique in situ advantage to investigate sink-to-sink transmission of bacteria through common drains. The two possible mechanisms for P-trap strainers becoming colonized are seeding of organisms from above and retrograde spread of organisms along common pipes in a hospital wastewater infrastructure. Here we demonstrate that it is possible for GFP-expressing E. coli to contaminate adjacent P-traps with just time and water given a standard U.S. code piping rise of 0.25 ft/ft. Sink-to-sink or retrograde transmission may explain the recurrence of pathogen colonization following intervention strategies like disinfection or replacement of plumbing .", "Here we demonstrate that it is possible for GFP-expressing E. coli to contaminate adjacent P-traps with just time and water given a standard U.S. code piping rise of 0.25 ft/ft. Sink-to-sink or retrograde transmission may explain the recurrence of pathogen colonization following intervention strategies like disinfection or replacement of plumbing . . Sink 3 was lowest on the slope in the drain line see Fig. 4 with arguably the most opportunity for reflux and retrograde wetting. Sink 1, on the other hand, was farthest away from the source sink 5 , and its P-trap had the greatest incline in the drain line connecting the sinks, which could perhaps contribute to the reasons there was no GFP-expressing E. coli colonization detected in it after 7 days.", "4 with arguably the most opportunity for reflux and retrograde wetting. Sink 1, on the other hand, was farthest away from the source sink 5 , and its P-trap had the greatest incline in the drain line connecting the sinks, which could perhaps contribute to the reasons there was no GFP-expressing E. coli colonization detected in it after 7 days. There has been more investigation about microbiologic dynamics of infectious viral particles such as those of severe acute respiratory syndrome SARS and Ebola viruses through premise plumbing systems . . . . However, the microbiology, sustainability, and dynamics might be very different, although the backflow and inoculation issues could have some parallels when comparing viruses to bacteria.", ". . However, the microbiology, sustainability, and dynamics might be very different, although the backflow and inoculation issues could have some parallels when comparing viruses to bacteria. As Enterobacteriaceae can either multiply or remain viable for long periods of time in biofilms coating the interior of P-traps and the connected plumbing, it may not be sustainable to target any intervention limited to a single isolated sink as a source of a particular pathogen. Data from different dispersal experiments suggest that although P-traps can act as the source or the reservoir of pathogens, the physical presence of the organism in the sink bowl or colonization of the strainer is necessary for the dispersal to occur. Colonization of strainers or drains reported in earlier studies .", "Data from different dispersal experiments suggest that although P-traps can act as the source or the reservoir of pathogens, the physical presence of the organism in the sink bowl or colonization of the strainer is necessary for the dispersal to occur. Colonization of strainers or drains reported in earlier studies . was perhaps a result of ascending biofilm growth from the P-trap to the strainer or introduction through contaminated fluids. Many of the studies used swab samples, which likely sampled the strainer rather than P-trap water . . Once the strainer was colonized, the water from the faucet resulted in GFP-expressing E. coli dispersion in the bowl and to the surrounding surfaces of up to 30 in.", ". Once the strainer was colonized, the water from the faucet resulted in GFP-expressing E. coli dispersion in the bowl and to the surrounding surfaces of up to 30 in. The range of dispersal . . Greater dispersal near the faucet may be attributed to the specific designs of the sink bowl and faucet in this study, which determine the contact angle of water impact. This is an important finding since many sinks in hospitals are similar in design, with faucet handles representing a high-touch surface for the sink users . . It can also be concluded from the dispersion experiments that secondary and successive dispersals would likely increase the degree and the scope of dispersion.", ". It can also be concluded from the dispersion experiments that secondary and successive dispersals would likely increase the degree and the scope of dispersion. There are several limitations to this work. First the use of similar sink bowls across these sinks only examines the dispersion pattern of this particular sink design. Similarly the sink-to-sink transmission may not be applicable to all wastewater plumbing systems as the fixtures on the pipe are very close together, unlike most layouts in health care settings. However, we speculate that transmission could occur on larger systems over greater time scales, especially if heavy nutrient and contamination loads were also included.", "Similarly the sink-to-sink transmission may not be applicable to all wastewater plumbing systems as the fixtures on the pipe are very close together, unlike most layouts in health care settings. However, we speculate that transmission could occur on larger systems over greater time scales, especially if heavy nutrient and contamination loads were also included. GFP-expressing E. coli is a laboratory surrogate, and the putative biofilms established in the short time frame of our experiments are unlikely to be as complex or stable as biofilms developed in a hospital wastewater system over many years. However, to address the monomicrobial dominance of the GFP-expressing E. coli added to the system, we kept the system open, and other environmental organisms were able to cocolonize in an attempt to mimic the hospital system. Another limitation was the need to add nutrients to the drain to ensure rapid and robust colonization. We are not clear how widespread the practice of disposing of dextrose-containing intravenous fluids or leftover beverages in the hand-wash sinks is; however, we have observed this practice, and anecdotally it appears to be relatively common in the United States.", "Another limitation was the need to add nutrients to the drain to ensure rapid and robust colonization. We are not clear how widespread the practice of disposing of dextrose-containing intravenous fluids or leftover beverages in the hand-wash sinks is; however, we have observed this practice, and anecdotally it appears to be relatively common in the United States. We also did not completely characterize the droplet sizes, nor do we demonstrate air sampling to understand if the dispersion is only droplet or if there are also aerosols that contain GFP-expressing E. coli. This would require additional testing and is planned as future work. In summary, this work for the first time better models the mechanisms of spread of multidrug-resistant pathogens arising from the sink drain and infecting patients. Droplet dispersion from the P-trap does not happen directly.", "In summary, this work for the first time better models the mechanisms of spread of multidrug-resistant pathogens arising from the sink drain and infecting patients. Droplet dispersion from the P-trap does not happen directly. Rather it is a multistage process: dispersal originates from the strainer and/or the bowl after growth of the biofilm up from the microbial reservoir of the P-trap. We also demonstrate sink-to-sink transmission via a common sanitary pipe. This work could have implications for patient safety, infection control, and interventions as well as the design of future hospital plumbing systems to eliminate this mode of transmission to vulnerable hospitalized patients. Sink gallery design.", "This work could have implications for patient safety, infection control, and interventions as well as the design of future hospital plumbing systems to eliminate this mode of transmission to vulnerable hospitalized patients. Sink gallery design. A dedicated sink gallery was set up to simulate hospital hand-washing sinks. The gallery was comprised of five sink modules assembled next to each other Fig. 4 . The five hand-wash sink stations were identical in bowl designs and dimensions and were modeled from the most common intensive care unit hand-washing sink type in the acute care hospital at the University of Virginia Medical Center. Partitions made of 24-in.-high Plexiglas sheet were installed between the sinks to prevent splatter and cross contamination.", "The five hand-wash sink stations were identical in bowl designs and dimensions and were modeled from the most common intensive care unit hand-washing sink type in the acute care hospital at the University of Virginia Medical Center. Partitions made of 24-in.-high Plexiglas sheet were installed between the sinks to prevent splatter and cross contamination. Each sink module was built with Corian integrated sink/countertops without an overflow and fitted with an 8-in. centerset 2-handle gooseneck faucet Elkay, Oak Brook, IL . The drain line Dearborn Brass-Oatey, Cleveland, OH . All of the fixtures were made of brass with chrome plating. Each of the sink P-traps was connected to a 3-in.", "All of the fixtures were made of brass with chrome plating. Each of the sink P-traps was connected to a 3-in. common cast-iron pipe sloping into a T-joint leading into the building sanitary line located behind sink 3 Fig. 4 . Inoculation, growth, and establishment of GFP-expressing E. coli in sink P-traps. For the GFP-expressing E. coli strain ATCC 25922GFP , the green fluorescent protein GFP gene is contained on a plasmid that also contains an ampicillin resistance gene. A single isolated colony of GFP-expressing E. coli grown from a Ϫ80°C stock was inoculated into 5 ml tryptic soy broth TSB Becton, Dickinson and Company, Sparks, MD containing 100 g/ml ampicillin ATCC medium 2855 .", "For the GFP-expressing E. coli strain ATCC 25922GFP , the green fluorescent protein GFP gene is contained on a plasmid that also contains an ampicillin resistance gene. A single isolated colony of GFP-expressing E. coli grown from a Ϫ80°C stock was inoculated into 5 ml tryptic soy broth TSB Becton, Dickinson and Company, Sparks, MD containing 100 g/ml ampicillin ATCC medium 2855 . The inoculum concentration and method varied for each experiment. For establishment of GFP-expressing E. coli in sink P-traps, new autoclaved P-traps were filled with 100 ml 0.1ϫ TSB and inoculated with ϳ10 3 CFU/ml GFPexpressing E. coli. Following inoculation, both ends of the P-traps were covered with perforated Parafilm Bemis, Inc., Oshkosh, WI and allowed to incubate at room temperature 22 Ϯ 2°C for 14 days to facilitate adherent bacterial growth. The medium in the P-trap was decanted and replaced with fresh 0.1ϫ TSB every 48 h. An aliquot of decanted medium and a swab sample from the inner surface of the P-trap were plated on tryptic soy agar Becton, Dickinson and Company, Sparks, MD plates containing 100 g/ml ampicillin TSA to monitor the growth of GFP-expressing E. coli in the P-traps.", "Following inoculation, both ends of the P-traps were covered with perforated Parafilm Bemis, Inc., Oshkosh, WI and allowed to incubate at room temperature 22 Ϯ 2°C for 14 days to facilitate adherent bacterial growth. The medium in the P-trap was decanted and replaced with fresh 0.1ϫ TSB every 48 h. An aliquot of decanted medium and a swab sample from the inner surface of the P-trap were plated on tryptic soy agar Becton, Dickinson and Company, Sparks, MD plates containing 100 g/ml ampicillin TSA to monitor the growth of GFP-expressing E. coli in the P-traps. TSA plates were incubated overnight at 37°C, and CFU fluorescing under UV light were enumerated. All preparatory culturing of GFP-expressing E. coli took place in a separate room from the sink gallery to avoid unintentional contamination. Installation of P-traps colonized with GFP-expressing E.coli. After the 14-day incubation, P-traps were fastened into the plumbing of the sinks Fig.", "Installation of P-traps colonized with GFP-expressing E.coli. After the 14-day incubation, P-traps were fastened into the plumbing of the sinks Fig. 5a . The remainder of the drain line was either autoclaved strainer, tailpipe, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI , maintaining at least 1 min of contact time. After the P-trap was installed, a daily regimen was followed in which 25 ml of TSB followed by 25 ml of 0.9% NaCl solution saline were added in the ratio 1:3 via the strainer Fig. 5b to mimic the potential nutrient exposure in the hospital.", "After the P-trap was installed, a daily regimen was followed in which 25 ml of TSB followed by 25 ml of 0.9% NaCl solution saline were added in the ratio 1:3 via the strainer Fig. 5b to mimic the potential nutrient exposure in the hospital. Sampling and enumeration of GFP-expressing E. coli. To monitor the growth of GFP-expressing E. coli in the plumbing, sampling ports were drilled along the length of the tailpiece between the P-trap and the strainer and the trap arm between the P-trap and the common line . These holes were fitted with size 00 silicone stoppers Cole-Parmer, Vernon Hills, IL Fig. 5a .", "These holes were fitted with size 00 silicone stoppers Cole-Parmer, Vernon Hills, IL Fig. 5a . Sterile cotton swabs Covidien, Mansfield, MA presoaked in saline were inserted through these sampling ports, and samples were collected by turning the swab in a circular motion on the inner surface ϳ20 cm 2 of the tailpipes. Sample swabs were pulse-vortexed in 3 ml saline, and serial dilutions were plated on TSA. The strainer, faucet aerator, and bowl surface were sampled with presoaked swabs and processed as described earlier. Sink-to-sink transmission of bacteria. To investigate sink-to-sink transmission of bacteria, a distal sink sink 5 Fig.", "Sink-to-sink transmission of bacteria. To investigate sink-to-sink transmission of bacteria, a distal sink sink 5 Fig. 4 was fitted with a P-trap inoculated with GFP-expressing E. coli. The effects of different inoculum concentrations of GFP-expressing E. coli-10 3 , 10 6 , and Ͼ10 10 CFU/ml colonized for 14 days -were investigated. Identification to the species level of fluorescent and nonfluorescent colonies identified from mixed pipe cultures was performed using a matrix-assisted laser desorption-ionization MALDI -time of flight MALDI-TOF mass spectrometer Vitek-MS; bioMérieux, Durham, NC . The wastewater paths of sinks 1 to 4 were either autoclaved strainer, tailpipe, P-traps, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI .", "Identification to the species level of fluorescent and nonfluorescent colonies identified from mixed pipe cultures was performed using a matrix-assisted laser desorption-ionization MALDI -time of flight MALDI-TOF mass spectrometer Vitek-MS; bioMérieux, Durham, NC . The wastewater paths of sinks 1 to 4 were either autoclaved strainer, tailpipe, P-traps, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI . Faucets on each of the five sinks were turned on simultaneously for 1 min, supplying water at a flow rate of 8 liters/min, once every 24 h for 7 days. No additional feed to any of the sinks was added during this period of 7 days. On days 0 and 7, P-traps on each of the five sinks were unfastened, and swab samples from the P-trap were collected and processed as described earlier. Dispersion measured using fluorescent microspheres.", "On days 0 and 7, P-traps on each of the five sinks were unfastened, and swab samples from the P-trap were collected and processed as described earlier. Dispersion measured using fluorescent microspheres. Fluoresbrite YO carboxylate microspheres Polysciences, Inc. which had a 1-m diameter and maximum excitation and emission of 529 nm and 546 nm, respectively, were used as a tracer in the preliminary experiments to understand droplet dispersion from the hand-wash sinks. To test whether microspheres could be dispersed from below the sink strainer, a 1-ml suspension of microspheres ϳ10 10 particles was injected through a strainer attached to a Hert 4.5-in. offset drain tailpiece typically used for wheelchair-accessible sinks American Standard, model 7723018.002 Fig. 5c .", "offset drain tailpiece typically used for wheelchair-accessible sinks American Standard, model 7723018.002 Fig. 5c . The vertical distance between the strainer and microsphere suspension injected into the tailpipe was ϳ4 in. Counter space around the sink bowl was thoroughly wiped with alcohol wipes Covidien Webcol 6818; Kendall , and polyester sheets precut to appropriate shapes were placed on the counter to cover the entire sink counter and labeled according to position Fig. 6a . The faucet was turned on for 5 min at a water flow rate of 1.8 to 3.0 liters/min.", "6a . The faucet was turned on for 5 min at a water flow rate of 1.8 to 3.0 liters/min. Polyester sheets were harvested and immediately analyzed using a ChemiDoc MP system Bio-Rad Laboratories, Inc. with an exposure time of 5 s. Fluorescent microspheres were enumerated from the digital micrographs using the Image Lab Software Bio-Rad Laboratories, Inc. . To test whether microspheres could be dispersed from the surface of the sink bowl, the sink bowl was evenly coated with a 20-ml microsphere suspension ϳ10 10 particles/ml using a disposable swab Sage Products, Inc., Cary, IL , and the dispersion experiment was repeated following the protocol described above. To ascertain there was no nonspecific background fluorescence in the sink and/or the water from the faucet, a control using the same protocol but without the fluorescent microspheres was performed before each experiment. Dispersion measured using GFP-expressing E.coli.", "To ascertain there was no nonspecific background fluorescence in the sink and/or the water from the faucet, a control using the same protocol but without the fluorescent microspheres was performed before each experiment. Dispersion measured using GFP-expressing E.coli. Dispersion using GFP-expressing E. coli was investigated in three experiments. To test whether live organisms in the P-trap could be dispersed by running water, ϳ10 10 CFU/ml GFP-expressing E. coli in saline was added to an autoclaved P-trap and fitted into the drain line that was preautoclaved strainer, tailpipe, and trap arms . Similarly, to test whether live organisms could be dispersed from the tailpieces of wheelchair-accessible sinks, a suspension of ϳ10 10 CFU/ml GFP-expressing E. coli was added with a syringe through the strainer into the Hert 4.5-ft offset drain tailpiece Fig. 5c .", "Similarly, to test whether live organisms could be dispersed from the tailpieces of wheelchair-accessible sinks, a suspension of ϳ10 10 CFU/ml GFP-expressing E. coli was added with a syringe through the strainer into the Hert 4.5-ft offset drain tailpiece Fig. 5c . Just as in the microsphere dispersion experiment, the vertical distance between the strainer and GFP-expressing E. coli suspension injected into the tailpipe was ϳ4 in. We next tested whether live organisms from the surface of the sink bowl could be dispersed by running water. The sink bowl surface was evenly coated with an approximately 20-ml suspension of 10 10 CFU/ml GFP-expressing E. coli. Finally, to mimic all of these conditions, a P-trap colonized with GFP-expressing E. coli for 14 days was installed, and a nutrient regimen Fig.", "The sink bowl surface was evenly coated with an approximately 20-ml suspension of 10 10 CFU/ml GFP-expressing E. coli. Finally, to mimic all of these conditions, a P-trap colonized with GFP-expressing E. coli for 14 days was installed, and a nutrient regimen Fig. 5b was followed for 14 days to intentionally promote the GFP-expressing E. coli colonization in the attached tailpipe and strainer. On day 15, the dispersion experiment was performed. Before each of the GFP-expressing E. coli dispersion experiments, the counter space was thoroughly disinfected with Caviwipes-1. TSA plates were then positioned on the sink counter surrounding the bowl and an extension platform Fig. 6b .", "TSA plates were then positioned on the sink counter surrounding the bowl and an extension platform Fig. 6b . Additional plates were attached to the sink bowl, faucets, Plexiglas partitions, and faucet handles using adhesive tape. TSA plates were also placed 3 m away from the sink as negative controls. The faucet was turned on for 5 min with a water flow rate of 1.8 to 3.0 liters/min. Lids of the TSA plates were removed only during faucet operation. Swab samples from the faucet aerators before and after operation were collected and plated on TSA.", "Lids of the TSA plates were removed only during faucet operation. Swab samples from the faucet aerators before and after operation were collected and plated on TSA. Prior to each dispersion experiment, 50 ml water from the faucet was also collected, and aliquots were plated to assess for the presence of GFP-expressing E. coli in source water and ensure cross contamination of GFP-expressing E. coli had not occurred. A control dispersion experiment was also performed using the same protocol prior to GFP-expressing E. coli inoculation in each case. Dispersion per defined area CFU per square centimeter was deduced by dividing the CFU counts in the TSA plate by the surface area of the TSA plate." ]

