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Variations in the physiological levels of glucose, lactate O2 and CO2 can provide an early indication of metabolic abnormalities that are usually progressive with weight gain associated with obesity. Although a number of metabolic trends related to obesity have been established, exact metabolite interrelationships and complexities as they pertain to obesity progression and the development of associated diseases are less well defined. In addition, obesity and associated metabolic changes appear to be patient-specific and this is largely uninvestigated. Metabolite imbalance resulting from obesity is known to lead to the development of a number of serious diseases including diabetes, cardiovascular diseases, cancer and respiratory depression. Over the past 5 years our team has been researching a revolutionary, totally implantable biosensor platform capable of continuously monitoring glucose. This miniaturized (0.5 x 0.5 x 5 mm) sensor platform is inserted subcutaneously through a needle and operated remotely through a personal digital assistant (PDA) device. One of the key components of this sensor platform is its coating, which is capable of controlling the tissue/implant interface by suppressing inflammation, fibrous encapsulation and inducing neo-angiogenesis. The proposed research intends to make a quantum-advance in metabolic monitoring by allowing simultaneous sensing of multiple analytes (i.e. glucose, lactate O2 and CO2). The trends of these analytes under various biological conditions (i.e. fed, fasting and light exercise) for normal and obese rats will be established for comparison with our proposed multi-analyte sensor. The close proximity of the four electrochemical sensors requires additional advances in device integration. By the end of the proposed three-year study, a first generation multi-analyte sensor will be realized along with coating optimization to permit one month in vivo operation. This sensor is envisioned to achieve a quantum leap in metabolic monitoring and will provide researchers in the field with the means to investigate the complexities of obesity and associated disease states. The small size and low cost will increase patient acceptance and compliance and will allow monitoring of large numbers of patients in a normal home environment and for extended periods of time. Such data will be extremely valuable to researchers in the field to assist in understanding the complexities and patient-specific nature of obesity. Moreover, we envision future integration of this multi-analyte sensory platform to provide closed-loop insulin/glucagon delivery for tight control of glucose levels in diabetic patients.

Human immunodeficiency virus type 1 (HIV-1), the etiological agent of acquired immunodeficiency syndrome (AIDS) and related complexes, displays a high degree of genetic and biological variability. The diversity of HIV-1 has implications for tracking the epidemiology of AIDS, for understanding the pathology of the virus, and for the development of therapeutic, preventive, and diagnostic reagents. There are important questions to be answered relating genetic differences to the biology of the virus, including its latency and its ability to infect and replicate in different cell types. Sequences involved in regulating most aspects of the HIV-1 life cycle, in particular viral gene expression and integration, are located within the long terminal repeats (LTRs) at the 5' and 3' ends of linear viral molecules. The direct contribution of genetic variability in cic-acting transcriptional regulatory elements within HIV-1 LTRs to phenotypic variability in vitro and in vivo has not been extensively evaluated. Integration into the host-cell genome involves covalent linkage between HIV-1 LTRs and cellular DNA. In general, unintergrated linear or circular DNA forms of retroviruses occur only as short-lived replicative intermediates. However, unintegrated viral forms accumulate in cultured cells infected with HIV-1 and appear to correlate with in vitro cytopathicity. The prevalence of unintegrated HIV-1 DNA within cells of infected individuals and its relationship, if any, to HIV-1 pathogenesis has not been determined. The proposed studies are specially aimed at: (1) evaluating directly in cells from infected individuals the extent of sequence heterogeneity within HIV-1 LTRs; (2) assessing the prevalence of unintegrated forms of HIV-1 in infected individuals; and (3) determining the biological effects of natural genetic variability in functional assays. An important aspect of the experimental design is evaluation of genetic diversity directly in infected individuals without selection for in vitro growth of HIV-1 isolates. The studies will focus on clinically ill pediatric AIDS patients and their asymptomatic, seropositive mothers to assess the generation of HIV-1 diversity over time between individuals with different clinical status infected with related viruses. Sequencing LTRs after enzymatic amplification using the polymerase chain reaction (PCR) will assess the extent of LTR variability. Transient expression of an indicator gene under control of variant LTRs will evaluate the biological effects of sequence diversity of LTR promoter function and cell-type specific gene expression. Proposed experiments are designed to test the hypotheses that natural genetic variability occurs in HIV-1 LTRS; that LTR diversity develops in vivo within and between individuals infected with related viruses; and that genetic diversity not only contributes to functional variability of LTRS, but is also associated within the clinical status of individuals infected with HIV-1.

Program Director/Principal Investigator (Last, First, Middle): Tolosa, Leah Project Description The Philippines is one of the planet's biodiversity hot spots. Of 6,000 endemic plants, it is believed that traditional doctors and herbalists use about 1,500 as medicinal plants of which only 1% has been characterized for their active components. Despite this paucity of information, in developing countries such as the Philippines where drugs imported from the developing world are beyond the purchasing power of the majority, herbal medicines remain as the main source of remedies for many ailments. Thus, the long-term objectives of this study is to definitively identify plants that are anti-diabetic based on bioassays for metabolite levels, as well as provide a novel method of quantifying efficacy of herbal medicines based on bioassay guided responses of experimental animals. The former will have an immediate impact in the availability of effective and affordable remedies for diabetes in the Philippines. The latter will provide a means of fact-based quality control of herbal medicines that at present is not always available even in the US. In order to arrive at these objectives, we propose to screen 10+ indigenous plants that have been reported to possess anti-diabetic properties. Screening will be executed at the University of the Philippines Diliman. The screening of crude extracts, fractions and isolates will be bioassay directed using a set of biosensors for glucose, glutamine and fatty acids. These biosensors are currently being developed in the Tolosa lab under project grant R01DK072465 for a device for continuous metabolite monitoring. Although the final device is not yet ready, the individual protein- based sensors can be used in an assay format to test for metabolites in blood. Thus, we propose to develop the protocols for measuring metabolite levels in Male Swiss Webster mice in response to a high glucose load. Using this bioassay protocol, we will screen and identify two plants with the most potent hypoglycemic activity from identified Philippine medicinal plants that have been folklorically reported for the treatment of diabetes. Crude extracts will be successively fractionated until the hypoglycemic constituents(s) are isolated from the two most potent Philippine medicinal plants. The chemical structures of these pure compounds will be determined by standard spectroscopic techniques. The novelty of this project is that rather than measuring a single metabolite (glucose), we will multiplex glucose, glutamine and fatty acids to see ratios and trends of these metabolites in response to a high glucose load, and then to the anti-diabetic principle(s). It should be interesting to see if patterns can be gleaned from these trends to further classify anti-diabetic compounds into groups based on their activity. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page

SIV strains containing HIV-1 env genes (SHIVenv) have been successfully employed to infect macaques through intravenous and mucosal routes. These macaque models have been crucial for studies on HIV pathogenesis, vaccine, and microbicide testing. However, few SHIVenv strains can maintain stable and prolonged infections. Several challenges are apparent in the testing of anti-HIV-1 microbicides and many of these stem from poor animal models to test efficacy. In the R21 proposal, we have outlined a system to construct and test the infectivity of SHIV based on the env and pol genes of subtype A, B, C, and D from acute/early infections. In aim 1, we will utilize a rapid yeast recombination cloning approach to shuttle approximately 400 HIV-1 env genes into an HIV-1NL4-3 or SIV backbones of mac239 and KB-9. The HIV-1 subtype A, C, and D env genes will be PCR amplified from the endocervix or blood of Ugandan and Zimbabwean women within three months or after three years of infection. Over 20 HIV-1 env chimeric viruses have already been constructed and tested using env genes from these patient samples. HIV and SIV env chimeric viruses will be included in subtype-specific pools if the clone is capable of replication on cell lines expressing human or rhesus CD4/CCR5 (respectively) and in human or rhesus PBMCs (respectively). In aim 2, the pathogenicity of these pools will then be accessed (1) using vaginal explants and (2) through vaginal exposure in macaques. The clones that establish infection in both the explant tissue and macaques can then be reconstituted into the "pathogenic" subtype A, B, C, and D pools for the microbicide studies described in the R33 section of this proposal (aim 3). First, we will determine if higher concentrations of cmpd167 or PSC- RANTES are required to inhibit the "pathogenic" subtype A, B, C, and D pools of HIV or SHIVs (as compared to the standard SHIVSF162-P3) in human or rhesus vaginal explant tissues. We determine the identity of any HIV or SHIV clone(s) that are capable of infection even in the presence of the drug. These specific HIV-1 clones (produced from original DNA clones) can then be tested for sensitivity to CMPD167 and PSC-RANTES and to determine if infection was related to drug resistance. Finally and most importantly, microbicides CMPD167 and PSC-RANTES will be vaginally applied to rhesus macaques prior to exposure with the "infectious" subtype A, B, C, and D pools as well as the standard SHIVSF162-P3. We suspect that the majority of the treated macaques will be protected from SHIVSF162-P3 infection. In contrast, the protective effects of the microbicides may be reduced and that in some animals, a slight delay in viremia (as compared to untreated animals) may be the result of infection by specific clone in the SHIV pool with reduced sensitivity to CMPD167 and PSC-RANTES. Vaginal microbicides provide an excellent method to protect women from HIV-1 infection but testing these products prior to human use remains a challenge. A monkey species (e.g. Rhesus macaques) and virus cousin of HIV-1 (SHIV) are used to test the level of protection by these compounds. In this proposal, we have designed new SHIVs that are more closely related to HIV-1 and provide more stringent testing of microbicides for future human use.