[ "There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein GFP -expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas <30 in.", "However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas <30 in. during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient.", "We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient. IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks.", "Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients. Text: D espite early reports . . . . .", "Text: D espite early reports . . . . . , the premise that hand-wash sink traps can act as reservoirs of bacteria that cause nosocomial infections has been frequently overlooked. There has recently been an alarming increase in sink-related outbreaks worldwide, with many reports establishing an observational link . . . . . . . . . A sink often operates as an open conduit to wastewater in a patient care area that is often in the same room as the patient. Health care establishments often invest in desperate interventions to deal with nosocomial outbreaks. The preferred method for addressing most of the environmentrelated transmission is to employ enhanced cleaning using chemical and physical agents . .", "The preferred method for addressing most of the environmentrelated transmission is to employ enhanced cleaning using chemical and physical agents . . Unfortunately, routine approaches are inefficient in completely eliminating drug-resistant Gammaproteobacteria in an inaccessible microbiologically active area such as a sink trap 6, . . . . . . The wet, humid, and relatively protected environment in a sink trap favors the formation of rich stable microbial communities . . These communities will be exposed to liquids and waste that are discarded in a sink and may include antimicrobials, discarded beverages, soap, presumably pathogenic bacteria from health care workers' hands, and other items.", ". These communities will be exposed to liquids and waste that are discarded in a sink and may include antimicrobials, discarded beverages, soap, presumably pathogenic bacteria from health care workers' hands, and other items. In short, sink traps could serve as a breeding ground for opportunistic and highly antimicrobial-resistant bacteria that cannot be easily cleaned or removed . . . . . . . There are many reports of a genetic association between pathogens found in sink traps and those found in patients . . However, surprisingly little work has been done to understand the microscale transmission dynamics.", ". However, surprisingly little work has been done to understand the microscale transmission dynamics. It was previously demonstrated using a suspension of fluorescent particles Glo Germ; Glo Germ Co., Moab, UT that material injected into the P-trap gets dispersed around a hand-washing sink . . This result however has not been replicated hitherto in the follow-up studies. Dispersion has never been investigated with living organisms.", ". This result however has not been replicated hitherto in the follow-up studies. Dispersion has never been investigated with living organisms. Ultimately, many details remain unaddressed surrounding the spread of Enterobacteriaceae in sink-trap wastewater systems: i can organisms grow retrograde from the P-trap water to the sink strainer, ii can organisms spread from one sink to another along the internal surfaces of pipes with shared drainage systems, and iii which portion of a colonized drain pipe results in dispersion into the sink bowl during a hand-washing event? We aim to better understand the dispersion dynamics of Gammaproteobacteria living in the wastewater of a sink strainer and P-trap into an area where patients and health care workers could be exposed. To study this dynamic, we used a surrogate organism that could be easily tracked while remaining in the Enterobacteriaceae family, where some of the most concerning threats in antimicrobial resistance are developing .", "We aim to better understand the dispersion dynamics of Gammaproteobacteria living in the wastewater of a sink strainer and P-trap into an area where patients and health care workers could be exposed. To study this dynamic, we used a surrogate organism that could be easily tracked while remaining in the Enterobacteriaceae family, where some of the most concerning threats in antimicrobial resistance are developing . . Growth and colonization of GFP-expressing E. coli in the P-trap. In the first 14 days following the installation of the P-trap with established green fluorescent protein GFP -expressing Escherichia coli and just water running from the faucet, GFPexpressing E. coli was not detected in the tailpipe beyond 1.5 in. above the liquid level in the P-trap.", "In the first 14 days following the installation of the P-trap with established green fluorescent protein GFP -expressing Escherichia coli and just water running from the faucet, GFPexpressing E. coli was not detected in the tailpipe beyond 1.5 in. above the liquid level in the P-trap. GFP-expressing E. coli, however, was found to be viable in the P-trap without any nutrients added. A nutrient regimen was then instituted to understand the influence of nutrients on mobility and upward growth. The addition of tryptic soy broth TSB promoted GFP-expressing E. coli growth as early as day 1, with growth observed in the tailpipe 2 in. above the liquid surface in the P-trap Table 1 .", "The addition of tryptic soy broth TSB promoted GFP-expressing E. coli growth as early as day 1, with growth observed in the tailpipe 2 in. above the liquid surface in the P-trap Table 1 . On day 7, the strainer ϳ8 in. above the liquid in the P-trap was found to be colonized with GFP-expressing E. coli. This translates to an average growth rate of 1 in./day along the length of the tailpipe with the addition of nutrients and without faucet operation. GFP-expressing E. coli was not detected in the faucet water. Sink-to-sink transmission of bacteria.", "GFP-expressing E. coli was not detected in the faucet water. Sink-to-sink transmission of bacteria. In these experiments, a flanking sink sink 5 was the only P-trap inoculated with GFP-expressing E. coli and therefore was the sole source for transmission to the connected sinks. Starting with a lower inoculum concentration 10 3 CFU/ml in sink 5, on day 7, GFP-expressing E. coli was detected in the sink 2 and sink 3 P-traps Fig. 1a . With inoculum concentrations of 10 6 CFU/ml and Ͼ10 10 CFU/ml in sink 5, all of the sink P-traps in the sink gallery with the exception of sink 1 were found to be colonized with GFP-expressing E. coli after 7 days Fig.", "1a . With inoculum concentrations of 10 6 CFU/ml and Ͼ10 10 CFU/ml in sink 5, all of the sink P-traps in the sink gallery with the exception of sink 1 were found to be colonized with GFP-expressing E. coli after 7 days Fig. 1b and c . Faucet water and aerators tested negative for GFP-expressing E. coli. Irrespective of the starting inoculum concentration, on day 7 the highest level of colonization was recorded in the sink 3 P-trap. After day 7, when the nutrient regimen described previously was followed for an additional 7 days in each of the sinks in the sink gallery with an inoculum concentration of Ͼ10 10 CFU/ml, GFP-expressing E. coli was detected in the strainers of sinks 2 and 3 on day 14.", "Irrespective of the starting inoculum concentration, on day 7 the highest level of colonization was recorded in the sink 3 P-trap. After day 7, when the nutrient regimen described previously was followed for an additional 7 days in each of the sinks in the sink gallery with an inoculum concentration of Ͼ10 10 CFU/ml, GFP-expressing E. coli was detected in the strainers of sinks 2 and 3 on day 14. This finding validated the upward growth and growth rate in the tailpipe when nutrients were added. Nonfluorescent colonies were occasionally observed in the P-trap water samples collected from the sinks, which were subsequently identified to be Pseudomonas sp. or Stenotrophomonas maltophilia, and fluorescent colonies were confirmed to be E. coli. Dispersion of microspheres from sinks.", "or Stenotrophomonas maltophilia, and fluorescent colonies were confirmed to be E. coli. Dispersion of microspheres from sinks. In the first dispersion experiment, when fluorescent microspheres were inoculated into the offset drain tailpiece only 4 in. below the strainer, no microspheres were detected on the polyester sheets placed on the counter space. However, when the sink bowl was coated with the microspheres, polyester sheets overlaid on the counter space captured the dispersed microspheres caused by the faucet operation. Dispersion was observed on almost all zones of the sink counter space Fig. 2 .", "Dispersion was observed on almost all zones of the sink counter space Fig. 2 . Relatively higher levels of dispersion were observed along the major and minor axes of the elliptical sink bowl zones 2, 5, 6, 9, 11, and 12 . Anterior corners of the sink counter space zones 4 and 7 , which were most distant from the impact of water in the sink bowl, received the lowest dispersion. Dispersion of GFP-expressing E. coli from sinks. Initially the P-trap alone was inoculated with GFP-expressing E. coli and carefully installed, keeping the tailpipe and strainer free of GFP-expressing E. coli before operating the faucets.", "Dispersion of GFP-expressing E. coli from sinks. Initially the P-trap alone was inoculated with GFP-expressing E. coli and carefully installed, keeping the tailpipe and strainer free of GFP-expressing E. coli before operating the faucets. No fluorescent CFU were observed on the plates placed on the counter or attached to the bowl surface after faucet operation. Similarly, no fluorescent CFU were detected when GFPexpressing E. coli was inoculated into the offset drain tailpiece only 4 in. below the strainer. Interestingly, when there was conspicuous water backup over the strainer as a result of a higher water flow rate from the faucet than the drainage rate from the P-trap, dispersal was detected on the plates attached to the bowl surface.", "below the strainer. Interestingly, when there was conspicuous water backup over the strainer as a result of a higher water flow rate from the faucet than the drainage rate from the P-trap, dispersal was detected on the plates attached to the bowl surface. The dispersion pattern recorded when the sink bowl was coated with GFPexpressing E. coli was comparable to the pattern recorded when the sink bowl was coated with fluorescent microspheres Fig. 2 . Dispersion was significantly higher along the axes zones 6, 9, 11, and 12 and lower at the corners of the sink counter space zones 4, 7, and 10 . In contrast, dispersion of GFP-expressing E. coli caused by the faucet operation was much more extensive when the strainer was allowed to be colonized with GFPexpressing E. coli prior to the dispersion experiment.", "Dispersion was significantly higher along the axes zones 6, 9, 11, and 12 and lower at the corners of the sink counter space zones 4, 7, and 10 . In contrast, dispersion of GFP-expressing E. coli caused by the faucet operation was much more extensive when the strainer was allowed to be colonized with GFPexpressing E. coli prior to the dispersion experiment. In addition to the sink counter space, we measured dispersion to the sink bowl, faucet, faucet handles, splatter shields, and the extended counter surface. Dispersion of GFP-expressing E. coli was highest on the plates attached to the sink bowl Fig. 3b . Further, dispersion was greater along the Ϫ\" and \"ϩ\" denote the absence and presence of GFP-expressing E. coli, respectively.", "3b . Further, dispersion was greater along the Ϫ\" and \"ϩ\" denote the absence and presence of GFP-expressing E. coli, respectively. minor axis of the sink bowl Fig. 3b , zones B3, B4, and B10 than along the major axis of the sink bowl, associated with a shorter distance from the strike point of the faucet water to the bowl along this axis. The next highest CFU count from the dispersal was recorded on the counter area near the faucets Fig. 3a , zones 12 and 11 . A similar pattern of higher dispersion near the faucets and lower dispersion at the corners of the counter space Fig.", "3a , zones 12 and 11 . A similar pattern of higher dispersion near the faucets and lower dispersion at the corners of the counter space Fig. 3a , zones 4, 7, and 10 was also observed using microspheres. Dispersion was also recorded in other zones of the counter space, on the Plexiglas splatter shields, faucets, faucet handles, and extended surface Fig. 3c . There were no GFP-expressing E. coli CFU recorded on plates placed beyond 30 in. from the strainer, demarcating the range of dispersion under these experimental conditions. Table 2 gives a summary of the total distribution loads recorded using fluorescent microspheres and GFP-expressing E. coli across each experiment.", "from the strainer, demarcating the range of dispersion under these experimental conditions. Table 2 gives a summary of the total distribution loads recorded using fluorescent microspheres and GFP-expressing E. coli across each experiment. The loads of dispersion on the sink counter were comparable when the sink bowl was coated with microspheres or GFP-expressing E. coli before the faucet operation. Although the dispersion load on the sink counter was lower when the sink strainer was colonized, it is interesting to note that the sink bowl received the highest dispersion. To mimic dispersion in a hospital setting, we first investigated whether GFPexpressing E. coli would establish consistent colonization in a sink trap as many other Gammaproteobacteria implicated in nosocomial outbreaks have done . .", "To mimic dispersion in a hospital setting, we first investigated whether GFPexpressing E. coli would establish consistent colonization in a sink trap as many other Gammaproteobacteria implicated in nosocomial outbreaks have done . . Many recent reports demonstrate that P-traps become colonized with highly consequential Gammaproteobacteria, which then results in nosocomial transmission . . The retained water in a sink P-trap is present to provide a water barrier to prevent off-gassing of sewer smell, but it may inadvertently provide favorable conditions for pathogenic and opportunistic antibiotic-resistant microorganisms to survive and develop resilient biofilms . . However, the mechanism of dispersal of the bacteria in the P-trap to patients or the surrounding health care area had not been fully elucidated.", ". However, the mechanism of dispersal of the bacteria in the P-trap to patients or the surrounding health care area had not been fully elucidated. We began with the hypothesis that the bacteria originate from the P-trap via droplet creation when the water from the faucet hits the P-trap water, thus contaminating the sink bowl and the surrounding area. The finding supporting this theory had been previously reported using Glo Germ particles . . However, in the present study with careful attention to avoid strainer and tail piece contamination, the dispersal directly from the sink P-trap with either microspheres or GFP-expressing E. coli could not be reproduced as previously reported . .", "However, in the present study with careful attention to avoid strainer and tail piece contamination, the dispersal directly from the sink P-trap with either microspheres or GFP-expressing E. coli could not be reproduced as previously reported . . Rather this work demonstrates a different, more staged mode of transmission from a P-trap reservoir to the sink and surrounding environment. GFP-expressing E. coli in the P-trap alone was sustained for 14 days but did not grow or mobilize up the tailpipe to the strainer with just intermittent water exposure. However, when nutrients were subsequently added to the system, the organisms rapidly grew up the tailpipe to the strainer at approximately an inch per day. In a real-world setting, motility of bacteria inside the tailpipe is restricted to relatively sporadic and brief wetting events in which swimming is an opportunity to colonize new surfaces.", "However, when nutrients were subsequently added to the system, the organisms rapidly grew up the tailpipe to the strainer at approximately an inch per day. In a real-world setting, motility of bacteria inside the tailpipe is restricted to relatively sporadic and brief wetting events in which swimming is an opportunity to colonize new surfaces. It is assumed that once established, the biofilm promotes the upward growth of GFP-expressing E. coli in the tailpipe at an accelerated rate. The nutrient regimen that promoted colonization in our model reflects our and others' observations of items commonly disposed of in hospital sinks intravenous fluids, feeding supplements, and leftover beverages . . Transmission of bacteria between sinks via a common pipe was a key finding in this study as this highlights the concept that premise plumbing may be a more continuous system with shared microbiology than a single isolated sink.", ". Transmission of bacteria between sinks via a common pipe was a key finding in this study as this highlights the concept that premise plumbing may be a more continuous system with shared microbiology than a single isolated sink. The sink gallery used in this study provided a unique in situ advantage to investigate sink-to-sink transmission of bacteria through common drains. The two possible mechanisms for P-trap strainers becoming colonized are seeding of organisms from above and retrograde spread of organisms along common pipes in a hospital wastewater infrastructure. Here we demonstrate that it is possible for GFP-expressing E. coli to contaminate adjacent P-traps with just time and water given a standard U.S. code piping rise of 0.25 ft/ft. Sink-to-sink or retrograde transmission may explain the recurrence of pathogen colonization following intervention strategies like disinfection or replacement of plumbing .", "Here we demonstrate that it is possible for GFP-expressing E. coli to contaminate adjacent P-traps with just time and water given a standard U.S. code piping rise of 0.25 ft/ft. Sink-to-sink or retrograde transmission may explain the recurrence of pathogen colonization following intervention strategies like disinfection or replacement of plumbing . . Sink 3 was lowest on the slope in the drain line see Fig. 4 with arguably the most opportunity for reflux and retrograde wetting. Sink 1, on the other hand, was farthest away from the source sink 5 , and its P-trap had the greatest incline in the drain line connecting the sinks, which could perhaps contribute to the reasons there was no GFP-expressing E. coli colonization detected in it after 7 days.", "4 with arguably the most opportunity for reflux and retrograde wetting. Sink 1, on the other hand, was farthest away from the source sink 5 , and its P-trap had the greatest incline in the drain line connecting the sinks, which could perhaps contribute to the reasons there was no GFP-expressing E. coli colonization detected in it after 7 days. There has been more investigation about microbiologic dynamics of infectious viral particles such as those of severe acute respiratory syndrome SARS and Ebola viruses through premise plumbing systems . . . . However, the microbiology, sustainability, and dynamics might be very different, although the backflow and inoculation issues could have some parallels when comparing viruses to bacteria.", ". . However, the microbiology, sustainability, and dynamics might be very different, although the backflow and inoculation issues could have some parallels when comparing viruses to bacteria. As Enterobacteriaceae can either multiply or remain viable for long periods of time in biofilms coating the interior of P-traps and the connected plumbing, it may not be sustainable to target any intervention limited to a single isolated sink as a source of a particular pathogen. Data from different dispersal experiments suggest that although P-traps can act as the source or the reservoir of pathogens, the physical presence of the organism in the sink bowl or colonization of the strainer is necessary for the dispersal to occur. Colonization of strainers or drains reported in earlier studies .", "Data from different dispersal experiments suggest that although P-traps can act as the source or the reservoir of pathogens, the physical presence of the organism in the sink bowl or colonization of the strainer is necessary for the dispersal to occur. Colonization of strainers or drains reported in earlier studies . was perhaps a result of ascending biofilm growth from the P-trap to the strainer or introduction through contaminated fluids. Many of the studies used swab samples, which likely sampled the strainer rather than P-trap water . . Once the strainer was colonized, the water from the faucet resulted in GFP-expressing E. coli dispersion in the bowl and to the surrounding surfaces of up to 30 in.", ". Once the strainer was colonized, the water from the faucet resulted in GFP-expressing E. coli dispersion in the bowl and to the surrounding surfaces of up to 30 in. The range of dispersal . . Greater dispersal near the faucet may be attributed to the specific designs of the sink bowl and faucet in this study, which determine the contact angle of water impact. This is an important finding since many sinks in hospitals are similar in design, with faucet handles representing a high-touch surface for the sink users . . It can also be concluded from the dispersion experiments that secondary and successive dispersals would likely increase the degree and the scope of dispersion.", ". It can also be concluded from the dispersion experiments that secondary and successive dispersals would likely increase the degree and the scope of dispersion. There are several limitations to this work. First the use of similar sink bowls across these sinks only examines the dispersion pattern of this particular sink design. Similarly the sink-to-sink transmission may not be applicable to all wastewater plumbing systems as the fixtures on the pipe are very close together, unlike most layouts in health care settings. However, we speculate that transmission could occur on larger systems over greater time scales, especially if heavy nutrient and contamination loads were also included.", "Similarly the sink-to-sink transmission may not be applicable to all wastewater plumbing systems as the fixtures on the pipe are very close together, unlike most layouts in health care settings. However, we speculate that transmission could occur on larger systems over greater time scales, especially if heavy nutrient and contamination loads were also included. GFP-expressing E. coli is a laboratory surrogate, and the putative biofilms established in the short time frame of our experiments are unlikely to be as complex or stable as biofilms developed in a hospital wastewater system over many years. However, to address the monomicrobial dominance of the GFP-expressing E. coli added to the system, we kept the system open, and other environmental organisms were able to cocolonize in an attempt to mimic the hospital system. Another limitation was the need to add nutrients to the drain to ensure rapid and robust colonization. We are not clear how widespread the practice of disposing of dextrose-containing intravenous fluids or leftover beverages in the hand-wash sinks is; however, we have observed this practice, and anecdotally it appears to be relatively common in the United States.", "Another limitation was the need to add nutrients to the drain to ensure rapid and robust colonization. We are not clear how widespread the practice of disposing of dextrose-containing intravenous fluids or leftover beverages in the hand-wash sinks is; however, we have observed this practice, and anecdotally it appears to be relatively common in the United States. We also did not completely characterize the droplet sizes, nor do we demonstrate air sampling to understand if the dispersion is only droplet or if there are also aerosols that contain GFP-expressing E. coli. This would require additional testing and is planned as future work. In summary, this work for the first time better models the mechanisms of spread of multidrug-resistant pathogens arising from the sink drain and infecting patients. Droplet dispersion from the P-trap does not happen directly.", "In summary, this work for the first time better models the mechanisms of spread of multidrug-resistant pathogens arising from the sink drain and infecting patients. Droplet dispersion from the P-trap does not happen directly. Rather it is a multistage process: dispersal originates from the strainer and/or the bowl after growth of the biofilm up from the microbial reservoir of the P-trap. We also demonstrate sink-to-sink transmission via a common sanitary pipe. This work could have implications for patient safety, infection control, and interventions as well as the design of future hospital plumbing systems to eliminate this mode of transmission to vulnerable hospitalized patients. Sink gallery design.", "This work could have implications for patient safety, infection control, and interventions as well as the design of future hospital plumbing systems to eliminate this mode of transmission to vulnerable hospitalized patients. Sink gallery design. A dedicated sink gallery was set up to simulate hospital hand-washing sinks. The gallery was comprised of five sink modules assembled next to each other Fig. 4 . The five hand-wash sink stations were identical in bowl designs and dimensions and were modeled from the most common intensive care unit hand-washing sink type in the acute care hospital at the University of Virginia Medical Center. Partitions made of 24-in.-high Plexiglas sheet were installed between the sinks to prevent splatter and cross contamination.", "The five hand-wash sink stations were identical in bowl designs and dimensions and were modeled from the most common intensive care unit hand-washing sink type in the acute care hospital at the University of Virginia Medical Center. Partitions made of 24-in.-high Plexiglas sheet were installed between the sinks to prevent splatter and cross contamination. Each sink module was built with Corian integrated sink/countertops without an overflow and fitted with an 8-in. centerset 2-handle gooseneck faucet Elkay, Oak Brook, IL . The drain line Dearborn Brass-Oatey, Cleveland, OH . All of the fixtures were made of brass with chrome plating. Each of the sink P-traps was connected to a 3-in.", "All of the fixtures were made of brass with chrome plating. Each of the sink P-traps was connected to a 3-in. common cast-iron pipe sloping into a T-joint leading into the building sanitary line located behind sink 3 Fig. 4 . Inoculation, growth, and establishment of GFP-expressing E. coli in sink P-traps. For the GFP-expressing E. coli strain ATCC 25922GFP , the green fluorescent protein GFP gene is contained on a plasmid that also contains an ampicillin resistance gene. A single isolated colony of GFP-expressing E. coli grown from a Ϫ80°C stock was inoculated into 5 ml tryptic soy broth TSB Becton, Dickinson and Company, Sparks, MD containing 100 g/ml ampicillin ATCC medium 2855 .", "For the GFP-expressing E. coli strain ATCC 25922GFP , the green fluorescent protein GFP gene is contained on a plasmid that also contains an ampicillin resistance gene. A single isolated colony of GFP-expressing E. coli grown from a Ϫ80°C stock was inoculated into 5 ml tryptic soy broth TSB Becton, Dickinson and Company, Sparks, MD containing 100 g/ml ampicillin ATCC medium 2855 . The inoculum concentration and method varied for each experiment. For establishment of GFP-expressing E. coli in sink P-traps, new autoclaved P-traps were filled with 100 ml 0.1ϫ TSB and inoculated with ϳ10 3 CFU/ml GFPexpressing E. coli. Following inoculation, both ends of the P-traps were covered with perforated Parafilm Bemis, Inc., Oshkosh, WI and allowed to incubate at room temperature 22 Ϯ 2°C for 14 days to facilitate adherent bacterial growth. The medium in the P-trap was decanted and replaced with fresh 0.1ϫ TSB every 48 h. An aliquot of decanted medium and a swab sample from the inner surface of the P-trap were plated on tryptic soy agar Becton, Dickinson and Company, Sparks, MD plates containing 100 g/ml ampicillin TSA to monitor the growth of GFP-expressing E. coli in the P-traps.", "Following inoculation, both ends of the P-traps were covered with perforated Parafilm Bemis, Inc., Oshkosh, WI and allowed to incubate at room temperature 22 Ϯ 2°C for 14 days to facilitate adherent bacterial growth. The medium in the P-trap was decanted and replaced with fresh 0.1ϫ TSB every 48 h. An aliquot of decanted medium and a swab sample from the inner surface of the P-trap were plated on tryptic soy agar Becton, Dickinson and Company, Sparks, MD plates containing 100 g/ml ampicillin TSA to monitor the growth of GFP-expressing E. coli in the P-traps. TSA plates were incubated overnight at 37°C, and CFU fluorescing under UV light were enumerated. All preparatory culturing of GFP-expressing E. coli took place in a separate room from the sink gallery to avoid unintentional contamination. Installation of P-traps colonized with GFP-expressing E.coli. After the 14-day incubation, P-traps were fastened into the plumbing of the sinks Fig.", "Installation of P-traps colonized with GFP-expressing E.coli. After the 14-day incubation, P-traps were fastened into the plumbing of the sinks Fig. 5a . The remainder of the drain line was either autoclaved strainer, tailpipe, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI , maintaining at least 1 min of contact time. After the P-trap was installed, a daily regimen was followed in which 25 ml of TSB followed by 25 ml of 0.9% NaCl solution saline were added in the ratio 1:3 via the strainer Fig. 5b to mimic the potential nutrient exposure in the hospital.", "After the P-trap was installed, a daily regimen was followed in which 25 ml of TSB followed by 25 ml of 0.9% NaCl solution saline were added in the ratio 1:3 via the strainer Fig. 5b to mimic the potential nutrient exposure in the hospital. Sampling and enumeration of GFP-expressing E. coli. To monitor the growth of GFP-expressing E. coli in the plumbing, sampling ports were drilled along the length of the tailpiece between the P-trap and the strainer and the trap arm between the P-trap and the common line . These holes were fitted with size 00 silicone stoppers Cole-Parmer, Vernon Hills, IL Fig. 5a .", "These holes were fitted with size 00 silicone stoppers Cole-Parmer, Vernon Hills, IL Fig. 5a . Sterile cotton swabs Covidien, Mansfield, MA presoaked in saline were inserted through these sampling ports, and samples were collected by turning the swab in a circular motion on the inner surface ϳ20 cm 2 of the tailpipes. Sample swabs were pulse-vortexed in 3 ml saline, and serial dilutions were plated on TSA. The strainer, faucet aerator, and bowl surface were sampled with presoaked swabs and processed as described earlier. Sink-to-sink transmission of bacteria. To investigate sink-to-sink transmission of bacteria, a distal sink sink 5 Fig.", "Sink-to-sink transmission of bacteria. To investigate sink-to-sink transmission of bacteria, a distal sink sink 5 Fig. 4 was fitted with a P-trap inoculated with GFP-expressing E. coli. The effects of different inoculum concentrations of GFP-expressing E. coli-10 3 , 10 6 , and Ͼ10 10 CFU/ml colonized for 14 days -were investigated. Identification to the species level of fluorescent and nonfluorescent colonies identified from mixed pipe cultures was performed using a matrix-assisted laser desorption-ionization MALDI -time of flight MALDI-TOF mass spectrometer Vitek-MS; bioMérieux, Durham, NC . The wastewater paths of sinks 1 to 4 were either autoclaved strainer, tailpipe, P-traps, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI .", "Identification to the species level of fluorescent and nonfluorescent colonies identified from mixed pipe cultures was performed using a matrix-assisted laser desorption-ionization MALDI -time of flight MALDI-TOF mass spectrometer Vitek-MS; bioMérieux, Durham, NC . The wastewater paths of sinks 1 to 4 were either autoclaved strainer, tailpipe, P-traps, and trap arms prior to installation or surface disinfected sink bowl, countertop, and faucets with Caviwipes-1 Meterx Research, Romulus, MI . Faucets on each of the five sinks were turned on simultaneously for 1 min, supplying water at a flow rate of 8 liters/min, once every 24 h for 7 days. No additional feed to any of the sinks was added during this period of 7 days. On days 0 and 7, P-traps on each of the five sinks were unfastened, and swab samples from the P-trap were collected and processed as described earlier. Dispersion measured using fluorescent microspheres.", "On days 0 and 7, P-traps on each of the five sinks were unfastened, and swab samples from the P-trap were collected and processed as described earlier. Dispersion measured using fluorescent microspheres. Fluoresbrite YO carboxylate microspheres Polysciences, Inc. which had a 1-m diameter and maximum excitation and emission of 529 nm and 546 nm, respectively, were used as a tracer in the preliminary experiments to understand droplet dispersion from the hand-wash sinks. To test whether microspheres could be dispersed from below the sink strainer, a 1-ml suspension of microspheres ϳ10 10 particles was injected through a strainer attached to a Hert 4.5-in. offset drain tailpiece typically used for wheelchair-accessible sinks American Standard, model 7723018.002 Fig. 5c .", "offset drain tailpiece typically used for wheelchair-accessible sinks American Standard, model 7723018.002 Fig. 5c . The vertical distance between the strainer and microsphere suspension injected into the tailpipe was ϳ4 in. Counter space around the sink bowl was thoroughly wiped with alcohol wipes Covidien Webcol 6818; Kendall , and polyester sheets precut to appropriate shapes were placed on the counter to cover the entire sink counter and labeled according to position Fig. 6a . The faucet was turned on for 5 min at a water flow rate of 1.8 to 3.0 liters/min.", "6a . The faucet was turned on for 5 min at a water flow rate of 1.8 to 3.0 liters/min. Polyester sheets were harvested and immediately analyzed using a ChemiDoc MP system Bio-Rad Laboratories, Inc. with an exposure time of 5 s. Fluorescent microspheres were enumerated from the digital micrographs using the Image Lab Software Bio-Rad Laboratories, Inc. . To test whether microspheres could be dispersed from the surface of the sink bowl, the sink bowl was evenly coated with a 20-ml microsphere suspension ϳ10 10 particles/ml using a disposable swab Sage Products, Inc., Cary, IL , and the dispersion experiment was repeated following the protocol described above. To ascertain there was no nonspecific background fluorescence in the sink and/or the water from the faucet, a control using the same protocol but without the fluorescent microspheres was performed before each experiment. Dispersion measured using GFP-expressing E.coli.", "To ascertain there was no nonspecific background fluorescence in the sink and/or the water from the faucet, a control using the same protocol but without the fluorescent microspheres was performed before each experiment. Dispersion measured using GFP-expressing E.coli. Dispersion using GFP-expressing E. coli was investigated in three experiments. To test whether live organisms in the P-trap could be dispersed by running water, ϳ10 10 CFU/ml GFP-expressing E. coli in saline was added to an autoclaved P-trap and fitted into the drain line that was preautoclaved strainer, tailpipe, and trap arms . Similarly, to test whether live organisms could be dispersed from the tailpieces of wheelchair-accessible sinks, a suspension of ϳ10 10 CFU/ml GFP-expressing E. coli was added with a syringe through the strainer into the Hert 4.5-ft offset drain tailpiece Fig. 5c .", "Similarly, to test whether live organisms could be dispersed from the tailpieces of wheelchair-accessible sinks, a suspension of ϳ10 10 CFU/ml GFP-expressing E. coli was added with a syringe through the strainer into the Hert 4.5-ft offset drain tailpiece Fig. 5c . Just as in the microsphere dispersion experiment, the vertical distance between the strainer and GFP-expressing E. coli suspension injected into the tailpipe was ϳ4 in. We next tested whether live organisms from the surface of the sink bowl could be dispersed by running water. The sink bowl surface was evenly coated with an approximately 20-ml suspension of 10 10 CFU/ml GFP-expressing E. coli. Finally, to mimic all of these conditions, a P-trap colonized with GFP-expressing E. coli for 14 days was installed, and a nutrient regimen Fig.", "The sink bowl surface was evenly coated with an approximately 20-ml suspension of 10 10 CFU/ml GFP-expressing E. coli. Finally, to mimic all of these conditions, a P-trap colonized with GFP-expressing E. coli for 14 days was installed, and a nutrient regimen Fig. 5b was followed for 14 days to intentionally promote the GFP-expressing E. coli colonization in the attached tailpipe and strainer. On day 15, the dispersion experiment was performed. Before each of the GFP-expressing E. coli dispersion experiments, the counter space was thoroughly disinfected with Caviwipes-1. TSA plates were then positioned on the sink counter surrounding the bowl and an extension platform Fig. 6b .", "TSA plates were then positioned on the sink counter surrounding the bowl and an extension platform Fig. 6b . Additional plates were attached to the sink bowl, faucets, Plexiglas partitions, and faucet handles using adhesive tape. TSA plates were also placed 3 m away from the sink as negative controls. The faucet was turned on for 5 min with a water flow rate of 1.8 to 3.0 liters/min. Lids of the TSA plates were removed only during faucet operation. Swab samples from the faucet aerators before and after operation were collected and plated on TSA.", "Lids of the TSA plates were removed only during faucet operation. Swab samples from the faucet aerators before and after operation were collected and plated on TSA. Prior to each dispersion experiment, 50 ml water from the faucet was also collected, and aliquots were plated to assess for the presence of GFP-expressing E. coli in source water and ensure cross contamination of GFP-expressing E. coli had not occurred. A control dispersion experiment was also performed using the same protocol prior to GFP-expressing E. coli inoculation in each case. Dispersion per defined area CFU per square centimeter was deduced by dividing the CFU counts in the TSA plate by the surface area of the TSA plate." ]

[ "Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.", "These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .", "Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .", "Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .", "Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.", "antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .", ". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .", ". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.", "Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.", "Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.", "The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.", "In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .", "Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .", "In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .", "Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.", ". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.", "Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .", "Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .", "Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.", ". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.", "Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.", ". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .", "The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.", "Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .", ". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .", "Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.", "choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .", "Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .", "Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.", ". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .", "Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.", "Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .", "Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .", "On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .", ". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript." ]

[ "Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.", "These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .", "Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .", "Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .", "Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.", "antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .", ". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .", ". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.", "Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.", "Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.", "The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.", "In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .", "Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .", "In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .", "Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.", ". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.", "Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .", "Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .", "Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.", ". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.", "Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.", ". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .", "The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.", "Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .", ". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .", "Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.", "choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .", "Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .", "Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.", ". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .", "Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.", "Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .", "Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .", "On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .", ". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript." ]

[ "Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.", "These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .", "Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .", "Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .", "Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.", "antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .", ". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .", ". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.", "Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.", "Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.", "The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.", "In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .", "Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .", "In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .", "Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.", ". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.", "Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .", "Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .", "Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.", ". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.", "Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.", ". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .", "The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.", "Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .", ". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .", "Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.", "choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .", "Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .", "Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.", ". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .", "Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.", "Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .", "Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .", "On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .", ". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript." ]

[ "Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.", "These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .", "Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .", "Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .", "Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.", "antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .", ". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .", ". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.", "Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.", "Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.", "The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.", "In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .", "Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .", "In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .", "Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.", ". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.", "Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .", "Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .", "Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.", ". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.", "Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.", ". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .", "The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.", "Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .", ". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .", "Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.", "choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .", "Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .", "Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.", ". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .", "Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.", "Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .", "Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .", "On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .", ". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript." ]

[ "Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.", "These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .", "Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .", "Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .", "Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.", "antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .", ". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .", ". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.", "Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.", "Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.", "The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.", "In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .", "Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .", "In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .", "Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.", ". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.", "Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .", "Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .", "Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.", ". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.", "Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.", ". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .", "The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.", "Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .", ". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .", "Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.", "choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .", "Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .", "Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.", ". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .", "Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.", "Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .", "Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .", "On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .", ". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript." ]

[ "Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.", "These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .", "Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .", "Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .", "Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.", "antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .", ". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .", ". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.", "Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.", "Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.", "The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.", "In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .", "Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .", "In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .", "Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.", ". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.", "Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .", "Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .", "Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.", ". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.", "Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.", ". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .", "The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.", "Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .", ". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .", "Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.", "choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .", "Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .", "Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.", ". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .", "Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.", "Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .", "Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .", "On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .", ". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript." ]

[ "Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.", "These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .", "Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .", "Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .", "Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.", "antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .", ". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .", ". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.", "Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.", "Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.", "The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.", "In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .", "Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .", "In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .", "Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.", ". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.", "Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .", "Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .", "Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.", ". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.", "Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.", ". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .", "The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.", "Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .", ". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .", "Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.", "choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .", "Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .", "Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.", ". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .", "Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.", "Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .", "Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .", "On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .", ". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript." ]

[ "Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.", "These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .", "Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .", "Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .", "Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.", "antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .", ". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .", ". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.", "Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.", "Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.", "The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.", "In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .", "Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .", "In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .", "Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.", ". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.", "Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .", "Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .", "Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.", ". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.", "Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.", ". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .", "The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.", "Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .", ". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .", "Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.", "choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .", "Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .", "Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.", ". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .", "Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.", "Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .", "Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .", "On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .", ". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript." ]

[ "Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.", "These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .", "Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .", "Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .", "Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.", "antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .", ". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .", ". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.", "Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.", "Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.", "The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.", "In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .", "Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .", "In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .", "Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.", ". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.", "Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .", "Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .", "Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.", ". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.", "Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.", ". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .", "The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.", "Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .", ". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .", "Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.", "choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .", "Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .", "Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.", ". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .", "Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.", "Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .", "Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .", "On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .", ". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript." ]

[ "Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor BCR -associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry.", "These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo. Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" .", "Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins antibodies contained in a solution in the serum of immunized animals must be identical to a cellular receptor \"for a really well-made key will not open different locks at the same time\" . . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies . . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . .", "Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification . . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells . . Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing .", "Together, these discoveries have allowed us to probe humoral immunity at the level of the antigen-specific B cell. Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing . vaccine candidates that elicit protective antibodies; . antibodies that prevent disease when given prophylactically; and . antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen.", "antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens. For HIV-1, the discovery of broadly neutralizing antibodies bnAbs that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies . . . . .", ". . . . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition . . For RSV, stabilized versions of the fusion F protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates . . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine . . . .", ". . . Like RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens 17-19 . Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases.", "Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection .. A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection .. Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells .. Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens. The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus .. Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens .. Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.", "Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice .. The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases. Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor BCR and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen.", "The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts 27-29 . On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased affinity and specificity for the cognate antigen .. Several general techniques are commonly used to identify antigen-specific B cells Table 1 . The B cell enzyme linked immunospot ELISPOT technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies.", "In the B cell ELISPOT, antibody secreting B cells ASCs present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . .", "Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of multiplexing the assay .. One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs .. Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate . . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis. Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . .", "In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates . . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody . . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells .", "Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells . . Advances in single cell next generation sequencing technology have allowed high throughput transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains . . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data.", ". In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection . . Flow cytometry is the most common method used for single cell analysis and isolation . . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen.", "Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . .", "Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression . . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation. Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells .", "Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells . . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome . . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population.", ". However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a \"decoy\" tetramer can be used to identify these contaminating B cells .. In this approach, the same fluorochrome used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer FRET .. Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated.", "Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind. One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry 33, . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry . . With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining.", ". With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts . . The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . .", "The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity . . Preincubation of B cells with increasing concentrations of a monomeric antigen prior to labeling with tetrameric antigen can also be used to further quantify binding affinity. Cells expressing high affinity BCRs will bind monomeric antigen at low concentrations, whereas low affinity BCRs will require higher concentrations of monomeric antigen to compete with and inhibit tetramer binding .. Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells.", "Individual cells can also be isolated by fluorescence activated cell sorting FACS for downstream analysis, including BCR sequencing and cloning, BCR affinity measurement, in vitro proliferation, and transcriptional profiling. Methods have recently been developed to further improve the sensitivity for detecting rare antigen-specific B cells. Magnetic nanoparticles conjugated to antibodies targeting the fluorochrome on the antigen of interest, allow for the enrichment of antigen-specific B cells prior to flow cytometry . . This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . .", ". This approach is particularly useful for detecting rare antigenspecific naïve B cells, autoreactive B cells, memory B cells, and plasmablasts . . The magnetic enrichment strategy allows for the analysis of significantly more cells in a shorter period of time by concentrating the cells of interest prior to flow cytometry Figure 1 . Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: .", "Notably, as with any method that seeks to identify a population of cells at a very low frequency, the background and noise inherent in the detection system is magnified with respect to the signal of interest, especially when that signal is weak. Therefore, to detect the antigen-specific population of interest, the following considerations are critical: . Using decoys to exclude B cells of unwanted specificities; . careful design of flow cytometry panels to avoid emission spillover into the channel for the antigen of interest; and . choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity.", "choosing the brightest fluorochromes, like R-phycoerythrin or allophycocyanin. In vivo methods to probe antigen-specific B cell responses in the presence of other antigen-presenting cells and T cell helpers, have increased our mechanistic understanding of the humoral immune response during vaccination, infection, and autoimmunity. Adoptively transferred B cells can be distinguished from recipient lymphocytes by taking advantage of mouse strains with allelic variations in CD45 or mice devoid of B cells. The adoptively transferred B cells can come from wildtype mice or from mice expressing transgenic BCRs Table 2 , and antigen-specific B cells can be analyzed using the techniques described above. Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits .", "Microscopy is another general technique that has been used to identify antigen-specific cells in vivo and offers the advantage of direct visualization. In the first reported application of this technique to demonstrate the cellular origin of antibodies in 1955, fluorescein-conjugated antibodies against ovalbumin and human immunoglobulin were used to stain tissue sections of the spleen from hyperimmune rabbits . . Since then, other groups have fluorescently labeled antigens to localize antigen-specific B cells by microscopy . . Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . .", "Advances in laser capture dissection microscopy, already used in the T cell field, also provide an opportunity for isolating individual antigen-specific B cells for downstream analysis, including sequencing and cloning of the BCR or transcriptional profiling . . However, antigen staining of BCRs in situ can be challenging depending on the binding of antigens from pathogens to other cellular receptors or an alteration of BCR specificity during tissue fixation or processing. Two-photon or multiphoton microscopy has the ability to resolve images at greater depths and with less photobleaching than confocal microscopy . . As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions.", ". As a result, this technology has allowed real-time imaging in living, intact lymphoid tissues of mice, permitting the direct in vivo observation of immune cell interactions. The dynamic movements and interactions of antigen-specific B cells can be studied in vivo by combining an adoptive transfer of individual B cells isolated by limiting dilution or FACS with two-photon microscopy . . Humanized mouse models are powerful tools for translating experiments in mice to applications in humans. Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . .", "Transgenic mice that produce humanized cytokines by knock-in replacement can be used to support human hematopoietic stem cells . . Transgenic mice with complete humanization of the mouse immunoglobulin loci provide an opportunity for recapitulating the breadth of the human B cell repertoire and serve as a valuable tool for therapeutic antibody discovery . . However, one caveat is that the allele frequencies found in the B cell repertoires of these mouse models may not necessarily recapitulate those found in humans . . Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells.", "Mass cytometry has the potential to provide further high-dimensional analysis of antigen-specific B cells. In this method, heavy metal ion tags rather than fluorochromes are used to label cells. Since data is collected as time-offlight mass spectrometry, up to 42 unique parameters can be simultaneously measured from a single sample without significant spillover between channels or the need for compensation. Mass cytometry with heavy metal-labeled tetramers can be constructed using streptavidin . . Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . .", "Mass cytometry with metal-labeled peptide-MHC tetramers has been used successfully to identify and characterize antigen-specific T cells, but to our knowledge has not yet been applied to antigen-specific B cells . . One limitation of this approach is that cells are unavailable for downstream analysis since they are vaporized by a plasma torch to atomize the ion tags. However, by simultaneously detecting many more surface markers and intracellular cytokines, transcription factors, and detecting more signaling molecules from individual cells than previously possible with traditional fluorescent labels, the application of mass cytometry with dimensionality reduction algorithms could help dissect the complexity of the B cell compartment, provide a higher resolution view of B cell development, and reveal novel subsets of antigen-specific B cells involved in mediating autoimmune diseases or protection against infection. On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . .", "On the horizon, single cell RNA-sequencing RNA-seq technologies have the potential to revolutionize the study of antigen-specific immune cells . . The ability to generate a library of tetramers with unique barcodes could allow the simultaneous examination of gene expression profiles from a large number of cells with different antigen specificities in a single experiment. Combining barcoded tetramers with oligonucleotide-conjugated antibodies and RNA-seq to simultaneously measure the protein and gene expression of antigen-specific cells could further increase the amount of unbiased multi-omic information about individual antigen-specific cells in normal and disease states and aid the rational design of vaccines and therapeutics . . . .", ". . . The ongoing analysis of antigen-specific B cell responses has led to the development of new diagnostic, therapeutic, and research reagents. Methods for studying antigen-specific B cell responses are being increasingly applied to tackle diseases like HIV, RSV, and autoimmune diseases, in which the immune response either fails to protect or clear disease, or where it enhances disease or is responsible for the disease itself. Considerable opportunities exist on the horizon for applying these methods to a myriad of diseases in which B cells play an active role. JB and JT reviewed the literature, generated figures and tables, and wrote the manuscript." ]

[ "Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them.", "However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 0.5% were confirmed by laboratory results. These confirmed cases 67.2% of which were males had a mean age of 43.23 years SD ± 22.8 . Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer.", "These confirmed cases 67.2% of which were males had a mean age of 43.23 years SD ± 22.8 . Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus MERS-CoV was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure .", "Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus MERS-CoV was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure . Since then, 27 countries have reported the presence of this virus, including the 12 countries of the Eastern Mediterranean region. Several outbreaks have occurred in multiple countries including Saudi Arabia, the United Arab Emirates and the Republic of Korea . Recent fatality rate CFR of 21% . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population .", "Recent fatality rate CFR of 21% . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population . e literature shows that MERS-CoV infects males more than females . e casefatality rate of men 52% is higher than that of women 23% . Males with a history of serious medical conditions are highly susceptible to this infection. Moreover, the mean age of infection in adults is 60 years . e mode of transmission is not entirely understood yet ; however, human-to-human and zoonotic sources of transmission have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans.", "e mode of transmission is not entirely understood yet ; however, human-to-human and zoonotic sources of transmission have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans. Interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings . Clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death . Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings . Studies have suggested an incubation period of 16 days with a mean of 5-6 days , while the median time until death is 11-13 days range 5-27 days among severely ill patients .", "Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings . Studies have suggested an incubation period of 16 days with a mean of 5-6 days , while the median time until death is 11-13 days range 5-27 days among severely ill patients . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction rRT-PCR assays . ere is no specific treatment for MERS-CoV. Like most viral infections, the treatment options are supportive and symptomatic . At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness .", "At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness . Such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. One must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided .", "Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided . Many studies have been conducted in recent years in Saudi Arabia to combat this deadly disease. A large multicentre study showed that it is nearly impossible to differentiate between patients of MERS-CoV and non-MERS-CoV just on the basis of clinical presentation . Another cohort study, which was hospital-based 17 cases vs. 82 controls , found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific .", "Another cohort study, which was hospital-based 17 cases vs. 82 controls , found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific . Physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing which usually takes between 12 and 24 hours . Identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers . Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period.", "Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. Both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained . Identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV.", "e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV. e nature of these markers has not been investigated in Saudi Arabia, and hence this study aims to identify them. A cross-sectional study was conducted at the regional laboratory and blood bank, located at Shumaisi Hospital in Riyadh, KSA. e laboratory has received the Central Blood Banks and Reference Laboratories Accreditation Program Saudi Central Board for Accreditation of Healthcare Institution CBAHI 2018 . Technique. Data were collected during the period of January 2017 to December 2017.", "Technique. Data were collected during the period of January 2017 to December 2017. All patients in Riyadh and Al-Qassim regions who had their samples tested at Riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. For the descriptive aim, we estimated the prevalence of MERS-CoV. For the analytical aim, a binary logistic regression model was developed. In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status yes/no , hospitals, and area of residence. Data were cross-checked with a labcomputerized database.", "In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status yes/no , hospitals, and area of residence. Data were cross-checked with a labcomputerized database. Further data were collected on demographic characteristics age and sex , underlying nationality, and health care status. We collected data from 25,400 cases, of which 23,646 suspected cases of MERS-CoV were included in the final analysis. Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics medians, interquartile ranges, minimum, and maximum were used to describe the distribution.", "Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics medians, interquartile ranges, minimum, and maximum were used to describe the distribution. A logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. At first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered.", "is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. A confirmed case is defined as a suspected case with laboratory confirmation of MERS-CoV infection . A total of 23,646 of MERS-CoV suspected cases were included in this study, of which 52.3% were males n � 12376 and 47.7% were females n � 11270 . e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032 , whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period .. A total of 23,646 suspected cases were included in the results.", "e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032 , whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period .. A total of 23,646 suspected cases were included in the results. Of the total suspected cases, 119 cases had been confirmed via laboratory results. All the confirmed cases are reported to MOH through HESN health electronic surveillance networks and to the World Health Organization WHO through the International Health Regulations IHR , National Focal Point of Saudi Arabia. We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases.", "We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. During the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of MERS-CoV infection cases has decreased in Riyadh and Al-Qassim regions in comparison to that of the last three years. From 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of Da'ar and Ahmed in their paper . e predominance of infection in males was also observed in another study pwefromed in KSA ., which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females .", "is also complements the findings reported by of Da'ar and Ahmed in their paper . e predominance of infection in males was also observed in another study pwefromed in KSA ., which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females . It is worth mentioning that Saudi Arabia defines age categories differently from the WHO children: 0-14, adult: otherwise . However, unlike the classification used in Saudi Arabia, we have followed the WHO categorization of age to differentiate between children/adolescents 0 to 19 years and adults 20 years and older as indicated in WHO reports for age-standardized population and in infectious diseases . is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points age of 41 and age of 60 .", "is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points age of 41 and age of 60 . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of MERS-CoV were reported among people aged 40 and above . In 2016, only 9 of 552 cases 1.6% of MERS-CoV infection were found among pediatric patients. Moreover, the study which was conducted in King Fahad Medical City in Riyadh KFMC between January 2012 and December 2013 did not report any MERS-CoV cases among children . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al.", "Moreover, the study which was conducted in King Fahad Medical City in Riyadh KFMC between January 2012 and December 2013 did not report any MERS-CoV cases among children . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al. between 2012 and 2016 suggests that the prevalence and distribution of MERS-CoV were the highest-risk in elderly aged 60 years or above . Similar to our results, this study also reported the highest number of confirmed cases during the summer season . Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers .", "Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers . Similarly, other studies also reported a lower prevalence in healthcare workers . Our data reported a higher prevalence of infection among Saudi nationals as compared with non-Saudi. Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only.", "Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only. In our study, we detected a low prevalence 0.5% . e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab.", "e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. In fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. To the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase AST .", "However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase AST . However, further investigations are needed to confirm our findings. One of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of MERS-CoV in the Riyadh and Al-Qassim regions. Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases.", "Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. In addition to this, quality assurance policies are implemented at HESN in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas two major regions in KSA .", "Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas two major regions in KSA . Given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. It must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed MERS-CoV with those with suspected MERS-CoV who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive.", "is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting MERS-CoV signs with a negative lab result may need retesting. Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific.", "Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. Future studies should aim to confirm such findings in other regions in Saudi Arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request.", "e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests." ]