Our previous immunohistochemical and biochemical studies of cerebral microvessels and cerebrovascular endothelial cultures showed the presence of phenylethanolamine-N-methyltransferase (PNMT) activity in both tissues. Since these extraneuronal tissues contain a catecholamine synthesizing enzyme which is responsible for conversion of norepinephrine toepinphrine, we extended these studies to determine whether vascular PNMT is indeed capable of producing epinephrine from norepinephrine. For this purpose a direct assay of endothelial epinephrine formed from norepinephrine was determined by using high pressure liquid chromatography. These studies, which are still in progress, have shown that the cultured cerebrovascular endothelium (2nd-4th generation) derived from dissociated cerebral microvascular fractions (obtained from rats) are capable of converting norepinephrine to epinephrine.

The of retinoblastoma susceptibility gene product (pRb) is a potent tumor supressor that is frequently mutated in numerous malignancies including leukemias. pRb and related proteins, p107 and p130 binds to and inactivates different members of the E2F family of transcription factors. In addition to E2F pRb can interact with other factors that modulate transcriptional activity of the pRb-E2F complexes such as: HBP1 (HMG-box- containing transcriptional repressor), hBRGl and hBRM (mammalian homologues of SNF2/SWI2 yeast transcription activator), and histone deacetylase (the enzyme involved in chromatin structure rearrangement) have all been reported to interact with pRb and to modulate its transcriptional activity. Binding of each of these modulators to pRb occur in an LXCXE motif-dependent manner. The LXCXE motif was originally found in certain viral oncoproteins (SV40 T antigen, HPV-16 E7, adenoviral E1A) that also are known to bind to pRb. Preliminary results from our laboratory raised a possibility that pRb may also interact with the HMG1/2 class of proteins. The HMG proteins are nuclear, non-histone structural elements of chromatin that contain an LXCXE motif. We propose to investigate whether the HMG class of proteins contributes to the pRb-dependent regulation of transcription. To address this question we will study specific association of HMG2 and pRB in vitro and in vivo model systems. To investigate the functional significance of HMG2/pRb interaction we will examine effect of HMG on pRb ability to repress transcription in model assays.

Epitaxis, otherwise known as nosebleed, is a common occurrence that typically responds to home treatment. Some cases, however, do not respond and a significant number of patients require emergency room treatment. In severe cases, hospital admittance and time in intensive care are necessary. Several methods of treatment have evolved for treatment of epistaxis: 1) gauze packing, 2) balloon and 3) desiccated sponge packs. Each strategy has strengths, but also weaknesses. Gauze packing decreases blood oxygen levels and is extremely painful, requiring administration of narcotics. Nasal balloons resist conformation to the intricacies of the nasal cavity. Desiccated sponges are solid and immalleable during insertion, often damaging the nasal mucosa. RBDC proposes to develop and test the Nasal Contour Pack (C-Pack). The C-Pack has a soft PVC tube base for easy insertion and removal with two thin, soft plastic bags mounted on top of the tube. Once inserted, the bags can be inflated to create sufficient pressure to stop the bleed. The soft bags will work into the various crevices, create even pressure, will insert easily, can be deflated for easy removal. It will be much less painful, will not require narcotics, and have a channel to provide supplemental oxygen. PROPOSED COMMERCIAL APPLICATIONS: Epistaxis remains one of the most common acute ENT disorders resulting in hospital admissions. Approximately 5-10% of the population experience an episode of active nasal bleed each year. Approximately 10% of these patients visit a physician and one in 10 will require hospitalization. A device that imposes none of the disadvantages of current technology, that can be inserted in an exact and simple manner, is effective as initial intervention and that could be produced at no greater expense than existing devices, would find a prominent place within the market.

A diverse set of agents including platelet-derived growth factor, fibroblast-derived factor, vasopression, phorbolesters, Ca++ ionophore A23187, and catecholamines have all been shown to inhibit insulin and epidermal growth factor (EGF) receptor binding activities. These agents in effect, desensitize the cells to the physiological actions of insulin and EGF. It is not surprising that hormones such as catecholamines inhibit insulin binding because of the direct antagonistic nature of these hormones with respect to the cell's metabolic responses. Thus, if one considers the binding of ligand to a cell surface receptor as the commitment step in a metabolic pathway, then the regulation of receptor binding activities should be expected. Further, if one assumes the ligand to be an allosteric regulator then a change in the intrinsic signalling mechanism of the receptors may also be regulated via modification of the ligand-receptor interaction. Our long term goal is to define the molecular mechanism by which these growth factor (insulin and EGF) receptor binding activities are coordinately regulated and determine what effect this has on their transmembrane signalling mechanisms. All the known actions of catecholamines, which are Beta-adrenergic receptor agonists are mediated thru cyclic AMP-dependent protein kinase (A kinase). Phorbolesters are known activators (substituting for diacylglycerol) of the Ca++, phospholipid-dependent protein kinase (C kinase). Vasopressin, although in some systems causing the elevation of cyclic AMP, primarily stimulates and increase in the intracellular Ca++ concentration. Treatment of isolated membranes or intact cells with platelet-derived growth factor leads to a dramatic increase in the phosphorylation state of membranes. Thus, for all the agents known to inhibit insulin and EGF receptor binding activities a circumstantial case can be made for the involvement of a kinase cascade. We plan to purify the insulin and EGF receptors both by conventional chromatography and by immunoprecipitation. We will then examine what effects these agents have on the phosphyrylation states of the receptors both in vivo and in vitro. The in vitro studies will involve purification of the A kinase and the C kinase and direct phosphorylation of the purified receptors. The effects of both the in vivo and in vitro phosphorylations will be examined with respect to receptor binding activities and intrinsic tyrosine kinase activities.

Project 1). Optokinetic and vestibular nystagmus interplay. We have found that some patients with cerebellar degeneration have a poor response. Most cerebellar patients have an increased or uninhibited caloric response. These two factors tend to make post-caloric nystagmus dominant to an induced OKN response in cerebellar lesions. In normal patients OKN is dominant to a post-caloric response. We wish to verify this hypothesis by examining patients with cerebellar degeneration and comparing them with controls. Project 2). Hyperventilation can cause disorientation in some patients. We wish to study the effects of hyperventilation in various positions and positioning procedures available to us. Project 3) We have numerous patients who have undergone a labyrinthectomy in the past. We wish to evaluate these to see the long term effects of central compensation, e.g. is nystagmus ever present to the unoperated side? How strong is directional preponderance as studied by hot and cold test limited to the remaining ear? Project 4). We wish to continue to examine the movements of animals (monkeys) who have had isolated lesions in the brain (mostly cerebellum); so far these findings have fitted in with clinical findings so that several eye movement syndromes are becoming apparent.

We recently performed studies to reduce the size of Inf2 to determine whether additional candidate genes emerged, and identified Tnf, Lta, Hsp70, and MHC class II genes as important determinants of susceptibility to ozone (O3)-induced airway inflammation. Ongoing studies are investigating the roles of Notch3 and Notch4, also located in Inf2, in O3-induced inflammation. Notch receptors are cell surface receptors important in development and immune cell differentiation. To test the roles of these genes in the model, wild type (B6129SF1, WT), Notch3 (Notch3-/-) and Notch4 (Notch4-/-) knockout mice were exposed to 0.3 ppm O3 or filtered air continuously for 6-48 hr. Immediately after exposure, airway inflammation and injury was assessed using protein concentration (a marker of lung permeability) and inflammatory cells in bronchoalveolar lavage fluid (BALF). O3 significantly increased BALF protein in all genotypes, but greater concentrations were found in Notch3-/- compared to WT (24, 48 hr), and concentrations were greater in Notch4-/- mice compared to Notch3-/- (24, 48 hr). Greater mean numbers of BALF neutrophils were found in Notch3-/- and Notch4-/- mice compared to WT (24, 48 hr). Expression of whole lung Tnf was significantly increased after O3 (24 hr) in all genotypes, and was greater in Notch3-/- and Notch4-/- compared to WT. Pre-treatment with the TNF inhibitor etanercept significantly attenuated the enhanced O3-induced BALF neutrophils in Notch3-/- and Notch4-/- relative to WT. O3-induced transcript expression of other Inf2 genes was not different between genotypes. We then used mRNA transcriptomics analyses to further investigate the role of Notch3/4. Statistical and visual data mining approaches identified differentially expressed genes basally e.g. Gbp1, Cntn1 and after O3 e.g. Ccl7, Il33 between WT and KO mice. Results are consistent with the hypothesis that Notch3 and Notch4 are susceptibility genes for O3-induced airway inflammation. Furthermore, results suggest an important interaction between Notch3, Notch4, and Tnf. These novel findings suggest Notch receptors protect against the innate immune inflammatory response to O3. In another study we have tested the hypothesis that the gene mannose binding lectin (MBL), which has a central role in the activation of the complement pathway of innate immunity, is necessary to elicit some of the pro-inflammatory events caused by ozone-mediated activation of the innate immune system. Our in vivo studies have shown, compared to wild type mice (Mbl+/+), there was significantly less neutrophilic infiltration in the lungs of mice with targeted deletion of Mbl (Mbl-/-) exposed to O3 (0.3 ppm) for 72 hours. We also found reduced levels of the neutrophil attractants MIP-2 and LIX at 48 hours post-exposure in Mbl-/- mice compared to Mbl+/+ mice. Microarray analyses have identified basal and post-exposure profiles and expression response profiles that differ between Mbl+/+ and Mbl-/- mice, providing insight to the mechanisms through which MBL modulates the pulmonary response to O3 inhalation. These novel studies are the first to identify a role for MBL in response of the lung to oxidative stress, and should lead to a better understanding of mechanisms of susceptibility. Children may be more at risk to air pollution than adults due to higher minute ventilation rates and activity levels outdoors, and continued lung development into adolescence. To study the impact of O3 exposure on the developing lung, we have collaborated with Dr. Edward Postlethwait (Univ Alabama, Birmingham) to study O3 effects on gene expression in infant rhesus macaque monkeys that were exposed to a regimen mimicking urban conditions and site and exposure duration samples were obtained for gene expression analysis. Primates were raised in filtered air (FA) and nighttime exposures to 0.5 ppm O3 conducted for 1 cycle (9 d FA followed by 8 hrs/d O3 for 5 d),11 cycles, or FA. Exposures ended at 180 d of age. Immediately post exposure, lungs were microdissected to obtain central axial airways (generation 8-10) and terminal bronchioles devoid of parenchyma, and stored in RNAlater. RNA was pooled to provide a single sample for each experimental group. Gene expression (Agilent rhesus monkey oligo microarrays) was analyzed initially by K-means clustering. Informative patterns were analyzed (Ingenuity) to identify interaction between differentially expressed genes. A number of informative patterns were identified. 1) Genes upregulated in axial and terminal bronchiole tissue after 1 cycle: inflammatory and immune responses (e.g. IL8, TNF). 2) Upregulated genes in axial tissue after 11 cycles: cellular inflammation and hematological system (IL1B, IL17, MAPK). 3) Genes differentially expressed in axial and terminal bronchioles irrespective of O3 exposure: developmental (ACAN, MUC2, CX3CL1) and immune function (IL17RD, FGF1, DAP1). In infant primates, the superimposition of injury and repair on growth and development results in anatomic and exposure specific alterations in gene expression that is likely coupled to the O3-induced structural, inflammatory, and biochemical effects also observed. Asthma is a known risk factor for acute ozone-associated respiratory disease. Ozone causes an immediate decrease in lung function and increased airway inflammation. The role of atopy and asthma in modulation of ozone-induced inflammation has not been determined. We sought to determine whether atopic status modulates ozone response phenotypes in human subjects. Fifty volunteers (25 healthy volunteers, 14 atopic nonasthmatic subjects, and 11 atopic asthmatic subjects not requiring maintenance therapy) underwent a 0.4-ppm ozone exposure protocol. Ozone response was determined based on changes in lung function and induced sputum composition, including airway inflammatory cell concentration, cell-surface markers, and cytokine and hyaluronic acid concentrations. All cohorts experienced similar decreases in lung function after ozone. Atopic and atopic asthmatic subjects had increased sputum neutrophil numbers and IL-8 levels after ozone exposure; values did not significantly change in healthy volunteers. After ozone exposure, atopic asthmatic subjects had significantly increased sputum IL-6 and IL-1beta levels and airway macrophage Toll-like receptor 4, FcepsilonRI, and CD23 expression; values in healthy volunteers and atopic nonasthmatic subjects showed no significant change. Atopic asthmatic subjects had significantly decreased IL-10 levels at baseline compared with healthy volunteers; IL-10 levels did not significantly change in any group with ozone. All groups had similar levels of hyaluronic acid at baseline, with increased levels after ozone exposure in atopic and atopic asthmatic subjects. Atopic asthmatic subjects have increased airway inflammatory responses to ozone. Increased Toll-like receptor 4 expression suggests a potential pathway through which ozone generates the inflammatory response in allergic asthmatic subjects but not in atopic subjects without asthma

Yale University has encountered rapid growth in two aspects of rodent-based research that require substantial expansion of rodent hazard containment space. First, the use of biohazardous agents is a significant and increasing component of rodent-based at Yale. Research with such agents has increased about 40 percent during the past five years. Most recently, Federal funding for biological hazards has substantially increased which suggests that the emergent trend in this area will continue and likely escalate. Second, the importation of genetically engineered mice (GEM) from investigators at other institutions-a scientifically essential component of contemporary research-requires quarantine and testing prior to releasing then into Yale colonies. Further, Yale is the site for a national metabolic phenotyping core which requires both quarantine and phenotyping. We are projecting to import over 400 cohorts this year subject to quarantine and testing and this number is increasing annually at an average of 18 percent. These two activities are currently packed into dispersed facilities, which are primarily used for housing specific pathogen-free (SPF) mice and are neither large enough in total capacity or configured appropriately for optimal containment safety and efficiency. Yale's rodent census has increased steadily since 1992 and has doubled within the past six years. The use of ventilated racks and, most recently, the opening of a new 57,000 square foot mouse facility, The Anlyan Center (TAG), has enabled decompression of overcrowded mouse1 rooms and consolidation of SPF mice. It has also vacated 10,000 gross square feet of space in an existing mouse holding facility in the Laboratory of Surgery, Obstetrics and Gynecology (LSOG). This application requests funds to help support the renovation for consolidation of housing and procedure space for rodents used in research requiring Biological Safety Level 2 (BL2) agents, chemical and radiologic hazards, quarantine, and phenotyping. The proposed renovation will allow LSOG to be used as an efficient, centralized, biosecure containment facility by converting underutilized storage into critically needed procedure space and by revamping existing, contiguous housing space that is readily adaptable for such use.