[ "Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them.", "However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 0.5% were confirmed by laboratory results. These confirmed cases 67.2% of which were males had a mean age of 43.23 years SD ± 22.8 . Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer.", "These confirmed cases 67.2% of which were males had a mean age of 43.23 years SD ± 22.8 . Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus MERS-CoV was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure .", "Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus MERS-CoV was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure . Since then, 27 countries have reported the presence of this virus, including the 12 countries of the Eastern Mediterranean region. Several outbreaks have occurred in multiple countries including Saudi Arabia, the United Arab Emirates and the Republic of Korea . Recent fatality rate CFR of 21% . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population .", "Recent fatality rate CFR of 21% . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population . e literature shows that MERS-CoV infects males more than females . e casefatality rate of men 52% is higher than that of women 23% . Males with a history of serious medical conditions are highly susceptible to this infection. Moreover, the mean age of infection in adults is 60 years . e mode of transmission is not entirely understood yet ; however, human-to-human and zoonotic sources of transmission have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans.", "e mode of transmission is not entirely understood yet ; however, human-to-human and zoonotic sources of transmission have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans. Interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings . Clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death . Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings . Studies have suggested an incubation period of 16 days with a mean of 5-6 days , while the median time until death is 11-13 days range 5-27 days among severely ill patients .", "Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings . Studies have suggested an incubation period of 16 days with a mean of 5-6 days , while the median time until death is 11-13 days range 5-27 days among severely ill patients . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction rRT-PCR assays . ere is no specific treatment for MERS-CoV. Like most viral infections, the treatment options are supportive and symptomatic . At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness .", "At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness . Such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. One must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided .", "Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided . Many studies have been conducted in recent years in Saudi Arabia to combat this deadly disease. A large multicentre study showed that it is nearly impossible to differentiate between patients of MERS-CoV and non-MERS-CoV just on the basis of clinical presentation . Another cohort study, which was hospital-based 17 cases vs. 82 controls , found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific .", "Another cohort study, which was hospital-based 17 cases vs. 82 controls , found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific . Physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing which usually takes between 12 and 24 hours . Identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers . Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period.", "Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. Both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained . Identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV.", "e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV. e nature of these markers has not been investigated in Saudi Arabia, and hence this study aims to identify them. A cross-sectional study was conducted at the regional laboratory and blood bank, located at Shumaisi Hospital in Riyadh, KSA. e laboratory has received the Central Blood Banks and Reference Laboratories Accreditation Program Saudi Central Board for Accreditation of Healthcare Institution CBAHI 2018 . Technique. Data were collected during the period of January 2017 to December 2017.", "Technique. Data were collected during the period of January 2017 to December 2017. All patients in Riyadh and Al-Qassim regions who had their samples tested at Riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. For the descriptive aim, we estimated the prevalence of MERS-CoV. For the analytical aim, a binary logistic regression model was developed. In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status yes/no , hospitals, and area of residence. Data were cross-checked with a labcomputerized database.", "In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status yes/no , hospitals, and area of residence. Data were cross-checked with a labcomputerized database. Further data were collected on demographic characteristics age and sex , underlying nationality, and health care status. We collected data from 25,400 cases, of which 23,646 suspected cases of MERS-CoV were included in the final analysis. Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics medians, interquartile ranges, minimum, and maximum were used to describe the distribution.", "Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics medians, interquartile ranges, minimum, and maximum were used to describe the distribution. A logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. At first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered.", "is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. A confirmed case is defined as a suspected case with laboratory confirmation of MERS-CoV infection . A total of 23,646 of MERS-CoV suspected cases were included in this study, of which 52.3% were males n � 12376 and 47.7% were females n � 11270 . e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032 , whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period .. A total of 23,646 suspected cases were included in the results.", "e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032 , whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period .. A total of 23,646 suspected cases were included in the results. Of the total suspected cases, 119 cases had been confirmed via laboratory results. All the confirmed cases are reported to MOH through HESN health electronic surveillance networks and to the World Health Organization WHO through the International Health Regulations IHR , National Focal Point of Saudi Arabia. We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases.", "We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. During the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of MERS-CoV infection cases has decreased in Riyadh and Al-Qassim regions in comparison to that of the last three years. From 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of Da'ar and Ahmed in their paper . e predominance of infection in males was also observed in another study pwefromed in KSA ., which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females .", "is also complements the findings reported by of Da'ar and Ahmed in their paper . e predominance of infection in males was also observed in another study pwefromed in KSA ., which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females . It is worth mentioning that Saudi Arabia defines age categories differently from the WHO children: 0-14, adult: otherwise . However, unlike the classification used in Saudi Arabia, we have followed the WHO categorization of age to differentiate between children/adolescents 0 to 19 years and adults 20 years and older as indicated in WHO reports for age-standardized population and in infectious diseases . is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points age of 41 and age of 60 .", "is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points age of 41 and age of 60 . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of MERS-CoV were reported among people aged 40 and above . In 2016, only 9 of 552 cases 1.6% of MERS-CoV infection were found among pediatric patients. Moreover, the study which was conducted in King Fahad Medical City in Riyadh KFMC between January 2012 and December 2013 did not report any MERS-CoV cases among children . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al.", "Moreover, the study which was conducted in King Fahad Medical City in Riyadh KFMC between January 2012 and December 2013 did not report any MERS-CoV cases among children . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al. between 2012 and 2016 suggests that the prevalence and distribution of MERS-CoV were the highest-risk in elderly aged 60 years or above . Similar to our results, this study also reported the highest number of confirmed cases during the summer season . Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers .", "Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers . Similarly, other studies also reported a lower prevalence in healthcare workers . Our data reported a higher prevalence of infection among Saudi nationals as compared with non-Saudi. Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only.", "Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only. In our study, we detected a low prevalence 0.5% . e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab.", "e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. In fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. To the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase AST .", "However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase AST . However, further investigations are needed to confirm our findings. One of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of MERS-CoV in the Riyadh and Al-Qassim regions. Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases.", "Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. In addition to this, quality assurance policies are implemented at HESN in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas two major regions in KSA .", "Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas two major regions in KSA . Given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. It must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed MERS-CoV with those with suspected MERS-CoV who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive.", "is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting MERS-CoV signs with a negative lab result may need retesting. Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific.", "Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. Future studies should aim to confirm such findings in other regions in Saudi Arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request.", "e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests." ]

[ "Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them.", "However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 0.5% were confirmed by laboratory results. These confirmed cases 67.2% of which were males had a mean age of 43.23 years SD ± 22.8 . Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer.", "These confirmed cases 67.2% of which were males had a mean age of 43.23 years SD ± 22.8 . Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus MERS-CoV was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure .", "Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus MERS-CoV was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure . Since then, 27 countries have reported the presence of this virus, including the 12 countries of the Eastern Mediterranean region. Several outbreaks have occurred in multiple countries including Saudi Arabia, the United Arab Emirates and the Republic of Korea . Recent fatality rate CFR of 21% . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population .", "Recent fatality rate CFR of 21% . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population . e literature shows that MERS-CoV infects males more than females . e casefatality rate of men 52% is higher than that of women 23% . Males with a history of serious medical conditions are highly susceptible to this infection. Moreover, the mean age of infection in adults is 60 years . e mode of transmission is not entirely understood yet ; however, human-to-human and zoonotic sources of transmission have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans.", "e mode of transmission is not entirely understood yet ; however, human-to-human and zoonotic sources of transmission have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans. Interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings . Clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death . Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings . Studies have suggested an incubation period of 16 days with a mean of 5-6 days , while the median time until death is 11-13 days range 5-27 days among severely ill patients .", "Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings . Studies have suggested an incubation period of 16 days with a mean of 5-6 days , while the median time until death is 11-13 days range 5-27 days among severely ill patients . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction rRT-PCR assays . ere is no specific treatment for MERS-CoV. Like most viral infections, the treatment options are supportive and symptomatic . At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness .", "At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness . Such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. One must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided .", "Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided . Many studies have been conducted in recent years in Saudi Arabia to combat this deadly disease. A large multicentre study showed that it is nearly impossible to differentiate between patients of MERS-CoV and non-MERS-CoV just on the basis of clinical presentation . Another cohort study, which was hospital-based 17 cases vs. 82 controls , found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific .", "Another cohort study, which was hospital-based 17 cases vs. 82 controls , found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific . Physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing which usually takes between 12 and 24 hours . Identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers . Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period.", "Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. Both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained . Identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV.", "e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV. e nature of these markers has not been investigated in Saudi Arabia, and hence this study aims to identify them. A cross-sectional study was conducted at the regional laboratory and blood bank, located at Shumaisi Hospital in Riyadh, KSA. e laboratory has received the Central Blood Banks and Reference Laboratories Accreditation Program Saudi Central Board for Accreditation of Healthcare Institution CBAHI 2018 . Technique. Data were collected during the period of January 2017 to December 2017.", "Technique. Data were collected during the period of January 2017 to December 2017. All patients in Riyadh and Al-Qassim regions who had their samples tested at Riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. For the descriptive aim, we estimated the prevalence of MERS-CoV. For the analytical aim, a binary logistic regression model was developed. In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status yes/no , hospitals, and area of residence. Data were cross-checked with a labcomputerized database.", "In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status yes/no , hospitals, and area of residence. Data were cross-checked with a labcomputerized database. Further data were collected on demographic characteristics age and sex , underlying nationality, and health care status. We collected data from 25,400 cases, of which 23,646 suspected cases of MERS-CoV were included in the final analysis. Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics medians, interquartile ranges, minimum, and maximum were used to describe the distribution.", "Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics medians, interquartile ranges, minimum, and maximum were used to describe the distribution. A logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. At first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered.", "is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. A confirmed case is defined as a suspected case with laboratory confirmation of MERS-CoV infection . A total of 23,646 of MERS-CoV suspected cases were included in this study, of which 52.3% were males n � 12376 and 47.7% were females n � 11270 . e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032 , whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period .. A total of 23,646 suspected cases were included in the results.", "e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032 , whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period .. A total of 23,646 suspected cases were included in the results. Of the total suspected cases, 119 cases had been confirmed via laboratory results. All the confirmed cases are reported to MOH through HESN health electronic surveillance networks and to the World Health Organization WHO through the International Health Regulations IHR , National Focal Point of Saudi Arabia. We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases.", "We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. During the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of MERS-CoV infection cases has decreased in Riyadh and Al-Qassim regions in comparison to that of the last three years. From 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of Da'ar and Ahmed in their paper . e predominance of infection in males was also observed in another study pwefromed in KSA ., which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females .", "is also complements the findings reported by of Da'ar and Ahmed in their paper . e predominance of infection in males was also observed in another study pwefromed in KSA ., which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females . It is worth mentioning that Saudi Arabia defines age categories differently from the WHO children: 0-14, adult: otherwise . However, unlike the classification used in Saudi Arabia, we have followed the WHO categorization of age to differentiate between children/adolescents 0 to 19 years and adults 20 years and older as indicated in WHO reports for age-standardized population and in infectious diseases . is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points age of 41 and age of 60 .", "is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points age of 41 and age of 60 . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of MERS-CoV were reported among people aged 40 and above . In 2016, only 9 of 552 cases 1.6% of MERS-CoV infection were found among pediatric patients. Moreover, the study which was conducted in King Fahad Medical City in Riyadh KFMC between January 2012 and December 2013 did not report any MERS-CoV cases among children . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al.", "Moreover, the study which was conducted in King Fahad Medical City in Riyadh KFMC between January 2012 and December 2013 did not report any MERS-CoV cases among children . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al. between 2012 and 2016 suggests that the prevalence and distribution of MERS-CoV were the highest-risk in elderly aged 60 years or above . Similar to our results, this study also reported the highest number of confirmed cases during the summer season . Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers .", "Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers . Similarly, other studies also reported a lower prevalence in healthcare workers . Our data reported a higher prevalence of infection among Saudi nationals as compared with non-Saudi. Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only.", "Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only. In our study, we detected a low prevalence 0.5% . e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab.", "e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. In fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. To the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase AST .", "However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase AST . However, further investigations are needed to confirm our findings. One of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of MERS-CoV in the Riyadh and Al-Qassim regions. Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases.", "Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. In addition to this, quality assurance policies are implemented at HESN in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas two major regions in KSA .", "Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas two major regions in KSA . Given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. It must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed MERS-CoV with those with suspected MERS-CoV who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive.", "is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting MERS-CoV signs with a negative lab result may need retesting. Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific.", "Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. Future studies should aim to confirm such findings in other regions in Saudi Arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request.", "e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests." ]

[ "Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them.", "However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 0.5% were confirmed by laboratory results. These confirmed cases 67.2% of which were males had a mean age of 43.23 years SD ± 22.8 . Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer.", "These confirmed cases 67.2% of which were males had a mean age of 43.23 years SD ± 22.8 . Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus MERS-CoV was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure .", "Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus MERS-CoV was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure . Since then, 27 countries have reported the presence of this virus, including the 12 countries of the Eastern Mediterranean region. Several outbreaks have occurred in multiple countries including Saudi Arabia, the United Arab Emirates and the Republic of Korea . Recent fatality rate CFR of 21% . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population .", "Recent fatality rate CFR of 21% . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population . e literature shows that MERS-CoV infects males more than females . e casefatality rate of men 52% is higher than that of women 23% . Males with a history of serious medical conditions are highly susceptible to this infection. Moreover, the mean age of infection in adults is 60 years . e mode of transmission is not entirely understood yet ; however, human-to-human and zoonotic sources of transmission have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans.", "e mode of transmission is not entirely understood yet ; however, human-to-human and zoonotic sources of transmission have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans. Interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings . Clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death . Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings . Studies have suggested an incubation period of 16 days with a mean of 5-6 days , while the median time until death is 11-13 days range 5-27 days among severely ill patients .", "Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings . Studies have suggested an incubation period of 16 days with a mean of 5-6 days , while the median time until death is 11-13 days range 5-27 days among severely ill patients . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction rRT-PCR assays . ere is no specific treatment for MERS-CoV. Like most viral infections, the treatment options are supportive and symptomatic . At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness .", "At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness . Such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. One must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided .", "Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided . Many studies have been conducted in recent years in Saudi Arabia to combat this deadly disease. A large multicentre study showed that it is nearly impossible to differentiate between patients of MERS-CoV and non-MERS-CoV just on the basis of clinical presentation . Another cohort study, which was hospital-based 17 cases vs. 82 controls , found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific .", "Another cohort study, which was hospital-based 17 cases vs. 82 controls , found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific . Physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing which usually takes between 12 and 24 hours . Identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers . Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period.", "Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. Both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained . Identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV.", "e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV. e nature of these markers has not been investigated in Saudi Arabia, and hence this study aims to identify them. A cross-sectional study was conducted at the regional laboratory and blood bank, located at Shumaisi Hospital in Riyadh, KSA. e laboratory has received the Central Blood Banks and Reference Laboratories Accreditation Program Saudi Central Board for Accreditation of Healthcare Institution CBAHI 2018 . Technique. Data were collected during the period of January 2017 to December 2017.", "Technique. Data were collected during the period of January 2017 to December 2017. All patients in Riyadh and Al-Qassim regions who had their samples tested at Riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. For the descriptive aim, we estimated the prevalence of MERS-CoV. For the analytical aim, a binary logistic regression model was developed. In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status yes/no , hospitals, and area of residence. Data were cross-checked with a labcomputerized database.", "In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status yes/no , hospitals, and area of residence. Data were cross-checked with a labcomputerized database. Further data were collected on demographic characteristics age and sex , underlying nationality, and health care status. We collected data from 25,400 cases, of which 23,646 suspected cases of MERS-CoV were included in the final analysis. Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics medians, interquartile ranges, minimum, and maximum were used to describe the distribution.", "Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics medians, interquartile ranges, minimum, and maximum were used to describe the distribution. A logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. At first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered.", "is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. A confirmed case is defined as a suspected case with laboratory confirmation of MERS-CoV infection . A total of 23,646 of MERS-CoV suspected cases were included in this study, of which 52.3% were males n � 12376 and 47.7% were females n � 11270 . e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032 , whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period .. A total of 23,646 suspected cases were included in the results.", "e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032 , whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period .. A total of 23,646 suspected cases were included in the results. Of the total suspected cases, 119 cases had been confirmed via laboratory results. All the confirmed cases are reported to MOH through HESN health electronic surveillance networks and to the World Health Organization WHO through the International Health Regulations IHR , National Focal Point of Saudi Arabia. We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases.", "We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. During the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of MERS-CoV infection cases has decreased in Riyadh and Al-Qassim regions in comparison to that of the last three years. From 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of Da'ar and Ahmed in their paper . e predominance of infection in males was also observed in another study pwefromed in KSA ., which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females .", "is also complements the findings reported by of Da'ar and Ahmed in their paper . e predominance of infection in males was also observed in another study pwefromed in KSA ., which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females . It is worth mentioning that Saudi Arabia defines age categories differently from the WHO children: 0-14, adult: otherwise . However, unlike the classification used in Saudi Arabia, we have followed the WHO categorization of age to differentiate between children/adolescents 0 to 19 years and adults 20 years and older as indicated in WHO reports for age-standardized population and in infectious diseases . is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points age of 41 and age of 60 .", "is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points age of 41 and age of 60 . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of MERS-CoV were reported among people aged 40 and above . In 2016, only 9 of 552 cases 1.6% of MERS-CoV infection were found among pediatric patients. Moreover, the study which was conducted in King Fahad Medical City in Riyadh KFMC between January 2012 and December 2013 did not report any MERS-CoV cases among children . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al.", "Moreover, the study which was conducted in King Fahad Medical City in Riyadh KFMC between January 2012 and December 2013 did not report any MERS-CoV cases among children . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al. between 2012 and 2016 suggests that the prevalence and distribution of MERS-CoV were the highest-risk in elderly aged 60 years or above . Similar to our results, this study also reported the highest number of confirmed cases during the summer season . Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers .", "Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers . Similarly, other studies also reported a lower prevalence in healthcare workers . Our data reported a higher prevalence of infection among Saudi nationals as compared with non-Saudi. Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only.", "Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only. In our study, we detected a low prevalence 0.5% . e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab.", "e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. In fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. To the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase AST .", "However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase AST . However, further investigations are needed to confirm our findings. One of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of MERS-CoV in the Riyadh and Al-Qassim regions. Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases.", "Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. In addition to this, quality assurance policies are implemented at HESN in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas two major regions in KSA .", "Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas two major regions in KSA . Given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. It must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed MERS-CoV with those with suspected MERS-CoV who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive.", "is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting MERS-CoV signs with a negative lab result may need retesting. Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific.", "Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. Future studies should aim to confirm such findings in other regions in Saudi Arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request.", "e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests." ]

[ "Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them.", "However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 0.5% were confirmed by laboratory results. These confirmed cases 67.2% of which were males had a mean age of 43.23 years SD ± 22.8 . Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer.", "These confirmed cases 67.2% of which were males had a mean age of 43.23 years SD ± 22.8 . Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus MERS-CoV was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure .", "Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus MERS-CoV was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure . Since then, 27 countries have reported the presence of this virus, including the 12 countries of the Eastern Mediterranean region. Several outbreaks have occurred in multiple countries including Saudi Arabia, the United Arab Emirates and the Republic of Korea . Recent fatality rate CFR of 21% . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population .", "Recent fatality rate CFR of 21% . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population . e literature shows that MERS-CoV infects males more than females . e casefatality rate of men 52% is higher than that of women 23% . Males with a history of serious medical conditions are highly susceptible to this infection. Moreover, the mean age of infection in adults is 60 years . e mode of transmission is not entirely understood yet ; however, human-to-human and zoonotic sources of transmission have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans.", "e mode of transmission is not entirely understood yet ; however, human-to-human and zoonotic sources of transmission have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans. Interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings . Clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death . Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings . Studies have suggested an incubation period of 16 days with a mean of 5-6 days , while the median time until death is 11-13 days range 5-27 days among severely ill patients .", "Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings . Studies have suggested an incubation period of 16 days with a mean of 5-6 days , while the median time until death is 11-13 days range 5-27 days among severely ill patients . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction rRT-PCR assays . ere is no specific treatment for MERS-CoV. Like most viral infections, the treatment options are supportive and symptomatic . At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness .", "At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness . Such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. One must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided .", "Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided . Many studies have been conducted in recent years in Saudi Arabia to combat this deadly disease. A large multicentre study showed that it is nearly impossible to differentiate between patients of MERS-CoV and non-MERS-CoV just on the basis of clinical presentation . Another cohort study, which was hospital-based 17 cases vs. 82 controls , found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific .", "Another cohort study, which was hospital-based 17 cases vs. 82 controls , found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific . Physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing which usually takes between 12 and 24 hours . Identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers . Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period.", "Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. Both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained . Identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV.", "e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV. e nature of these markers has not been investigated in Saudi Arabia, and hence this study aims to identify them. A cross-sectional study was conducted at the regional laboratory and blood bank, located at Shumaisi Hospital in Riyadh, KSA. e laboratory has received the Central Blood Banks and Reference Laboratories Accreditation Program Saudi Central Board for Accreditation of Healthcare Institution CBAHI 2018 . Technique. Data were collected during the period of January 2017 to December 2017.", "Technique. Data were collected during the period of January 2017 to December 2017. All patients in Riyadh and Al-Qassim regions who had their samples tested at Riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. For the descriptive aim, we estimated the prevalence of MERS-CoV. For the analytical aim, a binary logistic regression model was developed. In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status yes/no , hospitals, and area of residence. Data were cross-checked with a labcomputerized database.", "In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status yes/no , hospitals, and area of residence. Data were cross-checked with a labcomputerized database. Further data were collected on demographic characteristics age and sex , underlying nationality, and health care status. We collected data from 25,400 cases, of which 23,646 suspected cases of MERS-CoV were included in the final analysis. Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics medians, interquartile ranges, minimum, and maximum were used to describe the distribution.", "Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics medians, interquartile ranges, minimum, and maximum were used to describe the distribution. A logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. At first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered.", "is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. A confirmed case is defined as a suspected case with laboratory confirmation of MERS-CoV infection . A total of 23,646 of MERS-CoV suspected cases were included in this study, of which 52.3% were males n � 12376 and 47.7% were females n � 11270 . e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032 , whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period .. A total of 23,646 suspected cases were included in the results.", "e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032 , whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period .. A total of 23,646 suspected cases were included in the results. Of the total suspected cases, 119 cases had been confirmed via laboratory results. All the confirmed cases are reported to MOH through HESN health electronic surveillance networks and to the World Health Organization WHO through the International Health Regulations IHR , National Focal Point of Saudi Arabia. We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases.", "We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. During the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of MERS-CoV infection cases has decreased in Riyadh and Al-Qassim regions in comparison to that of the last three years. From 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of Da'ar and Ahmed in their paper . e predominance of infection in males was also observed in another study pwefromed in KSA ., which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females .", "is also complements the findings reported by of Da'ar and Ahmed in their paper . e predominance of infection in males was also observed in another study pwefromed in KSA ., which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females . It is worth mentioning that Saudi Arabia defines age categories differently from the WHO children: 0-14, adult: otherwise . However, unlike the classification used in Saudi Arabia, we have followed the WHO categorization of age to differentiate between children/adolescents 0 to 19 years and adults 20 years and older as indicated in WHO reports for age-standardized population and in infectious diseases . is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points age of 41 and age of 60 .", "is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points age of 41 and age of 60 . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of MERS-CoV were reported among people aged 40 and above . In 2016, only 9 of 552 cases 1.6% of MERS-CoV infection were found among pediatric patients. Moreover, the study which was conducted in King Fahad Medical City in Riyadh KFMC between January 2012 and December 2013 did not report any MERS-CoV cases among children . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al.", "Moreover, the study which was conducted in King Fahad Medical City in Riyadh KFMC between January 2012 and December 2013 did not report any MERS-CoV cases among children . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al. between 2012 and 2016 suggests that the prevalence and distribution of MERS-CoV were the highest-risk in elderly aged 60 years or above . Similar to our results, this study also reported the highest number of confirmed cases during the summer season . Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers .", "Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers . Similarly, other studies also reported a lower prevalence in healthcare workers . Our data reported a higher prevalence of infection among Saudi nationals as compared with non-Saudi. Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only.", "Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only. In our study, we detected a low prevalence 0.5% . e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab.", "e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. In fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. To the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase AST .", "However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase AST . However, further investigations are needed to confirm our findings. One of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of MERS-CoV in the Riyadh and Al-Qassim regions. Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases.", "Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. In addition to this, quality assurance policies are implemented at HESN in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas two major regions in KSA .", "Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas two major regions in KSA . Given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. It must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed MERS-CoV with those with suspected MERS-CoV who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive.", "is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting MERS-CoV signs with a negative lab result may need retesting. Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific.", "Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. Future studies should aim to confirm such findings in other regions in Saudi Arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request.", "e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests." ]