It is currently believed that vitamin A, retinol, is biologically inert and that its myriad of biological functions are exerted by active metabolites: the visual chromophore 11-cis-retinaldehyde, and retinoic acids, which regulate gene expression by activating specific nuclear hormone receptors. Surprisingly, our preliminary data suggest that retinol is a transcriptional regulator in its own right. Retinol circulates in blood bound to serum retinol- binding protein (RBP) but it must dissociate from the protein prior to entering target cells. It was recently shown that an integral plasma membrane protein termed STRA6 binds RBP and mediates the uptake of retinol into cells. Our preliminary results demonstrate however that, in addition to its function as a vitamin A transporter, STRA6 is a ligand-activated cell surface receptor which activates a JAK/STAT pathway in response to binding retinol-RBP. Specifically, the data indicate that, association of STRA6 with retinol-bound RBP results in phosphorylation and activation of STATs, which, in turn, induce the transcription of specific STAT target genes. Studies proposed here will address the hypothesis that retinol can regulate gene transcription by activating a signalling pathway mediated by an RBP/STRA6/STAT pathway. We further propose to elucidate its involvement of this pathway in regulation of lipid homeostasis and insulin responses and in control of cell growth and survival. The results of these studies may point at novel targets for therapeutic approaches in treatment of diabetes and cancer.

In addition to their essential role in the elicitation of immediate hypersensitivity reactions, mast cells have been implicated in the innate immune response to infectious bacteria. We found that human mast cells bind and internalize E. coli as assessed by both flow cytometric analysis and confocal microscopy. This process results in up-regulated activation-dependent gene expression by human mast cells as analyzed by DNA microarray assays. Transcripts up-regulated include those coding for enzymes, surface molecules, components of the cytoskeleton and molecules associated with signaling, the cell cycle, adhesion, and protein transport. More specifically, and relevant to innate immunity, genes encoding for TNF-alpha and three chemokines of the CC family e.g., I-309, MIP-3, and MIP-4 were found to be over-expressed following exposure of human mast cells to bacteria. We have analyzed Fyn-binding proteins in MC/9 mast cells to explore the Fyn-mediated signaling pathways, and identified 6 proteins that bind to Fyn including vimentin, pyruvate kinase, p62 ras-GAP associated phosphoprotein, SLP-76, HS-1, and FYB. After IgE-receptor mediated stimulation, binding of vimentin to Fyn was increased. Mast cells from vimentin-deficient mice showed enhanced mediator release and tyrosine-phosphorylation of intracellular proteins including NTAL and LAT.

In nursing home inspections conducted by state licensure and certification agencies, a physical restraint is defined as a device that is attached and cannot be easily removed by the resident which restricts freedom of movement and/or normal access to his/her body. In 1989, it was estimated that 44 percent of all residents in nursing homes were physically restrained. More recent research suggests that 13 percent of all residents in nursing homes are now being physically restrained. Because of the adverse consequences of restraint use, there is concern that physical restraints are still being overused. These adverse consequences include falls, nosocomial infections, pressure sores, agitation of residents, and cognitive decline. [unreadable] [unreadable] One area of research that remains to be investigated is the nexus between the use of physical restraints and mental health. Previous studies use limited statistical tests, such as correlations and t-tests, that could not account for potential biases, such as whether residents who become mentally disturbed are most likely to be restrained. We propose to use linear models of change that are less susceptible to this bias. These prior studies laid the groundwork of mental health decline and physical restraint use, but this issue remains to be investigated further. It is proposed to use the newly available Minimum Data Set (MDS), representing nursing home residents in Pennsylvania, to examine the association between use of physical restraints, cognitive decline, alterations in mood, behavior problems, and social engagement. [unreadable] [unreadable] Clearly, understanding whether restraints contribute to mental health problems is important in a mental health and quality-of-care context. If restraints contribute to a decline in mental health, a more powerful rationale for their careful use may develop, helping reduce restraint use. In these cases, the health and satisfaction of residents may also be improved. Presumably, with a concomitant reduction in residents with mental health problems, nursing homes may be better able to provide mental health (and other) services to impaired residents. [unreadable] [unreadable] [unreadable]

A comprehensive physical model will be developed for the effects of foreign ion substitution and laser irradiation on the dissolution kinetics of hydroxyapatite. Foreign ions to be studied are carbonate, fluoride, strontium and magnesium. The major hypothesis to be tested is that when these ions are incorporated into the apatite lattice, the free energy of the crystal (and consequently its solubility) is altered, even though the dissolution rate controlling phase may not change. Studies done with carbonate apatite samples suggest that there may be a linear free energy relationship between carbonate content and this crystal free energy. Specific hypotheses for individual ions are as follows. Carbonate is proposed to alter solubility but not participate in the rate controlling surface phase; fluoride, on the other hand, is thought to readily participate. The mechanism(s) by which strontium and magnesium act may be concentration dependent. Laser irradiation reduces solubility of hydroxyapatite, apparently by annealing away the more soluble crystalline dissolution sites. The interaction of foreign ion substitution on this effect will be characterized. The studies to be done during this project will collectively provide a broadly based mechanistic interpretation for foreign ion and laser effects. These proposed mechanisms will be directly relatable to crystal structure and energetics and therefore these studies will constitute a significant step toward an eventual quantitative atomic level understanding of foreign ion and laser effects.

Case fatality among persons hospitalized with hantavirus cardiopulmonary syndrome [HCPS] in Chile exceeds 50%, and there is not treatment with proven efficacy. Intravenous ribavirin is active against hantaviruses in vitro, and intravenous ribavirin administration decreased mortality in controlled trials in Hantaan-associated hemorrhagic fever with renal syndrome [HFRS]. A similar, placebo-controlled trial of ribavirin for the early treatment of suspected hantavirus infection has recently been initiated by the NIAID Collaborative Antiviral Study Group (G. Mertz, PI). As described in Project 3, there is strong evidence that cardiopulmonary shock phase of HCPS is immune mediated and that the severity of shock is associated with HLA type. In controlled trials done during the Korean conflict, early treatment with corticosteroid therapy (before the onset of shock) reduced the severity of shock and significantly reduced the severity of renal dysfunction. Recently physicians in Chile and Argentina have used high-dose, intravenous methylpredinsolone in persons with HCPS, and they believe it is effective when used before the onset of shock. We hypothesize that treatment with ribavirin and steroids may be additive or even synergistic. To determine whether ribavirin or steroid therapy reduces mortality in persons with HCPS, a controlled trial of intravenous ribavirin and intravenous corticosteroid treatment will be performed in persons with suspected hantavirus infection using a 2X2 factorial design with four study groups: placebo/placebo, ribavirin/placebo, prednisolone/placebo, and ribavirin/prednisolone. Entry criteria, drug dosing, measurements and endpoints will be consistent with those employed in the CASG-sponsored trial in the U.S. and Canada and, if possible, in trials under consideration in Argentina. As such, we will increase our ability to assess drug efficacy through meta-analyses or through direct comparison with adjustment for study site.

Cell surface adhesins mediate the first interactions of fungi with mammalian hosts, so adhesin-mediated binding is a prelude to differentiation, colonization, biofilm formation and pathogenic invasion. These events in turn lead to complications of morbidity and mortality, especially common in immunocompromised and chronic disease patients. The C. albicans Als adhesins are implicated in pathogenesis and biofilm formation. They bind to mammalian tissues, cause fungal cell aggregation, and also co-aggregate with other microbial pathogens to mediate polymicrobial infections. Als adhesins are also important in formation of persistent and drug-resistant biofilms in tissues and on indwelling devices. [unreadable] [unreadable] Our long-term goal is to understand the roles for cell adhesion proteins in fungal life cycles and pathogenesis. The central hypothesis of this proposal is that amyloid-forming sequences are a feature of biofilm-forming adhesins, and these sequences potentiate adhesion, fungal aggregation and host invasion. This hypothesis is based on our findings that Als5p causes adherence with amyloid-like features, that the purified Als5p can form authentic amyloids, and that bioinformatic analyses reveal amyloid-forming sequences in many biofilm-associated microbial adhesins. [unreadable] [unreadable] Three specific aims will test the amyloid/ biofilm hypothesis: (1) To determine the role of specific sequences in amyloid formation and microbial adherence, we will test the working hypothesis that the amyloid-forming sequences are essential for Als-mediated cellular aggregation. (2) We will test the hypothesis that these amyloid-forming sequences in Als proteins are essential for Als-initiated biofilm formation. (3) In Als proteins, glycosylated tandem repeats follow the amyloid-forming sequences, and these repeats greatly increase adhesion activity. We will therefore test the hypothesis that the peptide sequences and glycosylation patterns in the Als repeats modulate amyloid formation to promote cellular aggregation and biofilm formation. [unreadable] [unreadable] The proposed work is innovative in its conjunction of two important concepts: amyloid-like protein interactions and adherence of pathogens leading to biofilm formation. We are also the first group to work on structure and function of Thr-rich repeat sequences in pathogenic and other fungal adhesins. Completion of these aims will lead to better defined models for the initial events in biofilm formation, and will discover whether amyloid-forming sequences are essential for biofilm adherence in fungi. If the hypothesis is supported, the results will establish connections between searches for anti-biofilm and anti-amyloid therapeutic agents. [unreadable] [unreadable] [unreadable]

Alcohol use among youth is related to traffic crashes, suicides and assaults. A promising approach for preventing youth from consuming alcohol and experiencing these problems is to prevent youth from obtaining alcohol. The Complying with the Minimum Drinking Age Trial (CMDA) was a five-year multi-community quasi- experimental trial designed to examine the effectiveness of two interventions targeting alcohol establishments-owner/manager training and law enforcement compliance checks-to reduce the propensity to sell alcohol to underage youth. Results showed that law enforcement compliance checks have an immediate effect on propensity to sell alcohol to underage youth (reducing the propensity by 17%), but this effect decays either completely or by 50% depending on the type of establishment, within a three-month period. We propose to conduct a two-year study to analyze secondary data from the CMDA trial to assess whether a pattern of law enforcement compliance checks exists that maximizes effects of the checks for reducing illegal alcohol sales to underage youth. We will use state-of-the-art geospatial techniques and hierarchical regression models to assess: (1) spatial autocorrelation of propensity for alcohol establishments to sell alcohol to underage youth;(2) extent to which effects of law enforcement compliance checks diffuse across space to impact the propensity of neighboring alcohol establishments to sell to underage youth;and (3) simultaneous impact of compliance checks across both time and space, including possible interactive effects of the two. Results from the statistical models will be used to generate a pattern or patterns of law enforcement compliance checks that optimize the effects of these checks on preventing underage sales. PUBLIC HEALTH RELEVANCE: Communities are interested in identifying cost-efficient, research-based methods for reducing youth access to alcohol, and subsequently reducing alcohol problems and related costs. Law enforcement compliance checks have been identified as an effective deterrent to sales of alcohol to underage youth. Findings from the proposed study will indicate how far the deterrent effects of compliance checks diffuse geospatially, providing guidance on how many establishments need to be checked within specific neighborhoods and at what frequency.

DESCRIPTION: (Applicant's Description) Cancer is the second leading cause of death in the United States, accounting for nearly one quarter of total human mortality. Understanding the molecular changes that occur during the progression of human cancers could provide important data for prevention, early detection, diagnosis, and the development of optimal treatment protocols. The ability to produce a molecular fingerprint of each tumor may prove to be extremely important as histologically similar tumors may be the result of substantially different genetic changes. The Human Genome Project has provided important data and reagents that may serve as the basis for such an understanding. Analysis of the DNA sequence collected through the world-wide EST sequencing effort has allowed the identification of nearly 65,000 unique human transcripts and associated cDNA clones. Advances in technology also offer important new resources for the assessing the molecular state of a given tumor. cDNA microarraying is a recently developed technique that allows expression-level monitoring of as many as ten thousand individual transcripts on a single microscope slide. Laser capture microdissection allows the separation of tissue samples into unique cellular phenotypes. Used together with the cDNA and EST data that we now possess, these techniques should allow the identification of genes differentially expressed during tumor development and may allow the creation of novel technologies for evaluations of molecular alterations in tumors. The goal of this proposal is to use cDNA microarraying for the construction of molecular transcription profiles of tumors in a variety of tissues, focusing on breast, prostate, colon, lung, and ovary. Ultimately, we hope to develop a technique that will allow the rapid clinical analysis and characterization of human tumors through the microarray analysis of tissue-specific Reduced Expression Arrays.

PROJECT SUMMARY The overarching aim of the Northern New England Clinical and Translational Research Network (NNE-CTR) is to establish the necessary infrastructure to conduct high-quality clinical and translational research that will ultimately improve health outcomes for residents in this largely rural region. To achieve this ambitious aim, NNE-CTR leadership and staff will need to develop systems to continuously assess progress and identify opportunities for mid-course corrections. The Tracking and Evaluation Core (TEC) will provide the requisite infrastructure to ensure that the NNE-CTR can establish its own value and efficiency, and enact needed program improvements in a timely and effective manner. Led by a multidisciplinary team of evaluation, outcomes research and quality improvement experts, the functions of the TEC will be to: 1) identify meaningful and reliable performance metrics for the overall program, and the individual Key Component Activities; 2) continually track and monitor these metrics; and 3) rigorously analyze, synthesize, and disseminate performance information to NNE-CTR leadership and key stakeholders throughout the implementation process. Working collaboratively with Maine Medical Center and the University of Vermont, TEC staff will use a flexible, stakeholder-centered approach to facilitate the selection of performance metrics for each Core and the broader NNE-CTR initiative; implement and customize the REDCap data portal to support tracking and data collection; collect primary qualitative and quantitative data relevant to the conduct of clinical and translational research; routinely analyze and report performance data to the NNE-CTR leadership and key stakeholders; respond to evaluation consultation requests from other Cores; and produce summative reports of activities and outcomes of the IDeA-CTR award. The TEC activities will be grounded in the Center for Disease Control and Preventions' evaluation framework, which applies a population health model ideal for evaluating the health and systems-level outcomes of clinical and translational research. To complement the CDC model, the TEC Core will also adopt the ?systems evaluation partnership? model to assess a broad range of process and short- and long-term outcome measures. In addition, the TEC will apply quality improvement tools and techniques in tandem with more traditional evaluation methods to provide the NNE-CTR leadership and stakeholders with actionable rapid cycle feedback to help inform implementation efforts and identify opportunities for improvement. Through these activities, the TEC will facilitate transparency, promote data-informed decisions regarding the management of the initiative's many components, and thereby ensure the ultimate success of the overall program.