[ "Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them.", "However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 0.5% were confirmed by laboratory results. These confirmed cases 67.2% of which were males had a mean age of 43.23 years SD ± 22.8 . Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer.", "These confirmed cases 67.2% of which were males had a mean age of 43.23 years SD ± 22.8 . Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus MERS-CoV was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure .", "Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus MERS-CoV was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure . Since then, 27 countries have reported the presence of this virus, including the 12 countries of the Eastern Mediterranean region. Several outbreaks have occurred in multiple countries including Saudi Arabia, the United Arab Emirates and the Republic of Korea . Recent fatality rate CFR of 21% . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population .", "Recent fatality rate CFR of 21% . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population . e literature shows that MERS-CoV infects males more than females . e casefatality rate of men 52% is higher than that of women 23% . Males with a history of serious medical conditions are highly susceptible to this infection. Moreover, the mean age of infection in adults is 60 years . e mode of transmission is not entirely understood yet ; however, human-to-human and zoonotic sources of transmission have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans.", "e mode of transmission is not entirely understood yet ; however, human-to-human and zoonotic sources of transmission have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans. Interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings . Clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death . Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings . Studies have suggested an incubation period of 16 days with a mean of 5-6 days , while the median time until death is 11-13 days range 5-27 days among severely ill patients .", "Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings . Studies have suggested an incubation period of 16 days with a mean of 5-6 days , while the median time until death is 11-13 days range 5-27 days among severely ill patients . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction rRT-PCR assays . ere is no specific treatment for MERS-CoV. Like most viral infections, the treatment options are supportive and symptomatic . At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness .", "At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness . Such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. One must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided .", "Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided . Many studies have been conducted in recent years in Saudi Arabia to combat this deadly disease. A large multicentre study showed that it is nearly impossible to differentiate between patients of MERS-CoV and non-MERS-CoV just on the basis of clinical presentation . Another cohort study, which was hospital-based 17 cases vs. 82 controls , found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific .", "Another cohort study, which was hospital-based 17 cases vs. 82 controls , found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific . Physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing which usually takes between 12 and 24 hours . Identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers . Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period.", "Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. Both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained . Identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV.", "e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV. e nature of these markers has not been investigated in Saudi Arabia, and hence this study aims to identify them. A cross-sectional study was conducted at the regional laboratory and blood bank, located at Shumaisi Hospital in Riyadh, KSA. e laboratory has received the Central Blood Banks and Reference Laboratories Accreditation Program Saudi Central Board for Accreditation of Healthcare Institution CBAHI 2018 . Technique. Data were collected during the period of January 2017 to December 2017.", "Technique. Data were collected during the period of January 2017 to December 2017. All patients in Riyadh and Al-Qassim regions who had their samples tested at Riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. For the descriptive aim, we estimated the prevalence of MERS-CoV. For the analytical aim, a binary logistic regression model was developed. In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status yes/no , hospitals, and area of residence. Data were cross-checked with a labcomputerized database.", "In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status yes/no , hospitals, and area of residence. Data were cross-checked with a labcomputerized database. Further data were collected on demographic characteristics age and sex , underlying nationality, and health care status. We collected data from 25,400 cases, of which 23,646 suspected cases of MERS-CoV were included in the final analysis. Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics medians, interquartile ranges, minimum, and maximum were used to describe the distribution.", "Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics medians, interquartile ranges, minimum, and maximum were used to describe the distribution. A logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. At first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered.", "is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. A confirmed case is defined as a suspected case with laboratory confirmation of MERS-CoV infection . A total of 23,646 of MERS-CoV suspected cases were included in this study, of which 52.3% were males n � 12376 and 47.7% were females n � 11270 . e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032 , whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period .. A total of 23,646 suspected cases were included in the results.", "e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032 , whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period .. A total of 23,646 suspected cases were included in the results. Of the total suspected cases, 119 cases had been confirmed via laboratory results. All the confirmed cases are reported to MOH through HESN health electronic surveillance networks and to the World Health Organization WHO through the International Health Regulations IHR , National Focal Point of Saudi Arabia. We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases.", "We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. During the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of MERS-CoV infection cases has decreased in Riyadh and Al-Qassim regions in comparison to that of the last three years. From 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of Da'ar and Ahmed in their paper . e predominance of infection in males was also observed in another study pwefromed in KSA ., which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females .", "is also complements the findings reported by of Da'ar and Ahmed in their paper . e predominance of infection in males was also observed in another study pwefromed in KSA ., which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females . It is worth mentioning that Saudi Arabia defines age categories differently from the WHO children: 0-14, adult: otherwise . However, unlike the classification used in Saudi Arabia, we have followed the WHO categorization of age to differentiate between children/adolescents 0 to 19 years and adults 20 years and older as indicated in WHO reports for age-standardized population and in infectious diseases . is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points age of 41 and age of 60 .", "is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points age of 41 and age of 60 . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of MERS-CoV were reported among people aged 40 and above . In 2016, only 9 of 552 cases 1.6% of MERS-CoV infection were found among pediatric patients. Moreover, the study which was conducted in King Fahad Medical City in Riyadh KFMC between January 2012 and December 2013 did not report any MERS-CoV cases among children . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al.", "Moreover, the study which was conducted in King Fahad Medical City in Riyadh KFMC between January 2012 and December 2013 did not report any MERS-CoV cases among children . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al. between 2012 and 2016 suggests that the prevalence and distribution of MERS-CoV were the highest-risk in elderly aged 60 years or above . Similar to our results, this study also reported the highest number of confirmed cases during the summer season . Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers .", "Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers . Similarly, other studies also reported a lower prevalence in healthcare workers . Our data reported a higher prevalence of infection among Saudi nationals as compared with non-Saudi. Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only.", "Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only. In our study, we detected a low prevalence 0.5% . e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab.", "e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. In fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. To the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase AST .", "However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase AST . However, further investigations are needed to confirm our findings. One of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of MERS-CoV in the Riyadh and Al-Qassim regions. Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases.", "Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. In addition to this, quality assurance policies are implemented at HESN in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas two major regions in KSA .", "Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas two major regions in KSA . Given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. It must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed MERS-CoV with those with suspected MERS-CoV who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive.", "is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting MERS-CoV signs with a negative lab result may need retesting. Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific.", "Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. Future studies should aim to confirm such findings in other regions in Saudi Arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request.", "e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests." ]

[ "Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them.", "However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 0.5% were confirmed by laboratory results. These confirmed cases 67.2% of which were males had a mean age of 43.23 years SD ± 22.8 . Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer.", "These confirmed cases 67.2% of which were males had a mean age of 43.23 years SD ± 22.8 . Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus MERS-CoV was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure .", "Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus MERS-CoV was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure . Since then, 27 countries have reported the presence of this virus, including the 12 countries of the Eastern Mediterranean region. Several outbreaks have occurred in multiple countries including Saudi Arabia, the United Arab Emirates and the Republic of Korea . Recent fatality rate CFR of 21% . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population .", "Recent fatality rate CFR of 21% . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population . e literature shows that MERS-CoV infects males more than females . e casefatality rate of men 52% is higher than that of women 23% . Males with a history of serious medical conditions are highly susceptible to this infection. Moreover, the mean age of infection in adults is 60 years . e mode of transmission is not entirely understood yet ; however, human-to-human and zoonotic sources of transmission have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans.", "e mode of transmission is not entirely understood yet ; however, human-to-human and zoonotic sources of transmission have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans. Interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings . Clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death . Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings . Studies have suggested an incubation period of 16 days with a mean of 5-6 days , while the median time until death is 11-13 days range 5-27 days among severely ill patients .", "Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings . Studies have suggested an incubation period of 16 days with a mean of 5-6 days , while the median time until death is 11-13 days range 5-27 days among severely ill patients . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction rRT-PCR assays . ere is no specific treatment for MERS-CoV. Like most viral infections, the treatment options are supportive and symptomatic . At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness .", "At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness . Such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. One must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided .", "Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided . Many studies have been conducted in recent years in Saudi Arabia to combat this deadly disease. A large multicentre study showed that it is nearly impossible to differentiate between patients of MERS-CoV and non-MERS-CoV just on the basis of clinical presentation . Another cohort study, which was hospital-based 17 cases vs. 82 controls , found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific .", "Another cohort study, which was hospital-based 17 cases vs. 82 controls , found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific . Physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing which usually takes between 12 and 24 hours . Identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers . Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period.", "Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. Both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained . Identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV.", "e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV. e nature of these markers has not been investigated in Saudi Arabia, and hence this study aims to identify them. A cross-sectional study was conducted at the regional laboratory and blood bank, located at Shumaisi Hospital in Riyadh, KSA. e laboratory has received the Central Blood Banks and Reference Laboratories Accreditation Program Saudi Central Board for Accreditation of Healthcare Institution CBAHI 2018 . Technique. Data were collected during the period of January 2017 to December 2017.", "Technique. Data were collected during the period of January 2017 to December 2017. All patients in Riyadh and Al-Qassim regions who had their samples tested at Riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. For the descriptive aim, we estimated the prevalence of MERS-CoV. For the analytical aim, a binary logistic regression model was developed. In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status yes/no , hospitals, and area of residence. Data were cross-checked with a labcomputerized database.", "In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status yes/no , hospitals, and area of residence. Data were cross-checked with a labcomputerized database. Further data were collected on demographic characteristics age and sex , underlying nationality, and health care status. We collected data from 25,400 cases, of which 23,646 suspected cases of MERS-CoV were included in the final analysis. Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics medians, interquartile ranges, minimum, and maximum were used to describe the distribution.", "Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics medians, interquartile ranges, minimum, and maximum were used to describe the distribution. A logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. At first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered.", "is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. A confirmed case is defined as a suspected case with laboratory confirmation of MERS-CoV infection . A total of 23,646 of MERS-CoV suspected cases were included in this study, of which 52.3% were males n � 12376 and 47.7% were females n � 11270 . e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032 , whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period .. A total of 23,646 suspected cases were included in the results.", "e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032 , whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period .. A total of 23,646 suspected cases were included in the results. Of the total suspected cases, 119 cases had been confirmed via laboratory results. All the confirmed cases are reported to MOH through HESN health electronic surveillance networks and to the World Health Organization WHO through the International Health Regulations IHR , National Focal Point of Saudi Arabia. We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases.", "We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. During the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of MERS-CoV infection cases has decreased in Riyadh and Al-Qassim regions in comparison to that of the last three years. From 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of Da'ar and Ahmed in their paper . e predominance of infection in males was also observed in another study pwefromed in KSA ., which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females .", "is also complements the findings reported by of Da'ar and Ahmed in their paper . e predominance of infection in males was also observed in another study pwefromed in KSA ., which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females . It is worth mentioning that Saudi Arabia defines age categories differently from the WHO children: 0-14, adult: otherwise . However, unlike the classification used in Saudi Arabia, we have followed the WHO categorization of age to differentiate between children/adolescents 0 to 19 years and adults 20 years and older as indicated in WHO reports for age-standardized population and in infectious diseases . is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points age of 41 and age of 60 .", "is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points age of 41 and age of 60 . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of MERS-CoV were reported among people aged 40 and above . In 2016, only 9 of 552 cases 1.6% of MERS-CoV infection were found among pediatric patients. Moreover, the study which was conducted in King Fahad Medical City in Riyadh KFMC between January 2012 and December 2013 did not report any MERS-CoV cases among children . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al.", "Moreover, the study which was conducted in King Fahad Medical City in Riyadh KFMC between January 2012 and December 2013 did not report any MERS-CoV cases among children . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al. between 2012 and 2016 suggests that the prevalence and distribution of MERS-CoV were the highest-risk in elderly aged 60 years or above . Similar to our results, this study also reported the highest number of confirmed cases during the summer season . Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers .", "Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers . Similarly, other studies also reported a lower prevalence in healthcare workers . Our data reported a higher prevalence of infection among Saudi nationals as compared with non-Saudi. Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only.", "Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only. In our study, we detected a low prevalence 0.5% . e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab.", "e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. In fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. To the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase AST .", "However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase AST . However, further investigations are needed to confirm our findings. One of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of MERS-CoV in the Riyadh and Al-Qassim regions. Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases.", "Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. In addition to this, quality assurance policies are implemented at HESN in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas two major regions in KSA .", "Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas two major regions in KSA . Given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. It must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed MERS-CoV with those with suspected MERS-CoV who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive.", "is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting MERS-CoV signs with a negative lab result may need retesting. Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific.", "Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. Future studies should aim to confirm such findings in other regions in Saudi Arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request.", "e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests." ]

[ "Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them.", "However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 0.5% were confirmed by laboratory results. These confirmed cases 67.2% of which were males had a mean age of 43.23 years SD ± 22.8 . Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer.", "These confirmed cases 67.2% of which were males had a mean age of 43.23 years SD ± 22.8 . Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus MERS-CoV was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure .", "Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors. Text: A respiratory viral disease caused by the Middle East Respiratory Syndrome CoronaVirus MERS-CoV was first isolated in 2012, in a 60-year-old man who died in Jeddah, KSA due to severe acute pneumonia and multiple organ failure . Since then, 27 countries have reported the presence of this virus, including the 12 countries of the Eastern Mediterranean region. Several outbreaks have occurred in multiple countries including Saudi Arabia, the United Arab Emirates and the Republic of Korea . Recent fatality rate CFR of 21% . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population .", "Recent fatality rate CFR of 21% . Very limited evidence is available for exploring the epidemiology of this virus among the pediatric population . e literature shows that MERS-CoV infects males more than females . e casefatality rate of men 52% is higher than that of women 23% . Males with a history of serious medical conditions are highly susceptible to this infection. Moreover, the mean age of infection in adults is 60 years . e mode of transmission is not entirely understood yet ; however, human-to-human and zoonotic sources of transmission have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans.", "e mode of transmission is not entirely understood yet ; however, human-to-human and zoonotic sources of transmission have been documented in many studies. Dromedary camels are the major animal source of MERS-CoV transmission to humans. Interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings . Clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death . Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings . Studies have suggested an incubation period of 16 days with a mean of 5-6 days , while the median time until death is 11-13 days range 5-27 days among severely ill patients .", "Severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in ICUs across different healthcare settings . Studies have suggested an incubation period of 16 days with a mean of 5-6 days , while the median time until death is 11-13 days range 5-27 days among severely ill patients . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction rRT-PCR assays . ere is no specific treatment for MERS-CoV. Like most viral infections, the treatment options are supportive and symptomatic . At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness .", "At present, no vaccine exists for preventing the infections of MERS-CoV. e CDC indicated that preventative actions should be taken for any type of respiratory illness . Such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. One must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided .", "Repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. Intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided . Many studies have been conducted in recent years in Saudi Arabia to combat this deadly disease. A large multicentre study showed that it is nearly impossible to differentiate between patients of MERS-CoV and non-MERS-CoV just on the basis of clinical presentation . Another cohort study, which was hospital-based 17 cases vs. 82 controls , found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific .", "Another cohort study, which was hospital-based 17 cases vs. 82 controls , found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations . Similarly, two more single-centre case control studies reported that the presenting symptoms of MERS-CoV infection were not specific . Physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing which usually takes between 12 and 24 hours . Identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers . Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period.", "Mild symptomatic cases, which result in a positive PCR, may be isolated at home. Severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. Both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained . Identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV.", "e challenge is to flag these patients by some demographic markers, as the clinical presentation of MERS-CoV infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of MERS-CoV. e nature of these markers has not been investigated in Saudi Arabia, and hence this study aims to identify them. A cross-sectional study was conducted at the regional laboratory and blood bank, located at Shumaisi Hospital in Riyadh, KSA. e laboratory has received the Central Blood Banks and Reference Laboratories Accreditation Program Saudi Central Board for Accreditation of Healthcare Institution CBAHI 2018 . Technique. Data were collected during the period of January 2017 to December 2017.", "Technique. Data were collected during the period of January 2017 to December 2017. All patients in Riyadh and Al-Qassim regions who had their samples tested at Riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. For the descriptive aim, we estimated the prevalence of MERS-CoV. For the analytical aim, a binary logistic regression model was developed. In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status yes/no , hospitals, and area of residence. Data were cross-checked with a labcomputerized database.", "In this model, we included the risk factors of gender, age, seasons, nationality, healthcare status yes/no , hospitals, and area of residence. Data were cross-checked with a labcomputerized database. Further data were collected on demographic characteristics age and sex , underlying nationality, and health care status. We collected data from 25,400 cases, of which 23,646 suspected cases of MERS-CoV were included in the final analysis. Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics medians, interquartile ranges, minimum, and maximum were used to describe the distribution.", "Data were cleaned, entered, stored, and managed with an excel database and IBM SPSS Version 25. e statistical analyses consisted of descriptive counts and percentages. For those continuously scaled items, nonparametric statistics medians, interquartile ranges, minimum, and maximum were used to describe the distribution. A logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. At first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered.", "is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. To determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. A confirmed case is defined as a suspected case with laboratory confirmation of MERS-CoV infection . A total of 23,646 of MERS-CoV suspected cases were included in this study, of which 52.3% were males n � 12376 and 47.7% were females n � 11270 . e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032 , whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period .. A total of 23,646 suspected cases were included in the results.", "e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of MERS-CoV remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold A.OR: 3.11, 95% CI: 1.104-8.76, P value � 0.032 , whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of MERS-CoV infection laboratory-based data was conducted in Riyadh over a one-year period .. A total of 23,646 suspected cases were included in the results. Of the total suspected cases, 119 cases had been confirmed via laboratory results. All the confirmed cases are reported to MOH through HESN health electronic surveillance networks and to the World Health Organization WHO through the International Health Regulations IHR , National Focal Point of Saudi Arabia. We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases.", "We found that MERS-CoV infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. During the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of MERS-CoV infection cases has decreased in Riyadh and Al-Qassim regions in comparison to that of the last three years. From 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of Da'ar and Ahmed in their paper . e predominance of infection in males was also observed in another study pwefromed in KSA ., which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females .", "is also complements the findings reported by of Da'ar and Ahmed in their paper . e predominance of infection in males was also observed in another study pwefromed in KSA ., which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females . It is worth mentioning that Saudi Arabia defines age categories differently from the WHO children: 0-14, adult: otherwise . However, unlike the classification used in Saudi Arabia, we have followed the WHO categorization of age to differentiate between children/adolescents 0 to 19 years and adults 20 years and older as indicated in WHO reports for age-standardized population and in infectious diseases . is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points age of 41 and age of 60 .", "is categorization was also followed by Aly and his collaborators in their recent paper published in 2017 . Adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points age of 41 and age of 60 . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of MERS-CoV were reported among people aged 40 and above . In 2016, only 9 of 552 cases 1.6% of MERS-CoV infection were found among pediatric patients. Moreover, the study which was conducted in King Fahad Medical City in Riyadh KFMC between January 2012 and December 2013 did not report any MERS-CoV cases among children . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al.", "Moreover, the study which was conducted in King Fahad Medical City in Riyadh KFMC between January 2012 and December 2013 did not report any MERS-CoV cases among children . e study which was conducted across the Gulf countries for four years by Mahmoud Aly et al. between 2012 and 2016 suggests that the prevalence and distribution of MERS-CoV were the highest-risk in elderly aged 60 years or above . Similar to our results, this study also reported the highest number of confirmed cases during the summer season . Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers .", "Among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by Ahmad to estimate the survival rate in MERS-CoV globally prior to 26 January 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers . Similarly, other studies also reported a lower prevalence in healthcare workers . Our data reported a higher prevalence of infection among Saudi nationals as compared with non-Saudi. Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only.", "Another study also showed similar results but with a much higher percentage among Saudis, which may be due to the fact that it included Saudis from all regions . ere is no finding basis for comparison as such, because our study was focused on the Riyadh and Al Qassim regions only. In our study, we detected a low prevalence 0.5% . e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab.", "e estimated analytical sensitivity and specificity of the Real Star kit from Altona was reported to be 100% with no cross reactivity with other respiratory pathogens . Moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. In fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. To the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase AST .", "However, it is worth mentioning that Ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase AST . However, further investigations are needed to confirm our findings. One of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of MERS-CoV in the Riyadh and Al-Qassim regions. Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases.", "Secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in Saudi Arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. In addition to this, quality assurance policies are implemented at HESN in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas two major regions in KSA .", "Variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. According to our knowledge, this is one of the few studies that have specifically investigated MERS-CoV risk factors in the Riyadh and Al-Qassim areas two major regions in KSA . Given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. It must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed MERS-CoV with those with suspected MERS-CoV who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive.", "is might be the reason why we have found some significant demographic factors unlike other reports. In conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting MERS-CoV signs with a negative lab result may need retesting. Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific.", "Our study covered two main regions in Saudi Arabia and provides evidence that the MERS-CoV epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. Our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. Future studies should aim to confirm such findings in other regions in Saudi Arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request.", "e laboratory data used to support the findings of this study were provided by Riyadh Regional Laboratory under license and are not freely available. However, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests." ]