Chronic infection with hepatitis B virus (HBV) is a major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma (HCC) worldwide. Globally there are an estimated 400 million persons infected with HBV. In the United States, there are 1.25 million affected individuals and the epidemiology of the infection is changing with immigration of persons from endemic regions. The natural history of CHB also appears to be changing with an increasing prevalence of HBeAg negative chronic hepatitis B. Knowledge of the rate of progression between individuals with HBeAg positive and negative CHB is unknown. An equally important and related issue is the clinical assessment of disease severity. Unfortunately, there are no good laboratory markers of disease severity. Liver biopsy is the accepted gold standard for assessing disease severity and cirrhosis but is costly, invasive, and associated with complications, which often limits patient acceptability as well as being subject to sampling error ranging from 15-25%. Non-invasive methods to assess disease severity are highly desirable for practioners caring for patients with CHB. Despite the recent licensing of several new agents for treatment of CHB, therapy remains problematic due to the high rate of anti-viral drug resistance with nucleos(t)ide analogues and relatively poor response to interferon. Identifying the optimal regimen and defining the best parameters to monitor patients both on and off therapy are major unresolved issues. Given the number of chronically infected persons and the requirement for long-term therapy in many of these patients, newer agents with different therapeutic targets are needed, as well as cheaper, more effective regimens. Hypotheses/problems addressed: 1) Define the host, viral and environmental factors that determine the natural history and outcome of HBV infection. To study this problem, we have created a large database of untreated patients with chronic HBV (n350), which will be analyzed to identify factors that affect the natural history of chronic HBeAg positive and negative infections. This data will be used to develop a non-invasive model to predict fibrosis progression, in patients with CHB. We also plan to evaluate the role of transient ultrasound elastography (Fibroscan) to assess fibrosis stage in persons with CHB. These results will be compared to liver biopsy, MRI elastography and plasma will be stored for future proteomic analysis. The goal is to develop a series of blood and imaging tests that will obviate the need for liver biopsy in most patients with CHB. 2) Develop and evaluate novel, safer and more effective therapies for chronic viral hepatitis. Current therapy of CHB remains less than optimal. After one year of therapy, only 17-33% of HBeAg positive patients achieve HBeAg loss, a serologic marker of viral replication and 25-70% completely suppress viral replication. Consequently, relapse rates range from 30-50% if therapy is stopped after 48-52 weeks in HBeAg positive patients and approach 90% in HBeAg negative patients. There is limited information on the safety and efficacy of long-term therapy. In an effort to gain insight into the long-term benefit and more importantly safety of this agent, we conducted a long-term trial of lamivudine 100 mg daily for up to 10 years in patients who were able to maintain a normal alanine aminotransferase (ALT) level and complete viral suppression for a period of 4 years. In order to obtain data on long-term histologic outcome, patients underwent liver biopsy at baseline, 1, 4 and 8 years. Twenty-two of forty-three (6 HBeAg positive and 14 HBeAg negative) patients continue on therapy. This study revealed significant histologic improvement at 8 years including reversion of advanced fibrosis to normal findings. In addition, at 8-10 years clearance of HBsAg was observed in 25% of patients (rate at one year ranges from 0-2%) and no cases of hepatocellular carcinoma were observed. This data is important for advising patients on long-term prognosis with lamivudine therapy. Borrowing on the HIV paradigm that multiple agents that act at different sites of viral replication or have different resistant profiles may be more effective than monotherapy, we have conducted a randomized trial of lamivudine and adefovir versus adefovir monotherapy in patients with HBeAg positive and negative CHB with and without resistance to lamivudine. To date 42 patients have been enrolled into this trial. We have performed an analysis of the HBeAg positive, treatment nave cohort who have completed one year of therapy n=22. At last follow up, all 12 patients receiving combination therapy had normal ALT levels and 9 (75%) had lost HBeAg and had undetectable HBV DNA. In contrast, only 5 (50%) of the adefovir treated patients had normal ALT levels and only 4 (40%) lost HBeAg and had undetectable HBV DNA. One additional adefovir treated patient had viral rebound (≥1 log increase in HBV DNA from nadir) and was considered a treatment failure. No viral rebound, suggesting development of viral resistance was observed in the combination arm. Neither the combination of lamivudine and adefovir nor adefovir monotherapy were associated with significant side effects. These results suggest that combination therapy may be more appropriate for long-term use but the results need to be validated in larger study. These promising results have led us to evaluate the combination of tenofovir and emtricitabine compared to tenofovir for patients with CHB. Tenofovir is a nucleotide analogue that is very effective at suppressing HBV DNA and has an excellent resistance profile. This study will examine the long-term efficacy and safety of tenofovir and emtricitabine versus tenofovir alone in patients with HBeAg positive and negative CHB with the goals of maintaining long-term viral suppression ands preventing the emergence of viral resistance. Enrollment is planned to begin in September, 2007. Elucidate the viral pathogenesis of HBV infection and mechanisms of action of anti-viral therapy The course of CHB following the development of anti-viral resistance is highly variable with some patients showing continued viral suppression, some with an accelerated course and others who lie in-between. The reasons for this wide variation in outcome are unknown. We hypothesized that following initial viral breakthrough, compensatory mutations that do not alter sensitivity to lamivudine but instead affect viral replication may account for the different phenotypic presentations observed. To investigate this issue, we sequenced the entire polymerase region of HBV at three time points, (baseline, at time of viral breakthrough and last available follow-up from patients enrolled in our long-term lamivudine trial) in 18 patients with HBeAg positive CHB, genotypes A or C who developed lamivudine resistance. Additional mutations other than the rt M204V/I that confers resistance to lamivudine were found. Several interesting findings emerged from this study. First, whether a patient develops rtM204V/I, conferring resistance to lamivudine, appears to be HBV genotype dependent. Thus patients with genotype A were found to have predominantly rtM204V whilst patients with genotype C were more likely to have rtM204I. Second, compensatory mutations were observed more frequently in patients who progressed to cirrhosis compared to those who did not. We are now investigating the effects of these mutations on viral replication in genotype A and C replicative constructs. Furthermore, we are developing in-vitro assays to evaluate drug resistance using virus cloned from patients serum. This will permit cross-resistance monitoring to other antiviral agents and aid in managing patients with resistant HBV.

Ribulose bisphosphate carboxylase is the CO2-fixing enzyme in the Calvin cycle. It contains two types of subunits: the catalytic "large" subunit (Mr=55 kD) and the noncatalytic "small" subunit (Mr=ca 14 kD). The small subunit is synthesized in the cytoplasm as a precursor polypeptide, cleaved upon entry into the chloroplast, and assembled into holoenzyme. The large subunit is synthesized in the chloroplast, where it binds to a heterologous protein complex consisting of about 12 60 kD polypeptide subunits. This complex sediments at 29S in sucrose gradients and is well separated from the 18S carboxylase and from the 7S free large subunit pool and 3S small subunit pool. We propose to determine the mechanism of assembly of the enzyme by exploiting an in vitro assembly system we have developed from chloroplast extracts. In this system, large subunits can be released from the 29S complex by incubation with ATP, and their assembly into holoenzyme can be facilitated by nucleotides. We will fractionate the extract to separate the 29S complex from the free 7S large subunits and 3S small subunits to see if assembly can occur in the absence of the 29S complex. We will obtain de novo synthesized large subunits by in vitro transcription-translation of cloned large subunit genes, and add these to chloroplast extracts to see if they can assemble into holoenzyme either in the presence of the 29S complex or in its absence. We will also purify the 29S complex in bulk (it is an abundent protein) and study its association-dissociation behavior in vitro, both in isolation and together with either native or holoenzyme-derived large and small subunits. This may lead to the first in vitro re-assembly of this enzyme. The role of heterologous proteins in the assembly of eukaryotic oliogomeric proteins is a relatively unknown field, and is of general significance in cell and molecular biology. This carboxylase assembly system may contribute a lot to our understanding of this general phenomenon, because the components are soluble and well defined.

Mouse adenovirus type 1 (MAV-1) causes disease in the newborn or adult mouse by infecting endothelial cells and cells of the monocyte/macrophage lineage throughout the animal. Depending on the dose, virus strain, or host mouse strain, the outcome of infection can be inapparent infection, persistent infection, or death. The wealth of murine immunological reagents and inbred mouse strains, including immunodeficient mice, coupled with an easily manipulated virus causing several disease phenotypes, make MAV-1 an ideal model for studying viral pathogenesis in the natural host. This project will provide an understanding of (a) the role of the immune system in both control of viral infection and in producing viral disease, (b) the basis for infection by MAV-1 of two specific cell types, endothelial cells and macrophages, and (c) the contributions of early viral gene products to infection of cells in culture and mice. There are three specific aims: 1. Identify early immune response components affecting mouse susceptibility to infection. The role of B lymphocytes, natural killer cells, and monocytes/macrophages will be investigated using immunodeficient mice and depletions of specific cell types. 2. Determine whether MAV-1 infection induces gene expression changes that can alter effector function of endothelial and macrophage cells. Cellular genes, including interferon response, cytokine, and major histocompatibility complex gene class II genes will be characterized with respect to expression changes in target endothelial and macrophage cells by wild-type and early region 1A and 3 (E1A and E3) mutant viruses. The results of assays of in vitro and in vivo gene expression will be integrated to define host response pathways and strategies used by the virus to evade them. 3. Determine mechanisms by which viral early region genes contribute to MAV-1 infection. A biochemical assay will be used to identify host proteins that interact with MAV-1 E1A and E3, and these results will be incorporated into models developed in Aim 2. Viruses with mutations in E1A, E1B, and E3 will be used to infect immunodeficient mice to test specific hypotheses about early adenoviral gene function in vivo. These studies will further our knowledge of adenovirus biology, host immune mechanisms and viral immune evasion.

The Study to Help the AIDS Research Effort (SHARE) was funded by NIAID and NCI in 1983 to study the natural history of infection with human immunodeficiency virus, type 1 (HIV). SHARE, along with similar sites in Chicago, Pittsburgh, and Los Angeles, forms the Multicenter AIDS Cohort Study (MACS). MACS participants, including 1447 enrolled in SHARE, have been followed semiannually since 1984 and have provided questionnaire data, physical exam data, laboratory data (including HIV serostatus and T-cell subset measurements), and a large repository of plasma, serum, cryopreserved peripheral blood mononuclear cells and other specimens. Evaluating and following the prevalent and incident cases of HIV-infection in SHARE and the MACS, has provided key insights into risk factors for infection with HIV-1, monitoring and mechanisms of progression of HIV infection once it is established, host defense against HIV, genetic factors affecting HIV pathogenesis, and use and efficacy of different therapies for HIV infection and for opportunistic pathogens. SHARE and MACS have just completed the recruitment of 1326 additional participants, including 352 in SHARE, who are younger and more non-Caucasian than the cohort previously recruited. This application requests the continuation of the followup of the SHARE cohort from 2004 to 2008. Expected survival and continuation in the study of HIV-infected cohort members through this time period is 88%. Specific Aims of the renewal include: determine the long-term effectiveness of highly active antiretroviral therapy (HAART);define determinants of individual responses to HAART including host genetic characteristics, co-infections (hepatitis viruses, HHV-8), immune responses to HIV, adherence, race/ethnicity;define mechanisms of host resistance to HIV infection and progression of the infection;and serve as a platform for independently funded pathogenesis studies and other collaborative research, including virological and immunological mechanisms of HIV-1 pathogenesis, laboratory correlates of disease progression or non-progression, and biological interactions between co-infections and HIV. These aims can be addressed only with continued followup of this extremely well-characterized cohort. SHARE and the MACS should continue to play a leading role in studies designed to lead to better treatments and preventive vaccines for HIV infection.

The Specimen Acquisition and Pathology Core is a key component of the SPORE and will support the translational goals of SPORE Projects, as well as developmental projects funded through the SPORE. Dr. Porter's leadership will provide not only research and clinical breast pathology expertise but also integration of Core B activities with the SPORE overall, and developmental projects in particular. This coordination of specimen acquisition and distribution with promotion of new research will greatly aid translation of basic discoveries into experiments involving human samples. The Core augments the existing robust FHCRC/UW Cancer Consortium Breast Specimen Repository and Registry (BSRR) and will function to centralize specimen acquisition, processing, pathologic analysis and distribution of samples needed by SPORE projects. It will also provide a laboratory base for select biomarker assays in support of the major and developmental projects. Specifically the Core will: 1) Use the established and efficient BSRR to procure and distribute tissue and blood specimens for SPORE and developmental projects 2) Perform protocol-specific specimen processing for SPORE and developmental projects 3) Maintain a database that allows specimen tracking and distribution, linkage with relevant deidentified data, and data sharing with other projects and cores 4) Provide high-quality pathology support for SPORE and developmental projects including: [unreadable] Pathology review of tissues at various levels of complexity [unreadable] Histology sectioning and preparation of section slides for histologic evaluation [unreadable] Immunohistochemistry (IHC), and new antibody work-up and optimization [unreadable] IHC interpretation of both immunoperoxidase and immunofluorescence stains [unreadable] Laser capture microdissection (LCM) [unreadable] Tissue microarray (TMA) design, construction, sectioning, and imaging

The guinea pig was used as an extremely sensitive animal model to investigate structure-toxicity relationships in polyhalogenated aromatic hydrocarbons. Results differ from structure activity relationships associa.ed with the dioxin receptor. A protein binding model is being developed which we hope will show a better correlation with the observed structure-toxicity results.

Obesity has reached epidemic proportions and associated health consequences are alarming, but successful treatment remains a significant challenge, because the underlying causes are complex. In addition to the physiological energy and nutrient needs, external, environmental influences can drive appetite and eating through cognitive and hedonic processes, independently from hunger. Substantial progress has been made in our understanding the homeostatic regulation of food intake and body weight and the integration between physiological and central mechanisms within the hypothalamic and brainstem circuitries. Much less is known about the neural mechanisms mediating environmental influences, yet they are important in health and disease. Cues from the environment can become signals for food through associative learning, and based on that acquired ability can control feeding behavior. These cognitive processes enhance survival when they function in concert with homeostatic control and their stimulatory effects may have been adaptive in the past when energy resources were scarce. The developed world is rich in easily accessible palatable foods and stimulatory effects of omnipresent food cues are maladaptive, as they drive overeating and weight gain. Thus, determining the neural mechanisms underlying this cognitive, non-homeostatic motivation to eat is crucial for potential therapeutic interventions. The core components of the forebrain network underlying cue-induced feeding have been identified and include the basolateral area of the amygdala (BLA), the lateral hypothalamus and orexin/hypocretin (ORX) neurons, and the ventromedial prefrontal cortex (vmPFC). Much remains unknown about the temporal functional connectivity within this network and it is critical to determine which specific circuit and neurotransmitter system is the controller of food motivation at test. The proposed studies will utilize cutting-edge chemogenetic methods, DREADDs and Daun02 inactivation, and precise neuroanatomical and neurochemical techniques to establish a novel vmPFC circuitry with the anterior paraventricular nucleus of the thalamus (PVTa) and ORX receptor 1 signaling is the key controller?an on/off switch within the network?for cognitive food motivation (Aim 1 & 2). Another goal is to determine if this integrative function is mediated by the vmPFC neuronal ensemble plasticity, through dynamic communications with the BLA and PVTa (Aim 3). These mechanisms will be interrogated in behavioral preparations for cue- induced consumption (Aim 1) and persistent food seeking (context-mediated renewal of extinguished responding to food cues; Aim 2) in male and female rats. Sex differences in context renewal of food seeking were recently established and experiments here will test whether the vmPFC and PVTa are sites of sex- specific regulation via connections with the ventral hippocampal formation. The findings from these studies will establish key neurotransmitter and circuitry mechanisms mediating cognitive food motivation and potential sites of sex differences and novel targets for treatment of insatiable appetite and overeating.

A number of primary discrete emotions emerge over the first year of life. Little is known about the relation between facial signs of these affects and concomitant central nervous system processes. The research proposed in this application is designed to provide the first fundamental knowledge on this question. It is novel methodologically in combining the precision of detailed coding procedures for the measurement of facial affect with sophisticated techniques for the analysis of noninvasively recorded brain activity. Infants at 6 months and 12 months of age will be studied. The same affect eliciting conditions will be presented at each age. These include presentation of a sweet and sour taste, placement on the visual cliff and the mother smiling or frowning at her infant. Brain electrical activity from left and right sided leads in the frontal and parietal regions will be recorded. The research will focus on hemispheric asymmetry associated with the presence of different facial signs of discrete emotion. These findings will provide important new information on the relation between maturational changes in brain function and the emergence of different emotion systems over the first year of life.

It is generally believed that defocus blur provides the primary stimulus for the accommodative system. Our investigations now support the view that accommodation responds to a variety of both dioptric and nondioptric stimuli. The proposed experiments will examine these two categories of stimuli by concentrating on the following: 1) the chromatic cues that result from the chromatic aberration of the eye; and 2) changes in target size and apparent distance. The research involves the development of a versatile stimulus system that will be used along with an infrared recording optometer to determine the mode of action of the stimuli, their relative importance, and how they interact with other stimuli such as defocus blur. Specifically the aims of the experiments are to (1) determine the frequency response of the accommodative system to defocus blur while the effects of the chromatic aberration of the eye are manipulated by achromatizing lenses and monochromatic light; (2) determine the frequency response of the accommodative system to changes in the wavelength of light--the color of the target will change in a sinusoidal manner; (3) determine the frequency response of the accommodative system to defocus blur under isoluminant stimulus conditions; (4) examine the relative importance of target size and defocus blur as stimuli for accommodation by stepping the stimuli (size and blur) in opposite directions; (5) examine the relative importance of changing size and changes in apparent distance on the accommodative response; (6) examine the relative importance of size change and velocity of size change as stimuli by using ramp and step-ramp changes in target size. The investigation will confirm that accommodation responds to a variety of dioptric and non-dioptric stimuli and the findings may therefore have implications for the treatment of a range of vision anomalies such as accommodative infacility, myopia and amblyopia.

The purpose of this study is to investigate the safety, side effects and effectiveness of a new compound (534U87) as an additional treatment for partial seizures in adults and adolescents.