[ "Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos t ide synthesis pathways, resulting in the depletion or imbalance of d NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos t ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs.", "The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. Text: Nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular DNA/RNA polymerases . More recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α .", "Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α . Importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. The primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 .", "Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools.", "There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated.", "Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors.", "In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways .", "The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity.", "A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group.", "They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers .", "Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 .", "MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV .", "Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively .", "To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively . More recently, gemcitabine was shown to effectively suppress ZIKV infection and replication in human retinal pigment epithelium RPE cells, particularly at non-cytotoxic concentrations EC 50 of 0.01 µM vs. CC 50 of > 10 µM . ZIKV, a member of the Flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency .", "Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency . In a follow up study, Clouser et al. further reported the antiviral effect of gemcitabine against HIV-related retrovirus, murine leukemia virus MuLV , in vitro EC 50 of 1.6 nM and even in murine AIDS model . A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM .", "A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM . They also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on Sindbis virus and herpes simplex virus-1 HSV-1 >2 log reduction in virus titer but relatively weak effects on Semliki forest virus and human echovirus 6, and minimal effects on Bunyamwera virus, measles virus MeV , and vaccinia virus . The antiviral effect of gemcitabine on EVs, initially performed on Coxsackievirus B3 CVB3 , was found from screening FDA-approved drugs in CVB3 replicon-harboring Vero cells by our group EC 50 of 0.4 µM . Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model .", "Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model . In this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and the number of lung infiltrating lymphocytes. More recently, Zhang et al. also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases.", "also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases. Moreover, it is possible that gemcitabine is effective for other untested RNA viruses. Because gemcitabine is a deoxycytidine analog that interferes with DNA as well as RNA synthesis, DNA viruses may not be the exception. Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments.", "Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. This is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as MERS-CoV, SARS-CoV, and ZIKV, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. In this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents.", "Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. In this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. As an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, was reported against EVs such as CVB3 and EV71 . As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials.", "As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. Even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models . More extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis .", "Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis . Moreover, gemcitabine strongly induced the expression of several ISGs including CXCL10, IRF7, IRF9, IFIT1, and DDX58, which were the major effectors in the innate immunity that defended the host against the virus infection. These results were consistent with a previous report that gemcitabine stimulated the production of IFN-β and IFN-γ in IAV-infected RPE cells . Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, .", "Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, . Regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid MPA are inhibitors of inosine-5 -monophosphate IMP dehydrogenase IMPDH , which is a key enzyme of the purine biosynthesis pathway. These inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses .", "Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses . ISG activation occurred through an undefined mechanism that was different from the classical IFN signaling, intracellular dsRNA sensing pathway, Toll-like receptor and nuclear factor B pathways. More importantly, ribavirin-induced ISG activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting IMPDH inhibition-mediated ISG activation as an alternative innate immunity pathway. Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin .", "Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin . Similar results were obtained with several IMPDH1 or IMPDH2 inhibitors, with various affinities, that were custom-designed and synthesized . As shown in Table 2 , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase DHODH , an essential enzyme in de novo pyrimidine synthesis. Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV .", "Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV . DD264 enhanced the expression of several ISGs, which were almost completely suppressed by the addition of supplemented uridine, indicating DHODH inhibition-mediated ISG activation. Moreover, the antiviral activity of and ISG activation by DD264 required the interferon regulatory factor 1 IRF1 transcription factor, a master regulator of antiviral gene expression , which was consistent with the observation that the anti-HCV activity of MPA was partially mediated by IRF1 . In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 .", "In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 . However, whether ISG activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. The mechanism of nucleotide synthesis inhibitor-induced ISG activation is still presently unclear. Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al.", "Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al. clearly showed that ISG activation and anti-HEV activity induced by MPA or brequinar was not mediated by JAK . Second, IRF7 induction by ribavirin was not affected by knockdown of STAT1, while that of IFN-α was strongly affected under the same conditions . Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine .", "Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine . Moreover, the upregulation of DDX58 mRNAs induced by gemcitabine was not affected by IRF9 knockdown, which was contrary to the result that IFN-α-induced upregulation of DDX58 mRNAs was significantly suppressed under the same conditions. Consistent with above observations, there have been some reports that ISGs was induced in the absence of JAK1 or STAT1 activation . Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool.", "Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool. Inactivation of metabolic enzyme s itself or consequently altered nucleos t ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of ISGs, possibly through the relay of kinases and transcription factors. Based on the previously mentioned reports, this signal is less likely to be dependent on STAT1/2-IRF9 IFN-stimulated gene factor 3; ISGF3 , at least for gemcitabine, which is the major transcriptional complex in the IFN-induced JAK/STAT pathway. It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin .", "It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin . Despite the consensus of ISG activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of ISGs, at least with different patterns . Targeting an enzyme in which pathways purine or pyrimidine synthesis or steps early/late and de novo/salvage produce different levels of intermediates and nucleos t ides will consequently result in diverse outcomes of ISG activations. There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation.", "There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation. Systematic analyses of signaling kinases, IRFs, and STATs using siRNA knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos t ides should therefore clarify the underlying molecular mechanisms of ISG activation by purine/pyrimidine biosynthesis inhibitors. As newly emerging or re-emerged viruses such as SARS-CoV, MERS-CoV, and ZIKV have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs.", "In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. Nucleoside analogs probably induce different subsets of ISGs, at least with a different pattern, leading to various combinations of ISGs and resulting antiviral outcomes. Moreover, according to Schoggins et al., different viruses are affected by distinct subsets of ISGs and some ISGs such as IRF1, MB21D1, HPSE, DDX58, MDA, and IFITM3 act broadly on various viruses . Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives.", "Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives. Nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. Careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered." ]

[ "Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos t ide synthesis pathways, resulting in the depletion or imbalance of d NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos t ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs.", "The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. Text: Nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular DNA/RNA polymerases . More recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α .", "Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α . Importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. The primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 .", "Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools.", "There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated.", "Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors.", "In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways .", "The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity.", "A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group.", "They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers .", "Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 .", "MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV .", "Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively .", "To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively . More recently, gemcitabine was shown to effectively suppress ZIKV infection and replication in human retinal pigment epithelium RPE cells, particularly at non-cytotoxic concentrations EC 50 of 0.01 µM vs. CC 50 of > 10 µM . ZIKV, a member of the Flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency .", "Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency . In a follow up study, Clouser et al. further reported the antiviral effect of gemcitabine against HIV-related retrovirus, murine leukemia virus MuLV , in vitro EC 50 of 1.6 nM and even in murine AIDS model . A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM .", "A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM . They also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on Sindbis virus and herpes simplex virus-1 HSV-1 >2 log reduction in virus titer but relatively weak effects on Semliki forest virus and human echovirus 6, and minimal effects on Bunyamwera virus, measles virus MeV , and vaccinia virus . The antiviral effect of gemcitabine on EVs, initially performed on Coxsackievirus B3 CVB3 , was found from screening FDA-approved drugs in CVB3 replicon-harboring Vero cells by our group EC 50 of 0.4 µM . Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model .", "Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model . In this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and the number of lung infiltrating lymphocytes. More recently, Zhang et al. also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases.", "also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases. Moreover, it is possible that gemcitabine is effective for other untested RNA viruses. Because gemcitabine is a deoxycytidine analog that interferes with DNA as well as RNA synthesis, DNA viruses may not be the exception. Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments.", "Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. This is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as MERS-CoV, SARS-CoV, and ZIKV, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. In this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents.", "Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. In this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. As an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, was reported against EVs such as CVB3 and EV71 . As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials.", "As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. Even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models . More extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis .", "Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis . Moreover, gemcitabine strongly induced the expression of several ISGs including CXCL10, IRF7, IRF9, IFIT1, and DDX58, which were the major effectors in the innate immunity that defended the host against the virus infection. These results were consistent with a previous report that gemcitabine stimulated the production of IFN-β and IFN-γ in IAV-infected RPE cells . Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, .", "Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, . Regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid MPA are inhibitors of inosine-5 -monophosphate IMP dehydrogenase IMPDH , which is a key enzyme of the purine biosynthesis pathway. These inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses .", "Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses . ISG activation occurred through an undefined mechanism that was different from the classical IFN signaling, intracellular dsRNA sensing pathway, Toll-like receptor and nuclear factor B pathways. More importantly, ribavirin-induced ISG activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting IMPDH inhibition-mediated ISG activation as an alternative innate immunity pathway. Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin .", "Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin . Similar results were obtained with several IMPDH1 or IMPDH2 inhibitors, with various affinities, that were custom-designed and synthesized . As shown in Table 2 , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase DHODH , an essential enzyme in de novo pyrimidine synthesis. Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV .", "Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV . DD264 enhanced the expression of several ISGs, which were almost completely suppressed by the addition of supplemented uridine, indicating DHODH inhibition-mediated ISG activation. Moreover, the antiviral activity of and ISG activation by DD264 required the interferon regulatory factor 1 IRF1 transcription factor, a master regulator of antiviral gene expression , which was consistent with the observation that the anti-HCV activity of MPA was partially mediated by IRF1 . In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 .", "In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 . However, whether ISG activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. The mechanism of nucleotide synthesis inhibitor-induced ISG activation is still presently unclear. Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al.", "Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al. clearly showed that ISG activation and anti-HEV activity induced by MPA or brequinar was not mediated by JAK . Second, IRF7 induction by ribavirin was not affected by knockdown of STAT1, while that of IFN-α was strongly affected under the same conditions . Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine .", "Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine . Moreover, the upregulation of DDX58 mRNAs induced by gemcitabine was not affected by IRF9 knockdown, which was contrary to the result that IFN-α-induced upregulation of DDX58 mRNAs was significantly suppressed under the same conditions. Consistent with above observations, there have been some reports that ISGs was induced in the absence of JAK1 or STAT1 activation . Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool.", "Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool. Inactivation of metabolic enzyme s itself or consequently altered nucleos t ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of ISGs, possibly through the relay of kinases and transcription factors. Based on the previously mentioned reports, this signal is less likely to be dependent on STAT1/2-IRF9 IFN-stimulated gene factor 3; ISGF3 , at least for gemcitabine, which is the major transcriptional complex in the IFN-induced JAK/STAT pathway. It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin .", "It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin . Despite the consensus of ISG activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of ISGs, at least with different patterns . Targeting an enzyme in which pathways purine or pyrimidine synthesis or steps early/late and de novo/salvage produce different levels of intermediates and nucleos t ides will consequently result in diverse outcomes of ISG activations. There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation.", "There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation. Systematic analyses of signaling kinases, IRFs, and STATs using siRNA knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos t ides should therefore clarify the underlying molecular mechanisms of ISG activation by purine/pyrimidine biosynthesis inhibitors. As newly emerging or re-emerged viruses such as SARS-CoV, MERS-CoV, and ZIKV have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs.", "In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. Nucleoside analogs probably induce different subsets of ISGs, at least with a different pattern, leading to various combinations of ISGs and resulting antiviral outcomes. Moreover, according to Schoggins et al., different viruses are affected by distinct subsets of ISGs and some ISGs such as IRF1, MB21D1, HPSE, DDX58, MDA, and IFITM3 act broadly on various viruses . Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives.", "Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives. Nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. Careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered." ]

[ "Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos t ide synthesis pathways, resulting in the depletion or imbalance of d NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos t ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs.", "The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. Text: Nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular DNA/RNA polymerases . More recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α .", "Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α . Importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. The primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 .", "Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools.", "There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated.", "Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors.", "In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways .", "The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity.", "A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group.", "They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers .", "Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 .", "MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV .", "Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively .", "To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively . More recently, gemcitabine was shown to effectively suppress ZIKV infection and replication in human retinal pigment epithelium RPE cells, particularly at non-cytotoxic concentrations EC 50 of 0.01 µM vs. CC 50 of > 10 µM . ZIKV, a member of the Flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency .", "Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency . In a follow up study, Clouser et al. further reported the antiviral effect of gemcitabine against HIV-related retrovirus, murine leukemia virus MuLV , in vitro EC 50 of 1.6 nM and even in murine AIDS model . A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM .", "A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM . They also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on Sindbis virus and herpes simplex virus-1 HSV-1 >2 log reduction in virus titer but relatively weak effects on Semliki forest virus and human echovirus 6, and minimal effects on Bunyamwera virus, measles virus MeV , and vaccinia virus . The antiviral effect of gemcitabine on EVs, initially performed on Coxsackievirus B3 CVB3 , was found from screening FDA-approved drugs in CVB3 replicon-harboring Vero cells by our group EC 50 of 0.4 µM . Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model .", "Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model . In this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and the number of lung infiltrating lymphocytes. More recently, Zhang et al. also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases.", "also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases. Moreover, it is possible that gemcitabine is effective for other untested RNA viruses. Because gemcitabine is a deoxycytidine analog that interferes with DNA as well as RNA synthesis, DNA viruses may not be the exception. Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments.", "Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. This is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as MERS-CoV, SARS-CoV, and ZIKV, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. In this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents.", "Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. In this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. As an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, was reported against EVs such as CVB3 and EV71 . As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials.", "As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. Even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models . More extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis .", "Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis . Moreover, gemcitabine strongly induced the expression of several ISGs including CXCL10, IRF7, IRF9, IFIT1, and DDX58, which were the major effectors in the innate immunity that defended the host against the virus infection. These results were consistent with a previous report that gemcitabine stimulated the production of IFN-β and IFN-γ in IAV-infected RPE cells . Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, .", "Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, . Regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid MPA are inhibitors of inosine-5 -monophosphate IMP dehydrogenase IMPDH , which is a key enzyme of the purine biosynthesis pathway. These inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses .", "Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses . ISG activation occurred through an undefined mechanism that was different from the classical IFN signaling, intracellular dsRNA sensing pathway, Toll-like receptor and nuclear factor B pathways. More importantly, ribavirin-induced ISG activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting IMPDH inhibition-mediated ISG activation as an alternative innate immunity pathway. Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin .", "Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin . Similar results were obtained with several IMPDH1 or IMPDH2 inhibitors, with various affinities, that were custom-designed and synthesized . As shown in Table 2 , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase DHODH , an essential enzyme in de novo pyrimidine synthesis. Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV .", "Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV . DD264 enhanced the expression of several ISGs, which were almost completely suppressed by the addition of supplemented uridine, indicating DHODH inhibition-mediated ISG activation. Moreover, the antiviral activity of and ISG activation by DD264 required the interferon regulatory factor 1 IRF1 transcription factor, a master regulator of antiviral gene expression , which was consistent with the observation that the anti-HCV activity of MPA was partially mediated by IRF1 . In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 .", "In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 . However, whether ISG activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. The mechanism of nucleotide synthesis inhibitor-induced ISG activation is still presently unclear. Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al.", "Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al. clearly showed that ISG activation and anti-HEV activity induced by MPA or brequinar was not mediated by JAK . Second, IRF7 induction by ribavirin was not affected by knockdown of STAT1, while that of IFN-α was strongly affected under the same conditions . Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine .", "Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine . Moreover, the upregulation of DDX58 mRNAs induced by gemcitabine was not affected by IRF9 knockdown, which was contrary to the result that IFN-α-induced upregulation of DDX58 mRNAs was significantly suppressed under the same conditions. Consistent with above observations, there have been some reports that ISGs was induced in the absence of JAK1 or STAT1 activation . Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool.", "Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool. Inactivation of metabolic enzyme s itself or consequently altered nucleos t ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of ISGs, possibly through the relay of kinases and transcription factors. Based on the previously mentioned reports, this signal is less likely to be dependent on STAT1/2-IRF9 IFN-stimulated gene factor 3; ISGF3 , at least for gemcitabine, which is the major transcriptional complex in the IFN-induced JAK/STAT pathway. It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin .", "It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin . Despite the consensus of ISG activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of ISGs, at least with different patterns . Targeting an enzyme in which pathways purine or pyrimidine synthesis or steps early/late and de novo/salvage produce different levels of intermediates and nucleos t ides will consequently result in diverse outcomes of ISG activations. There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation.", "There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation. Systematic analyses of signaling kinases, IRFs, and STATs using siRNA knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos t ides should therefore clarify the underlying molecular mechanisms of ISG activation by purine/pyrimidine biosynthesis inhibitors. As newly emerging or re-emerged viruses such as SARS-CoV, MERS-CoV, and ZIKV have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs.", "In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. Nucleoside analogs probably induce different subsets of ISGs, at least with a different pattern, leading to various combinations of ISGs and resulting antiviral outcomes. Moreover, according to Schoggins et al., different viruses are affected by distinct subsets of ISGs and some ISGs such as IRF1, MB21D1, HPSE, DDX58, MDA, and IFITM3 act broadly on various viruses . Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives.", "Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives. Nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. Careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered." ]

[ "Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos t ide synthesis pathways, resulting in the depletion or imbalance of d NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos t ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs.", "The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos t ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. Text: Nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular DNA/RNA polymerases . More recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α .", "Some nucleoside analog drugs targeting specific viral polymerases acyclovir for herpesviruses, zidovudine for human immunodeficiency virus HIV , and sofosbuvir for hepatitis C virus HCV have been successful in clinical trials and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α . Importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. The primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 .", "Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools.", "There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated.", "Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors.", "In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations Figure 1 . Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways .", "The other mechanism is based on the modulation of cellular nucleos t ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos t ide synthesis pathways . By targeting metabolic enzymes s , nucleoside analogs block the natural flow of nucleos t ide synthesis and consequently cause the depletion or imbalance of d NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity.", "A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes ISGs . Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group.", "They have been well-summarized in a previous report . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers .", "Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 .", "MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 . The mechanism of antiviral effect of nucleos t ide analogs. Nucleos t ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV .", "Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus MERS-CoV , severe acute respiratory syndrome coronavirus SARS-CoV , Zika virus ZIKV , HCV, poliovirus PV , influenza A virus IAV , HIV, and enteroviruses EV . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively .", "To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV EC 50 of 1.2 µM and 4.9 µM, respectively . More recently, gemcitabine was shown to effectively suppress ZIKV infection and replication in human retinal pigment epithelium RPE cells, particularly at non-cytotoxic concentrations EC 50 of 0.01 µM vs. CC 50 of > 10 µM . ZIKV, a member of the Flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency .", "Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively , which were lower concentrations than those used in cancer therapy . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency . In a follow up study, Clouser et al. further reported the antiviral effect of gemcitabine against HIV-related retrovirus, murine leukemia virus MuLV , in vitro EC 50 of 1.6 nM and even in murine AIDS model . A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM .", "A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. EC 50 of 0.068 µM . They also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on Sindbis virus and herpes simplex virus-1 HSV-1 >2 log reduction in virus titer but relatively weak effects on Semliki forest virus and human echovirus 6, and minimal effects on Bunyamwera virus, measles virus MeV , and vaccinia virus . The antiviral effect of gemcitabine on EVs, initially performed on Coxsackievirus B3 CVB3 , was found from screening FDA-approved drugs in CVB3 replicon-harboring Vero cells by our group EC 50 of 0.4 µM . Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model .", "Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 EV71 and human rhinoviruses HRVs EC 50 s of 1 and 1-5 µM, respectively . In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model . In this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and the number of lung infiltrating lymphocytes. More recently, Zhang et al. also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases.", "also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells EC 50 of 0.3 µM . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases. Moreover, it is possible that gemcitabine is effective for other untested RNA viruses. Because gemcitabine is a deoxycytidine analog that interferes with DNA as well as RNA synthesis, DNA viruses may not be the exception. Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments.", "Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. This is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as MERS-CoV, SARS-CoV, and ZIKV, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. In this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents.", "Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. In this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. As an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, was reported against EVs such as CVB3 and EV71 . As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials.", "As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. Even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models . More extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis .", "Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis . Moreover, gemcitabine strongly induced the expression of several ISGs including CXCL10, IRF7, IRF9, IFIT1, and DDX58, which were the major effectors in the innate immunity that defended the host against the virus infection. These results were consistent with a previous report that gemcitabine stimulated the production of IFN-β and IFN-γ in IAV-infected RPE cells . Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, .", "Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 6, 10, . Regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid MPA are inhibitors of inosine-5 -monophosphate IMP dehydrogenase IMPDH , which is a key enzyme of the purine biosynthesis pathway. These inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses .", "Both have antiviral activities against viruses such as HCV, hepatitis E virus HEV , MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus WNV , Chikungunya virus CHIKV , and IAV , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses . ISG activation occurred through an undefined mechanism that was different from the classical IFN signaling, intracellular dsRNA sensing pathway, Toll-like receptor and nuclear factor B pathways. More importantly, ribavirin-induced ISG activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting IMPDH inhibition-mediated ISG activation as an alternative innate immunity pathway. Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin .", "Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin . Similar results were obtained with several IMPDH1 or IMPDH2 inhibitors, with various affinities, that were custom-designed and synthesized . As shown in Table 2 , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase DHODH , an essential enzyme in de novo pyrimidine synthesis. Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV .", "Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element ISRE -stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV . DD264 enhanced the expression of several ISGs, which were almost completely suppressed by the addition of supplemented uridine, indicating DHODH inhibition-mediated ISG activation. Moreover, the antiviral activity of and ISG activation by DD264 required the interferon regulatory factor 1 IRF1 transcription factor, a master regulator of antiviral gene expression , which was consistent with the observation that the anti-HCV activity of MPA was partially mediated by IRF1 . In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 .", "In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 . However, whether ISG activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. The mechanism of nucleotide synthesis inhibitor-induced ISG activation is still presently unclear. Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al.", "Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal . First, Wang et al. clearly showed that ISG activation and anti-HEV activity induced by MPA or brequinar was not mediated by JAK . Second, IRF7 induction by ribavirin was not affected by knockdown of STAT1, while that of IFN-α was strongly affected under the same conditions . Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine .", "Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine . Moreover, the upregulation of DDX58 mRNAs induced by gemcitabine was not affected by IRF9 knockdown, which was contrary to the result that IFN-α-induced upregulation of DDX58 mRNAs was significantly suppressed under the same conditions. Consistent with above observations, there have been some reports that ISGs was induced in the absence of JAK1 or STAT1 activation . Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool.", "Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the d NTP pool. Inactivation of metabolic enzyme s itself or consequently altered nucleos t ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of ISGs, possibly through the relay of kinases and transcription factors. Based on the previously mentioned reports, this signal is less likely to be dependent on STAT1/2-IRF9 IFN-stimulated gene factor 3; ISGF3 , at least for gemcitabine, which is the major transcriptional complex in the IFN-induced JAK/STAT pathway. It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin .", "It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin . Despite the consensus of ISG activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of ISGs, at least with different patterns . Targeting an enzyme in which pathways purine or pyrimidine synthesis or steps early/late and de novo/salvage produce different levels of intermediates and nucleos t ides will consequently result in diverse outcomes of ISG activations. There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation.", "There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation. Systematic analyses of signaling kinases, IRFs, and STATs using siRNA knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos t ides should therefore clarify the underlying molecular mechanisms of ISG activation by purine/pyrimidine biosynthesis inhibitors. As newly emerging or re-emerged viruses such as SARS-CoV, MERS-CoV, and ZIKV have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs.", "In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. Nucleoside analogs probably induce different subsets of ISGs, at least with a different pattern, leading to various combinations of ISGs and resulting antiviral outcomes. Moreover, according to Schoggins et al., different viruses are affected by distinct subsets of ISGs and some ISGs such as IRF1, MB21D1, HPSE, DDX58, MDA, and IFITM3 act broadly on various viruses . Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives.", "Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives. Nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. Careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered." ]

[ "BACKGROUND: Rabbit haemorrhagic disease virus RHDV , as the pathogeny of Rabbit haemorrhagic disease, can cause a highly infectious and often fatal disease only affecting wild and domestic rabbits. Recent researches revealed that it, as one number of the Caliciviridae, has some specialties in its genome, its reproduction and so on. RESULTS: In this report, we firstly analyzed its genome and two open reading frameworks ORFs from this aspect of codon usage bias. Our researches indicated that mutation pressure rather than natural is the most important determinant in RHDV with high codon bias, and the codon usage bias is nearly contrary between ORF1 and ORF2, which is maybe one of factors regulating the expression of VP60 encoding by ORF1 and VP10 encoding by ORF2 . Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. CONCLUSIONS: We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV.", "Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. CONCLUSIONS: We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV. Text: Synonymous codons are not used randomly . The variation of codon usage among ORFs in different organisms is accounted by mutational pressure and translational selection as two main factors . Levels and causes of codon usage bias are available to understand viral evolution and the interplay between viruses and the immune response .", "The variation of codon usage among ORFs in different organisms is accounted by mutational pressure and translational selection as two main factors . Levels and causes of codon usage bias are available to understand viral evolution and the interplay between viruses and the immune response . Thus, many organisms such as bacteria, yeast, Drosophila, and mammals, have been studied in great detail up on codon usage bias and nucleotide composition . However, same researches in viruses, especially in animal viruses, have been less studied. It has been observed that codon usage bias in human RNA viruses is related to mutational pressure, G +C content, the segmented nature of the genome and the route of transmission of the virus . For some vertebrate DNA viruses, genome-wide mutational pressure is regarded as the main determinant of codon usage rather than natural selection for specific coding triplets .", "It has been observed that codon usage bias in human RNA viruses is related to mutational pressure, G +C content, the segmented nature of the genome and the route of transmission of the virus . For some vertebrate DNA viruses, genome-wide mutational pressure is regarded as the main determinant of codon usage rather than natural selection for specific coding triplets . Analysis of the bovine papillomavirus type 1 BPV1 late genes has revealed a relationship between codon usage and tRNA availability . In the mammalian papillomaviruses, it has been proposed that differences from the average codon usage frequencies in the host genome strongly influence both viral replication and gene expression . Codon usage may play a key role in regulating latent versus productive infection in Epstein-Barr virus . Recently, it was reported that codon usage is an important driving force in the evolution of astroviruses and small DNA viruses .", "Codon usage may play a key role in regulating latent versus productive infection in Epstein-Barr virus . Recently, it was reported that codon usage is an important driving force in the evolution of astroviruses and small DNA viruses . Clearly, studies of synonymous codon usage in viruses can reveal much about the molecular evolution of viruses or individual genes. Such information would be relevant in understanding the regulation of viral gene expression. Up to now, little codon usage analysis has been performed on Rabbit haemorrhagic disease virus RHDV , which is the pathogen causing Rabbit haemorrhagic disease RHD , also known as rabbit calicivirus disease RCD or viral haemorrhagic disease VHD , a highly infectious and often fatal disease that affects wild and domestic rabbits. Although the virus infects only rabbits, RHD continues to cause serious problems in different parts of the world.", "Up to now, little codon usage analysis has been performed on Rabbit haemorrhagic disease virus RHDV , which is the pathogen causing Rabbit haemorrhagic disease RHD , also known as rabbit calicivirus disease RCD or viral haemorrhagic disease VHD , a highly infectious and often fatal disease that affects wild and domestic rabbits. Although the virus infects only rabbits, RHD continues to cause serious problems in different parts of the world. RHDV is a single positive stranded RNA virus without envelope, which contains two open reading frames ORFs separately encoding a predicted polyprotein and a minor structural protein named VP10 . After the hydrolysis of self-coding 3C-like cysteinase, the polyprotein was finally hydrolyzed into 8 cleavage products including 7 nonstructural proteins and 1 structural protein named as VP60 . Studies on the phylogenetic relationship of RHDVs showed only one serotype had been isolated, and no genotyping for RHDV was reported. It reported that the VP10 was translated with an efficiency of 20% of the preceding ORF1 .", "Studies on the phylogenetic relationship of RHDVs showed only one serotype had been isolated, and no genotyping for RHDV was reported. It reported that the VP10 was translated with an efficiency of 20% of the preceding ORF1 . In order to better understand the characteristics of the RHDV genome and to reveal more information about the viral genome, we have analyzed the codon usage and dinucleotide composition. In this report, we sought to address the following issues concerning codon usage in RHDV: i the extent and causes of codon bias in RHDV; ii A possible genotyping of RHDV; iii Codon usage bias as a factor reducing the expression of VP10 and iiii the evolution of the ORFs. The 30 available complete RNA sequences of RHDV were obtained from GenBank randomly in January 2011. The serial number SN , collection dates, isolated areas and GenBank accession numbers are listed in Table 1 .", "The 30 available complete RNA sequences of RHDV were obtained from GenBank randomly in January 2011. The serial number SN , collection dates, isolated areas and GenBank accession numbers are listed in Table 1 . To investigate the characteristics of synonymous codon usage without the influence of amino acid composition, RSCU values of each codon in a ORF of RHDV were calculated according to previous reports 2 Sharp, Tuohy et al. 1986 as the followed formula: Where g ij is the observed number of the ith codon for jth amino acid which has n i type of synonymous codons. The codons with RSCU value higher than 1.0 have positive codon usage bias, while codons with value lower than 1.0 has relative negative codon usage bias. As RSCU values of some codons are nearly equal to 1.0, it means that these codons are chosen equally and randomly.", "The codons with RSCU value higher than 1.0 have positive codon usage bias, while codons with value lower than 1.0 has relative negative codon usage bias. As RSCU values of some codons are nearly equal to 1.0, it means that these codons are chosen equally and randomly. The index GC3s means the fraction of the nucleotides G+C at the synonymous third codon position, excluding Met, Trp, and the termination codons. The ENC, as the best estimator of absolute synonymous codon usage bias , was calculated for the quantification of the codon usage bias of each ORF . The predicted values of ENC were calculated as ENC = 2 + s + 29 where s represents the given G+C 3 % value. The values of ENC can also be obtained by EMBOSS CHIPS program .", "The predicted values of ENC were calculated as ENC = 2 + s + 29 where s represents the given G+C 3 % value. The values of ENC can also be obtained by EMBOSS CHIPS program . Analyses were conducted with the Nei-Gojobori model , involving 30 nucleotide sequences. All positions containing gaps and missing data were eliminated. The values of dn, ds and ω dn/ds were calculated in MEGA4.0 . Multivariate statistical analysis can be used to explore the relationships between variables and samples. In this study, correspondence analysis was used to investigate the major trend in codon usage variation among ORFs.", "Multivariate statistical analysis can be used to explore the relationships between variables and samples. In this study, correspondence analysis was used to investigate the major trend in codon usage variation among ORFs. In this study, the complete coding region of each ORF was represented as a 59 dimensional vector, and each dimension corresponds to the RSCU value of one sense codon excluding Met, Trp, and the termination codons . Correlation analysis was used to identify the relationship between nucleotide composition and synonymous codon usage pattern . This analysis was implemented based on the Spearman's rank correlation analysis way. All statistical processes were carried out by with statistical software SPSS 17.0 for windows.", "This analysis was implemented based on the Spearman's rank correlation analysis way. All statistical processes were carried out by with statistical software SPSS 17.0 for windows. The values of nucleotide contents in complete coding region of all 30 RHDV genomes were analyzed and listed in Table 2 and Table 3 . Evidently, C+G % content of the ORF1 fluctuated from 50.889 to 51.557 with a mean value of 51.14557, and C+G % content of the ORF2 were ranged from 35.593 to 40.113 with a mean value of 37.6624, which were indicating that nucleotides A and U were the major elements of ORF2 against ORF1. Comparing the values of A 3 %, U 3 %, C 3 % and G 3 %, it is clear that C 3 % was distinctly high and A 3 % was the lowest of all in ORF1 of RHDV, while U 3 % was distinctly high and C 3 % was the lowest of all in ORF2 of Table 2 Identified nucleotide contents in complete coding region length > 250 bps in the ORF1 of RHDV 30 isolates genome Table 4 . Most preferentially used codons in ORF1 were C-ended or G-ended codons except Ala, Pro and Ser, however, A-ended or G-ended codons were preferred as the content of ORF2.", "Comparing the values of A 3 %, U 3 %, C 3 % and G 3 %, it is clear that C 3 % was distinctly high and A 3 % was the lowest of all in ORF1 of RHDV, while U 3 % was distinctly high and C 3 % was the lowest of all in ORF2 of Table 2 Identified nucleotide contents in complete coding region length > 250 bps in the ORF1 of RHDV 30 isolates genome Table 4 . Most preferentially used codons in ORF1 were C-ended or G-ended codons except Ala, Pro and Ser, however, A-ended or G-ended codons were preferred as the content of ORF2. In addition, the dn, ds and ω dN/dS values of ORF1 were separately 0.014, 0.338 and 0.041, and the values of ORF2 were 0.034, 0.103 and 0.034, respectively. The ω values of two ORFs in RHDV genome are generally low, indicating that the RHDV whole genome is subject to relatively strong selective constraints. COA was used to investigate the major trend in codon usage variation between two ORFs of all 30 RHDV selected for this study. After COA for RHDV Genome, one major trend in the first axis f' 1 which accounted for 42.967% of the total variation, and another major trend in the second axis f' 2 which accounted for 3.632% of the total variation.", "COA was used to investigate the major trend in codon usage variation between two ORFs of all 30 RHDV selected for this study. After COA for RHDV Genome, one major trend in the first axis f' 1 which accounted for 42.967% of the total variation, and another major trend in the second axis f' 2 which accounted for 3.632% of the total variation. The coordinate of the complete coding region of each ORF was plotted in Figure 1 defining by the first and second principal axes. It is clear that coordinate of each ORF is relatively isolated. Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group marked as Group 1 is To estimate whether the evolution of RHDV genome on codon usage was regulated by mutation pressure or natural selection, the A%, U%, C%, G% and C+G % were compared with A 3 %, U 3 %, C 3 %, G 3 % and C 3 +G 3 %, respectively Table 5 . There is a complex correlation among nucleotide compositions.", "Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group marked as Group 1 is To estimate whether the evolution of RHDV genome on codon usage was regulated by mutation pressure or natural selection, the A%, U%, C%, G% and C+G % were compared with A 3 %, U 3 %, C 3 %, G 3 % and C 3 +G 3 %, respectively Table 5 . There is a complex correlation among nucleotide compositions. In detail, A 3 %, U 3 %, C 3 % and G 3 % have a significant negative correlation with G%, C%, U% and A% and positive correlation with A%, U%, C% and G%, respectively. It suggests that nucleotide constraint may influence synonymous codon usage patterns. However, A 3 % has non-correlation with U% and C%, and U 3 % has noncorrelation with A% and G%, respectively, which haven't indicated any peculiarity about synonymous codon usage. Furthermore, C 3 % and G 3 % have non-correlation with A%, G% and U%, C%, respectively, indicating these data don't reflect the true feature of synonymous codon usage as well.", "However, A 3 % has non-correlation with U% and C%, and U 3 % has noncorrelation with A% and G%, respectively, which haven't indicated any peculiarity about synonymous codon usage. Furthermore, C 3 % and G 3 % have non-correlation with A%, G% and U%, C%, respectively, indicating these data don't reflect the true feature of synonymous codon usage as well. Therefore, linear regression analysis was implemented to analyze the correlation between synonymous codon usage bias and nucleotide compositions. Details of correlation analysis between the first two principle axes f' 1 and f' 2 of each RHDV genome in COA and nucleotide contents were listed in Table 6 . In surprise, only f2 values are closely related to base nucleotide A and G content on the third codon position only, suggesting that nucleotide A and G is a factor influencing the synonymous codon usage pattern of RHDV genome. However, f' 1 value has non-correlation with base nucleotide contents on the third codon position; it is observably suggest that codon usage patterns in RHDV were probably influenced by other factors, such as the second structure of viral genome and limits of host.", "In surprise, only f2 values are closely related to base nucleotide A and G content on the third codon position only, suggesting that nucleotide A and G is a factor influencing the synonymous codon usage pattern of RHDV genome. However, f' 1 value has non-correlation with base nucleotide contents on the third codon position; it is observably suggest that codon usage patterns in RHDV were probably influenced by other factors, such as the second structure of viral genome and limits of host. In spite of that, compositional constraint is a factor shaping the pattern of synonymous codon usage in RHDV genome. Figure 1 A plot of value of the first and second axis of RHDV genome in COA. The first axis f' 1 accounts for 42.967% of the total variation, and the second axis f' 2 accounts for 3.632% of the total variation. Table 5 Summary of correlation analysis between the A, U, C, G contents and A 3 , U 3 , C 3 , G 3 contents in all selected samples There have been more and more features that are unique to RHDV within the family Caliciviridae, including its single host tropism, its genome and its VP10 as a structural protein with unknown function.", "The first axis f' 1 accounts for 42.967% of the total variation, and the second axis f' 2 accounts for 3.632% of the total variation. Table 5 Summary of correlation analysis between the A, U, C, G contents and A 3 , U 3 , C 3 , G 3 contents in all selected samples There have been more and more features that are unique to RHDV within the family Caliciviridae, including its single host tropism, its genome and its VP10 as a structural protein with unknown function. After we analyzed synonymous codon usage in RHDV Table 2 , we obtained several conclusions and conjectures as followed. 4.1 Mutational bias as a main factor leading to synonymous codon usage variation ENC-plot, as a general strategy, was utilized to investigate patterns of synonymous codon usage. The ENC-plots of ORFs constrained only by a C 3 +G 3 composition will lie on or just below the curve of the predicted values . ENC values of RHDV genomes were plotted against its corresponding C 3 +G 3 %.", "The ENC-plots of ORFs constrained only by a C 3 +G 3 composition will lie on or just below the curve of the predicted values . ENC values of RHDV genomes were plotted against its corresponding C 3 +G 3 %. All of the spots lie below the curve of the predicted values, as shown in Figure 2 , suggesting that the codon usage bias in all these 30 RHDV genomes is principally influenced by the mutational bias. As we know, the efficiency of gene expression is influenced by regulator sequences or elements and codon usage bias. It reported that the RNA sequence of the 3terminal 84 nucleotides of ORF1were found to be crucial for VP10 expression instead of the encoded peptide. VP10 coding by ORF2 has been reported as a low expressive structural protein against VP60 coding by ORF1 .", "It reported that the RNA sequence of the 3terminal 84 nucleotides of ORF1were found to be crucial for VP10 expression instead of the encoded peptide. VP10 coding by ORF2 has been reported as a low expressive structural protein against VP60 coding by ORF1 . And its efficiency of translation is only 20% of VP60. According to results showed by Table 4 , it revealed the differences in codon usage patterns of two ORFs, which is a possible factor reducing the expression of VP10. Although VP10 encoded by ORF2, as a minor structural protein with unknown functions, has been described by LIU as a nonessential protein for virus infectivity, the ω Figure 2 Effective number of codons used in each ORF plotted against the GC3s. The continuous curve plots the relationship between GC3s and ENC in the absence of selection.", "Although VP10 encoded by ORF2, as a minor structural protein with unknown functions, has been described by LIU as a nonessential protein for virus infectivity, the ω Figure 2 Effective number of codons used in each ORF plotted against the GC3s. The continuous curve plots the relationship between GC3s and ENC in the absence of selection. All of spots lie below the expected curve. value of ORF2 suggests VP10 plays an important role in the certain stage of whole RHDV lifecycle. After combining with low expression and ω value of VP10, we conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. This mechanism has been confirmed in various positive-chain RNA viruses, including coxsackievirus, dengue virus, equine arterivirus, footand-mouth disease virus, hepatitis C virus, poliovirus, rhinovirus, and severe acute respiratory syndrome , although the details remain elusive.", "After combining with low expression and ω value of VP10, we conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. This mechanism has been confirmed in various positive-chain RNA viruses, including coxsackievirus, dengue virus, equine arterivirus, footand-mouth disease virus, hepatitis C virus, poliovirus, rhinovirus, and severe acute respiratory syndrome , although the details remain elusive. As preceding description, ENC reflects the evolution of codon usage variation and nucleotide composition to some degree. After the correlation analysis of ENC values between ORF1 and ORF2 Table 7 , the related coefficient of ENC values of two ORFs is 0.230, and p value is 0.222 more than 0.05. These data revealed that no correlation existed in ENC values of two ORFs, indicating that codon usage patterns and evolution of two ORFs are separated each other. Further, this information maybe helps us well understand why RSCU and ENC between two ORFs are quite different.", "These data revealed that no correlation existed in ENC values of two ORFs, indicating that codon usage patterns and evolution of two ORFs are separated each other. Further, this information maybe helps us well understand why RSCU and ENC between two ORFs are quite different. Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group marked as Group 1 is positive value and the other one marked as Group 2 is negative value. And all of those strains isolated before 2000 belonged to Group 2, including Italy-90, RHDV-V351, RHDV-FRG, BS89, RHDV-SD and M67473.1. Although RHDV has been reported as only one type, this may be a reference on dividing into two genotypes. In this report, we firstly analyzed its genome and two open reading frameworks ORFs from this aspect of codon usage bias.", "Although RHDV has been reported as only one type, this may be a reference on dividing into two genotypes. In this report, we firstly analyzed its genome and two open reading frameworks ORFs from this aspect of codon usage bias. Our researches indicated that mutation pressure rather than natural is the most important determinant in RHDV with high codon bias, and the codon usage bias is nearly contrary between ORF1 and ORF2, which is maybe one of factors regulating the expression of VP60 encoding by ORF1 and VP10 encoding by ORF2 . Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV.", "We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV. All the results will guide the next researches on the RHDV as a reference." ]

[ "BACKGROUND: Rabbit haemorrhagic disease virus RHDV , as the pathogeny of Rabbit haemorrhagic disease, can cause a highly infectious and often fatal disease only affecting wild and domestic rabbits. Recent researches revealed that it, as one number of the Caliciviridae, has some specialties in its genome, its reproduction and so on. RESULTS: In this report, we firstly analyzed its genome and two open reading frameworks ORFs from this aspect of codon usage bias. Our researches indicated that mutation pressure rather than natural is the most important determinant in RHDV with high codon bias, and the codon usage bias is nearly contrary between ORF1 and ORF2, which is maybe one of factors regulating the expression of VP60 encoding by ORF1 and VP10 encoding by ORF2 . Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. CONCLUSIONS: We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV.", "Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. CONCLUSIONS: We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV. Text: Synonymous codons are not used randomly . The variation of codon usage among ORFs in different organisms is accounted by mutational pressure and translational selection as two main factors . Levels and causes of codon usage bias are available to understand viral evolution and the interplay between viruses and the immune response .", "The variation of codon usage among ORFs in different organisms is accounted by mutational pressure and translational selection as two main factors . Levels and causes of codon usage bias are available to understand viral evolution and the interplay between viruses and the immune response . Thus, many organisms such as bacteria, yeast, Drosophila, and mammals, have been studied in great detail up on codon usage bias and nucleotide composition . However, same researches in viruses, especially in animal viruses, have been less studied. It has been observed that codon usage bias in human RNA viruses is related to mutational pressure, G +C content, the segmented nature of the genome and the route of transmission of the virus . For some vertebrate DNA viruses, genome-wide mutational pressure is regarded as the main determinant of codon usage rather than natural selection for specific coding triplets .", "It has been observed that codon usage bias in human RNA viruses is related to mutational pressure, G +C content, the segmented nature of the genome and the route of transmission of the virus . For some vertebrate DNA viruses, genome-wide mutational pressure is regarded as the main determinant of codon usage rather than natural selection for specific coding triplets . Analysis of the bovine papillomavirus type 1 BPV1 late genes has revealed a relationship between codon usage and tRNA availability . In the mammalian papillomaviruses, it has been proposed that differences from the average codon usage frequencies in the host genome strongly influence both viral replication and gene expression . Codon usage may play a key role in regulating latent versus productive infection in Epstein-Barr virus . Recently, it was reported that codon usage is an important driving force in the evolution of astroviruses and small DNA viruses .", "Codon usage may play a key role in regulating latent versus productive infection in Epstein-Barr virus . Recently, it was reported that codon usage is an important driving force in the evolution of astroviruses and small DNA viruses . Clearly, studies of synonymous codon usage in viruses can reveal much about the molecular evolution of viruses or individual genes. Such information would be relevant in understanding the regulation of viral gene expression. Up to now, little codon usage analysis has been performed on Rabbit haemorrhagic disease virus RHDV , which is the pathogen causing Rabbit haemorrhagic disease RHD , also known as rabbit calicivirus disease RCD or viral haemorrhagic disease VHD , a highly infectious and often fatal disease that affects wild and domestic rabbits. Although the virus infects only rabbits, RHD continues to cause serious problems in different parts of the world.", "Up to now, little codon usage analysis has been performed on Rabbit haemorrhagic disease virus RHDV , which is the pathogen causing Rabbit haemorrhagic disease RHD , also known as rabbit calicivirus disease RCD or viral haemorrhagic disease VHD , a highly infectious and often fatal disease that affects wild and domestic rabbits. Although the virus infects only rabbits, RHD continues to cause serious problems in different parts of the world. RHDV is a single positive stranded RNA virus without envelope, which contains two open reading frames ORFs separately encoding a predicted polyprotein and a minor structural protein named VP10 . After the hydrolysis of self-coding 3C-like cysteinase, the polyprotein was finally hydrolyzed into 8 cleavage products including 7 nonstructural proteins and 1 structural protein named as VP60 . Studies on the phylogenetic relationship of RHDVs showed only one serotype had been isolated, and no genotyping for RHDV was reported. It reported that the VP10 was translated with an efficiency of 20% of the preceding ORF1 .", "Studies on the phylogenetic relationship of RHDVs showed only one serotype had been isolated, and no genotyping for RHDV was reported. It reported that the VP10 was translated with an efficiency of 20% of the preceding ORF1 . In order to better understand the characteristics of the RHDV genome and to reveal more information about the viral genome, we have analyzed the codon usage and dinucleotide composition. In this report, we sought to address the following issues concerning codon usage in RHDV: i the extent and causes of codon bias in RHDV; ii A possible genotyping of RHDV; iii Codon usage bias as a factor reducing the expression of VP10 and iiii the evolution of the ORFs. The 30 available complete RNA sequences of RHDV were obtained from GenBank randomly in January 2011. The serial number SN , collection dates, isolated areas and GenBank accession numbers are listed in Table 1 .", "The 30 available complete RNA sequences of RHDV were obtained from GenBank randomly in January 2011. The serial number SN , collection dates, isolated areas and GenBank accession numbers are listed in Table 1 . To investigate the characteristics of synonymous codon usage without the influence of amino acid composition, RSCU values of each codon in a ORF of RHDV were calculated according to previous reports 2 Sharp, Tuohy et al. 1986 as the followed formula: Where g ij is the observed number of the ith codon for jth amino acid which has n i type of synonymous codons. The codons with RSCU value higher than 1.0 have positive codon usage bias, while codons with value lower than 1.0 has relative negative codon usage bias. As RSCU values of some codons are nearly equal to 1.0, it means that these codons are chosen equally and randomly.", "The codons with RSCU value higher than 1.0 have positive codon usage bias, while codons with value lower than 1.0 has relative negative codon usage bias. As RSCU values of some codons are nearly equal to 1.0, it means that these codons are chosen equally and randomly. The index GC3s means the fraction of the nucleotides G+C at the synonymous third codon position, excluding Met, Trp, and the termination codons. The ENC, as the best estimator of absolute synonymous codon usage bias , was calculated for the quantification of the codon usage bias of each ORF . The predicted values of ENC were calculated as ENC = 2 + s + 29 where s represents the given G+C 3 % value. The values of ENC can also be obtained by EMBOSS CHIPS program .", "The predicted values of ENC were calculated as ENC = 2 + s + 29 where s represents the given G+C 3 % value. The values of ENC can also be obtained by EMBOSS CHIPS program . Analyses were conducted with the Nei-Gojobori model , involving 30 nucleotide sequences. All positions containing gaps and missing data were eliminated. The values of dn, ds and ω dn/ds were calculated in MEGA4.0 . Multivariate statistical analysis can be used to explore the relationships between variables and samples. In this study, correspondence analysis was used to investigate the major trend in codon usage variation among ORFs.", "Multivariate statistical analysis can be used to explore the relationships between variables and samples. In this study, correspondence analysis was used to investigate the major trend in codon usage variation among ORFs. In this study, the complete coding region of each ORF was represented as a 59 dimensional vector, and each dimension corresponds to the RSCU value of one sense codon excluding Met, Trp, and the termination codons . Correlation analysis was used to identify the relationship between nucleotide composition and synonymous codon usage pattern . This analysis was implemented based on the Spearman's rank correlation analysis way. All statistical processes were carried out by with statistical software SPSS 17.0 for windows.", "This analysis was implemented based on the Spearman's rank correlation analysis way. All statistical processes were carried out by with statistical software SPSS 17.0 for windows. The values of nucleotide contents in complete coding region of all 30 RHDV genomes were analyzed and listed in Table 2 and Table 3 . Evidently, C+G % content of the ORF1 fluctuated from 50.889 to 51.557 with a mean value of 51.14557, and C+G % content of the ORF2 were ranged from 35.593 to 40.113 with a mean value of 37.6624, which were indicating that nucleotides A and U were the major elements of ORF2 against ORF1. Comparing the values of A 3 %, U 3 %, C 3 % and G 3 %, it is clear that C 3 % was distinctly high and A 3 % was the lowest of all in ORF1 of RHDV, while U 3 % was distinctly high and C 3 % was the lowest of all in ORF2 of Table 2 Identified nucleotide contents in complete coding region length > 250 bps in the ORF1 of RHDV 30 isolates genome Table 4 . Most preferentially used codons in ORF1 were C-ended or G-ended codons except Ala, Pro and Ser, however, A-ended or G-ended codons were preferred as the content of ORF2.", "Comparing the values of A 3 %, U 3 %, C 3 % and G 3 %, it is clear that C 3 % was distinctly high and A 3 % was the lowest of all in ORF1 of RHDV, while U 3 % was distinctly high and C 3 % was the lowest of all in ORF2 of Table 2 Identified nucleotide contents in complete coding region length > 250 bps in the ORF1 of RHDV 30 isolates genome Table 4 . Most preferentially used codons in ORF1 were C-ended or G-ended codons except Ala, Pro and Ser, however, A-ended or G-ended codons were preferred as the content of ORF2. In addition, the dn, ds and ω dN/dS values of ORF1 were separately 0.014, 0.338 and 0.041, and the values of ORF2 were 0.034, 0.103 and 0.034, respectively. The ω values of two ORFs in RHDV genome are generally low, indicating that the RHDV whole genome is subject to relatively strong selective constraints. COA was used to investigate the major trend in codon usage variation between two ORFs of all 30 RHDV selected for this study. After COA for RHDV Genome, one major trend in the first axis f' 1 which accounted for 42.967% of the total variation, and another major trend in the second axis f' 2 which accounted for 3.632% of the total variation.", "COA was used to investigate the major trend in codon usage variation between two ORFs of all 30 RHDV selected for this study. After COA for RHDV Genome, one major trend in the first axis f' 1 which accounted for 42.967% of the total variation, and another major trend in the second axis f' 2 which accounted for 3.632% of the total variation. The coordinate of the complete coding region of each ORF was plotted in Figure 1 defining by the first and second principal axes. It is clear that coordinate of each ORF is relatively isolated. Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group marked as Group 1 is To estimate whether the evolution of RHDV genome on codon usage was regulated by mutation pressure or natural selection, the A%, U%, C%, G% and C+G % were compared with A 3 %, U 3 %, C 3 %, G 3 % and C 3 +G 3 %, respectively Table 5 . There is a complex correlation among nucleotide compositions.", "Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group marked as Group 1 is To estimate whether the evolution of RHDV genome on codon usage was regulated by mutation pressure or natural selection, the A%, U%, C%, G% and C+G % were compared with A 3 %, U 3 %, C 3 %, G 3 % and C 3 +G 3 %, respectively Table 5 . There is a complex correlation among nucleotide compositions. In detail, A 3 %, U 3 %, C 3 % and G 3 % have a significant negative correlation with G%, C%, U% and A% and positive correlation with A%, U%, C% and G%, respectively. It suggests that nucleotide constraint may influence synonymous codon usage patterns. However, A 3 % has non-correlation with U% and C%, and U 3 % has noncorrelation with A% and G%, respectively, which haven't indicated any peculiarity about synonymous codon usage. Furthermore, C 3 % and G 3 % have non-correlation with A%, G% and U%, C%, respectively, indicating these data don't reflect the true feature of synonymous codon usage as well.", "However, A 3 % has non-correlation with U% and C%, and U 3 % has noncorrelation with A% and G%, respectively, which haven't indicated any peculiarity about synonymous codon usage. Furthermore, C 3 % and G 3 % have non-correlation with A%, G% and U%, C%, respectively, indicating these data don't reflect the true feature of synonymous codon usage as well. Therefore, linear regression analysis was implemented to analyze the correlation between synonymous codon usage bias and nucleotide compositions. Details of correlation analysis between the first two principle axes f' 1 and f' 2 of each RHDV genome in COA and nucleotide contents were listed in Table 6 . In surprise, only f2 values are closely related to base nucleotide A and G content on the third codon position only, suggesting that nucleotide A and G is a factor influencing the synonymous codon usage pattern of RHDV genome. However, f' 1 value has non-correlation with base nucleotide contents on the third codon position; it is observably suggest that codon usage patterns in RHDV were probably influenced by other factors, such as the second structure of viral genome and limits of host.", "In surprise, only f2 values are closely related to base nucleotide A and G content on the third codon position only, suggesting that nucleotide A and G is a factor influencing the synonymous codon usage pattern of RHDV genome. However, f' 1 value has non-correlation with base nucleotide contents on the third codon position; it is observably suggest that codon usage patterns in RHDV were probably influenced by other factors, such as the second structure of viral genome and limits of host. In spite of that, compositional constraint is a factor shaping the pattern of synonymous codon usage in RHDV genome. Figure 1 A plot of value of the first and second axis of RHDV genome in COA. The first axis f' 1 accounts for 42.967% of the total variation, and the second axis f' 2 accounts for 3.632% of the total variation. Table 5 Summary of correlation analysis between the A, U, C, G contents and A 3 , U 3 , C 3 , G 3 contents in all selected samples There have been more and more features that are unique to RHDV within the family Caliciviridae, including its single host tropism, its genome and its VP10 as a structural protein with unknown function.", "The first axis f' 1 accounts for 42.967% of the total variation, and the second axis f' 2 accounts for 3.632% of the total variation. Table 5 Summary of correlation analysis between the A, U, C, G contents and A 3 , U 3 , C 3 , G 3 contents in all selected samples There have been more and more features that are unique to RHDV within the family Caliciviridae, including its single host tropism, its genome and its VP10 as a structural protein with unknown function. After we analyzed synonymous codon usage in RHDV Table 2 , we obtained several conclusions and conjectures as followed. 4.1 Mutational bias as a main factor leading to synonymous codon usage variation ENC-plot, as a general strategy, was utilized to investigate patterns of synonymous codon usage. The ENC-plots of ORFs constrained only by a C 3 +G 3 composition will lie on or just below the curve of the predicted values . ENC values of RHDV genomes were plotted against its corresponding C 3 +G 3 %.", "The ENC-plots of ORFs constrained only by a C 3 +G 3 composition will lie on or just below the curve of the predicted values . ENC values of RHDV genomes were plotted against its corresponding C 3 +G 3 %. All of the spots lie below the curve of the predicted values, as shown in Figure 2 , suggesting that the codon usage bias in all these 30 RHDV genomes is principally influenced by the mutational bias. As we know, the efficiency of gene expression is influenced by regulator sequences or elements and codon usage bias. It reported that the RNA sequence of the 3terminal 84 nucleotides of ORF1were found to be crucial for VP10 expression instead of the encoded peptide. VP10 coding by ORF2 has been reported as a low expressive structural protein against VP60 coding by ORF1 .", "It reported that the RNA sequence of the 3terminal 84 nucleotides of ORF1were found to be crucial for VP10 expression instead of the encoded peptide. VP10 coding by ORF2 has been reported as a low expressive structural protein against VP60 coding by ORF1 . And its efficiency of translation is only 20% of VP60. According to results showed by Table 4 , it revealed the differences in codon usage patterns of two ORFs, which is a possible factor reducing the expression of VP10. Although VP10 encoded by ORF2, as a minor structural protein with unknown functions, has been described by LIU as a nonessential protein for virus infectivity, the ω Figure 2 Effective number of codons used in each ORF plotted against the GC3s. The continuous curve plots the relationship between GC3s and ENC in the absence of selection.", "Although VP10 encoded by ORF2, as a minor structural protein with unknown functions, has been described by LIU as a nonessential protein for virus infectivity, the ω Figure 2 Effective number of codons used in each ORF plotted against the GC3s. The continuous curve plots the relationship between GC3s and ENC in the absence of selection. All of spots lie below the expected curve. value of ORF2 suggests VP10 plays an important role in the certain stage of whole RHDV lifecycle. After combining with low expression and ω value of VP10, we conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. This mechanism has been confirmed in various positive-chain RNA viruses, including coxsackievirus, dengue virus, equine arterivirus, footand-mouth disease virus, hepatitis C virus, poliovirus, rhinovirus, and severe acute respiratory syndrome , although the details remain elusive.", "After combining with low expression and ω value of VP10, we conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. This mechanism has been confirmed in various positive-chain RNA viruses, including coxsackievirus, dengue virus, equine arterivirus, footand-mouth disease virus, hepatitis C virus, poliovirus, rhinovirus, and severe acute respiratory syndrome , although the details remain elusive. As preceding description, ENC reflects the evolution of codon usage variation and nucleotide composition to some degree. After the correlation analysis of ENC values between ORF1 and ORF2 Table 7 , the related coefficient of ENC values of two ORFs is 0.230, and p value is 0.222 more than 0.05. These data revealed that no correlation existed in ENC values of two ORFs, indicating that codon usage patterns and evolution of two ORFs are separated each other. Further, this information maybe helps us well understand why RSCU and ENC between two ORFs are quite different.", "These data revealed that no correlation existed in ENC values of two ORFs, indicating that codon usage patterns and evolution of two ORFs are separated each other. Further, this information maybe helps us well understand why RSCU and ENC between two ORFs are quite different. Interestingly, we found that relatively isolated spots from ORF2 tend to cluster into two groups: the ordinate value of one group marked as Group 1 is positive value and the other one marked as Group 2 is negative value. And all of those strains isolated before 2000 belonged to Group 2, including Italy-90, RHDV-V351, RHDV-FRG, BS89, RHDV-SD and M67473.1. Although RHDV has been reported as only one type, this may be a reference on dividing into two genotypes. In this report, we firstly analyzed its genome and two open reading frameworks ORFs from this aspect of codon usage bias.", "Although RHDV has been reported as only one type, this may be a reference on dividing into two genotypes. In this report, we firstly analyzed its genome and two open reading frameworks ORFs from this aspect of codon usage bias. Our researches indicated that mutation pressure rather than natural is the most important determinant in RHDV with high codon bias, and the codon usage bias is nearly contrary between ORF1 and ORF2, which is maybe one of factors regulating the expression of VP60 encoding by ORF1 and VP10 encoding by ORF2 . Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV.", "We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV. All the results will guide the next researches on the RHDV as a reference." ]

[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]

[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]

[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]

[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]

[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]

[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]

[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]

[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]

[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]

[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]

[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]

[ "AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 .", "Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL p<0.05 . Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl p<0.05 . The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle.", "CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period . Its etiology involves bacteria commonly Escherichia coli , viruses rota and coronavirus , parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine .", "As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations . Hepcidin plays a fundamental role in the regulation of Fe metabolism , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells .", "On the other hand, by participating in redox reactions leading to the production of reactive oxygen species ROSs , Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented .", "There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases .", "Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress . Free radicals play a significant role in the pathogenesis of many diseases . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 .", "This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status TAS , total oxidant status TOS , and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee MAKU-HADYEK-Submission: 2014/77 . The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature hypo-or hyperthermia , mucosal hyperemia, and full sclera and hematological increase in white blood cell WBC count examinations; the animals were suspected to have septicemia . The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases.", "The animals were given standard treatment antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent . For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France .", "Serum hepcidin Mybiosource ® , TAS Rel Assay Diagnostics ® , and TOS Rel Assay Diagnostics ® were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological WBC, lymphocyte LYM , red blood cells RBC , mean corpuscular volume MCV , and hematocrit HCT analysis was performed on blood counter VG-MS4e ® , Melet Schloesıng, France . The results were evaluated using the t-test in the SPSS ® SPSS 20, USA statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1.", "The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment Table-1 . The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 .", "The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment Table-2 . This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose . The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC.", "The hematological parameters WBC, HCT, LYM, and MCV evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted .", "The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis.", "As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs .", "These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs . Free iron has toxic characteristics as it catalyses the production of ROSs and thus causes oxidative stress . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage .", "The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage . The host cells initiate the antioxidant system in case of exposure to oxidative stress . Kabu et al. reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves .", "reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress malondialdehyde were higher and antioxidant parameters superoxide dismutase , TAS were lower in diarrheic calves . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion.", "Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis.", "EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript." ]

[ "BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection.", "RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature.", "Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide.", "Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms .", "HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases.", "In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus.", "The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ .", "This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously .", "Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported .", "Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016.", "All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 .", "All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 .", "The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1.", "1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 .", "Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively.", "The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 .", "ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports .", "To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 .", "Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this.", "Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 .", "The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region.", "Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 .", "The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model.", "To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 .", "Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 .", "It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation.", "First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections." ]

[ "BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection.", "RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature.", "Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide.", "Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms .", "HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases.", "In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus.", "The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ .", "This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously .", "Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported .", "Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016.", "All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 .", "All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 .", "The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1.", "1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 .", "Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively.", "The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 .", "ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports .", "To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 .", "Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this.", "Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 .", "The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region.", "Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 .", "The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model.", "To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 .", "Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 .", "It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation.", "First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections." ]

[ "BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection.", "RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature.", "Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide.", "Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms .", "HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases.", "In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus.", "The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ .", "This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously .", "Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported .", "Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016.", "All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 .", "All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 .", "The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1.", "1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 .", "Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively.", "The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 .", "ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports .", "To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 .", "Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this.", "Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 .", "The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region.", "Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 .", "The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model.", "To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 .", "Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 .", "It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation.", "First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections." ]

[ "BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection.", "RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature.", "Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide.", "Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms .", "HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases.", "In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus.", "The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ .", "This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously .", "Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported .", "Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016.", "All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 .", "All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 .", "The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1.", "1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 .", "Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively.", "The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 .", "ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports .", "To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 .", "Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this.", "Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 .", "The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region.", "Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 .", "The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model.", "To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 .", "Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 .", "It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation.", "First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections." ]

[ "BACKGROUND: Human bocavirus 1 HBoV1 is an important cause of acute respiratory illness ARI , yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients ≤14 years old , with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection.", "RESULTS: Of the 11,399 patients tested, 5606 49.2% were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 2.2% were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients 45.2%, 112/248 . A significant difference in the prevalence of HBoV1 was found in patients in different age groups p < 0.001 , and the peak prevalence was found in patients aged 7–12 months 4.7%, 56/1203 . Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature.", "Two HBoV1 prevalence peaks were found in summer between June and September and winter between November and December . The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide.", "Text: Human bocavirus 1 HBoV1 , which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness ARI , according to different studies . Serological and nucleic acid test results are generally consistent , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms .", "HBoV1 can cause both upper respiratory illness URI and lower respiratory illness LRI . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications . In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases.", "In some cases, patients develop severe respiratory injury symptoms, which can be fatal . HBoV1 can be detected in fecal samples , blood samples , urine , cerebrospinal fluid , river water and sewage , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus.", "The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children ≤14 years old hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ .", "This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′ . Inclusion criteria were pediatric patients ≤14 years old who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously .", "Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae MP , and Chlamydophila pneumoniae CP . The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature °C , mean relative humidity % , rainfall mm , mean wind speed m/s , mean air pressure hPa , mean vapor pressure hPa , sunshine duration h . Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit Qiagen, Shanghai, China , respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported .", "Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously . Common respiratory pathogens, including respiratory syncytial virus RSV , influenza A virus InfA , influenza B virus InfB , four types of parainfluenza PIV1-4 , adenovirus ADV , enterovirus EV , human metapneumovirus HMPV , four strains of human coronavirus HCoV-229E, OC43, NL63 and HKU1 , human rhinovirus HRV , MP and CP were detected simultaneously as previously reported . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 SPSS Inc., Chicago, IL, USA . Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016.", "All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients ≤14 years old hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 7361:4038 and the median age was 1.75 years interquartile range 0.75-3.83 . Overall, 86.5% 9857/11399 of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 .", "All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 49.2% were positive for one or more of those pathogens Table 1 , and had a median age of 1.50 years interquartile range 0.67-3.00 . The male-to-female ratioes were 1.94: 1 3698:1908 in pathogen-positive patients and 1.72: 1 3663:2130 in pathogen-negative patients p = 0.002 . Two hundred forty-eight of 11,399 patients 2.2% tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 45.2% were co-infected with other pathogens, most frequently with RSV 11.7%, 29/248 Table 1 . The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 .", "The median age was 1 year interquartile range 0.75-1.83 . The male-to-female ratio was 2.54:1 178:70 in HBoV1-positive patients and 1.81:1 7183:3968 in HBoV1-negative patients p = 0.019 . To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups p < 0.001 and the peak prevalence was found in patients aged 7-12 months 4.7%, 56/1203 Fig. 1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1.", "1 . In this study, we monitored the prevalence of HBoV1 in patients ≤14 years old hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′ , has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C mean ± standard deviation , humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 .", "Between 2009 and 2016, the mean temperature from May to September was greater than 25°C Fig. 3 . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure adjusted R 2 = 0.793, p < 0.001 and mean vapor pressure adjusted R 2 = 0.929, p < 0.001 , which linearly associated with mean temperature, and rainfall adjusted R 2 = 0.278, p < 0.001 , which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively.", "The effect of temperature had a delay therefore mean temperature in the preceding month mean temperature 1 month before was also included as an independent variable in the analysis Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature coefficient = 0.259 in the current temperature model p = 0.002 , coefficient = 0.328 in mean temperature in the preceding month model p < 0.001 . Conversely, HBoV1 prevalence was negatively correlated with relative humidity coefficient = − 0.126 in the current temperature model p = 0.024 , coefficient = − 0.083 in the temperature delay model p = 0.039 Table 2 . ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 .", "ARI is one of the most common human diseases, predominantly caused by different respiratory viruses . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas 43 . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports .", "To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children ≤14 years old , hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period Table 1 . In the current study, 86.5% 9857/11399 of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports . Overall, 49.2% 5606/11399 of patients tested positive for one or more respiratory pathogens, 2.2% 248/11399 of patients were tested with HBoV1 infection Table 1 . A higher prevalence of HBoV1 was detected in male patients compared with female patients p = 0.019 , consistent with previous reports . Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 .", "Co-infection with HBoV1 and other pathogens is common . In our study, 45.2% 112/248 of HBoV1-positive patients also tested positive for other pathogens Table 1 . This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference Fig. 2 . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this.", "Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages p < 0.001 . The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 .", "The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months Fig. 1 , consistent with previous serological and epidemiological reports on the virus 8-11, 15, 16, 39, 44 . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region.", "Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period Fig. 3 . In this study, two HBoV1 peaks were observed Fig. 2 . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 .", "The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C Fig. 3 . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring , as well as from tropical regions, such as India, where no obvious epidemic season has been found . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model.", "To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature or mean temperature in the preceding month , mean relative humidity, mean wind speed and sunshine duration as the independent variables Table 2 . Both regression models were established p < 0.001 and the adjusted R 2 value 0.373 of the temperature dorp 1 month model was greater than the adjusted R 2 value 0.231 of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity Table 2 . In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 .", "Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou , which may be related to Guangzhou high humidity mean monthly relative humidity was 77.2 ± 7.3% Fig. 3 . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 .", "It might be partly related to the relatively higher mean relative humidity during this period Fig. 3 . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study p > 0.05 Table 2 . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation.", "First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections." ]