Human temporal bones obtained at autopsy from patients with various forms of sensorineural deafness are to be studied by microdissection and phase-contrast examination of surface preparations for correlation of audiological and pathological findings. Similar studies will be carried out in monkeys treated with ototoxic aminoglycosides and other drugs, for which complete behavioral audiograms have been obtained before, during, and after treatment. The patas monkey will be studied as a model for drug ototoxicity in man, because it alone among laboratory animals has been found to be susceptible to the ototoxic action of dihydrostreptomycin at dose-levels similar to those formerly used in treating human tuberculosis. Structure-activity relationships for various aminoglycosides, enzymatic modifications, and fragments, will be explored in guinea pigs, as will the possible relationship between salicylate ototoxicity and prostaglandin effects on the inner ear microvasculature. The question of potentiation of cochlear injury by combinations of aminoglycoside treatment and noise will be explored in monkeys. Temporary and permanent threshold shifts and accompanying cochlear changes will be examined in monkeys exposed to recorded industrial noise from automotive factories and to impulsive noise from power-actuated tools used in the construction industry.

How do proteins fold into their unique three-dimensional structures? How does the three dimensional structure of a protein give rise to function? Can proteins be designed, either from first principles or through reconstruction of natural protein substructures? Can low molecular weight and highly stable mimics of natural proteins be designed? This meeting will describe progress towards understanding these questions that lie at the heart of modern molecular biology. In-recent years folding motifs have been recognized in a variety of gene regulatory proteins, and their structures are being determined by a combination of predictive methods, NMR, and crystallography. Considerable progress with the folding problem has been made in vitro studies as well as whole cells. Enormous progress has been made in understanding the structures and functions of membrane proteins. We have begun to study and manipulate the repertoire of biological molecular recognition using immunoglobulins and related molecules. This meeting should bring together chemists, molecular biologists, and cellular biologists to review recent advances in these areas, and discuss the future of this multidisciplinary field.

In this proposal, we request a second renewal of our Chemistry-Biology Interface (CBI) Predoctoral Training Grant. In 1995 we launched a new interdisciplinary training program at the University of Massachusetts that built on existing strengths and formalized the faculty's commitment to collaboration between the physical and life sciences. We implemented a CBI curriculum that meshed with the requirements of the three participating graduate programs, Chemistry, Molecular & Cellular Biology, and Polymer Science & Engineering, yet enabled students with either chemical or biological backgrounds to supplement training in their home discipline with training in the complementary discipline. [unreadable] [unreadable] During the current funding period, our CBI Training Program has grown substantially: the number of participating departments has grown from 3 to 4, the number of CBI Training Faculty has grown from 16 to 19 and most notably, the number of formally affiliated students has increased from 14 to 61. The addition of Chemical Engineering builds our strengths in quantitative training and in biomaterials, and adds new opportunities in bioengineering. We anticipate continued growth of our CBI Program as additional departments, notably Physics and Computer Science, move into CBI-related areas. The CBI Program currently provides NIH support for six predoctoral students, and a University match supports one additional graduate student working at the chemistry-biology interface. To continue to foster the growing strength of the CBI program as it expands into new disciplines and trains increasing numbers of students, we request a modest increase in the number of NIH-funded Traineeships in years 3-5 of the of the grant period. [unreadable] [unreadable] The interdisciplinary training we provide is exemplified by the development of a new "Drug Design" course that is now an integral part of the CBI curriculum. In this popular course, students are provided with background lectures in chemistry and biology to bring them to a common ground, followed by weekly seminars by speakers from the pharmaceutical industry to introduce students to the full range of topics in chemistry and biology that are critical for successful design and development of new drugs. With this type of training in the methods and intellectual framework of both chemistry and biology, our CBI graduates are equipped to pose and solve significant biomedical questions in their future work. [unreadable] [unreadable] [unreadable]

A "caged" molecule is a photosensitive, but temporarily inert, precursor of a biologically active molecule. Light absorption transforms the precursor into a molecule with full bioactivity. Because a light beam can be easily focused and steered, and photochemical reactions are extremely fast, caged molecules are versatile tools for using light to manipulate biology with exceptionally high spatial and temporal resolution. The long-term objective is to develop a broad spectrum of novel photochemical tools that will enable cellular physiologists to use light to probe and control dynamic signaling processes in living cells and tissues. The proposed research has four foci: 1) Develop new "cages" that a) are strongly activated by light, b) show fast kinetics of product release on photolysis, c) give high yield of product when photolyzed, and d) are chemically and metabolically stable in the absence of light. 2) Develop new probes for cellular signaling, specifically cage neurotransmitters, lipid messengers, as well as probes that permit "photochemical knock-out" of neurotransmitter receptors and transporters, and ion channels. 3) Develop and optimize two probes of intracellular calcium signaling: a caged calcium and a caged "anti- calcium" for using light to rapidly generate and ablate intracellular calcium signals, respectively. 4) Apply the developed probes to cellular physiology research in the areas of signal processing by nerve terminals and dendrites, synaptic plasticity, and calcium regulation of cell excitability.

The aim of this research is to compare preoperative, initial and later postoperative psychological capacities of patients with hemispherectomy for infantile hemiplegia and for brain tumor to provide data on the principles underlying the organization, disorganization and reorganization of cerebral functions in the development of the brain. Accumulating data have demonstrated systematic differences between effects of left vs. right hemispherectomy for neoplastic or traumatic lesions in adults. Left "dominant" hemispherectomy results in marked persisting impairment of speech, reading, verbal reasoning and auditory memory, but comprehension of speech is relatively spared, and nonverbal visual ideational, construction and memory functions are intact. Right hemispherectomy results in a reciprocal syndrome of selective impairment of nonlanguage functions, but language and verbal reasoning capacities are intact. The development of superior language, verbal and nonverbal reasoning capacities following left hemispherectomy for epileptogenic lesions incurred at birth in this research reflects the "functional plasticity" of the infant brain. Systematic studies of effects of hemispherectomy for lesions incurred at different stages in the development of the brain will provide data for assessing 1) the nature, rate, and extent to which the "functional plasticity" of the infant brain diminishes with advancing age; 2) the extent to which each hemisphere participates in certain language and nonlanguage functions and specializes in others as the brain matures; and 3) the limitations and principles underlying reorganization of cerebral functions following removals of either hemisphere for lesions incurred at different stages in the development of the brain.

Chronic Traumatic Encephalopathy (CTE) is a progressive neurodegenerative disease that occurs as a consequence of repetitive mild traumatic brain injury (mTBI). CTE causes personality and behavioral changes, executive dysfunction and memory loss and is characterized by the accumulation of phosphorylated tau protein in the brain. The ongoing wars in Iraq and Afghanistan have resulted in a large cohort of OEF/OIF veterans with mTBI from blast exposure who may be at increased risk for CTE. Presently, CTE can only be diagnosed at autopsy and there are no known clinical biomarkers. The development of biomarkers to diagnose this disorder in living individuals would be a major scientific and clinical advance. We hypothesize that multiple combat blast-induced mTBIs cause altered tau metabolism indicative of preclinical CTE. We expect that increased tau biomarkers will be detected in cerebrospinal fluid (CSF) and in brain using advanced brain imaging techniques in veterans with a history of multiple blast mTBIs. In addition, recent data from our Translational Research Center for TBI and Stress Disorders (TRACTS) show that veteran with significant blast exposure exhibit greater rates and more severe post-traumatic stress disorder (PTSD) than veterans not exposed to blast neurotrauma. This suggests that the same individuals at risk for PTSD may also be at high risk for CTE. This project will draw its sample from the TRACTS cohort, which presently consists of 200 OEF/OIF veterans and will increase to ~400 during the proposed study period. History of blast exposure and TBI are documented via a semi-structured interview using the Boston Assessment of TBI: Lifetime (BAT-L). PTSD and other psychiatric illnesses are diagnosed using DSM-IV criteria. The proposed study will investigate the correlative relationship between mTBI metrics (number, severity, distance from blast) and neurocognitive measures of attention and executive function, phosphorylated tau pathology, axonal fiber tract damage, and structural brain changes. We will recruit two groups of 30 participants (total sample size of 60) from the TRACTS cohort that differ maximally in their history of combat TBI: (1) two or more blast mTBIs with associated cognitive symptoms (i.e., acute change in mental status, post-traumatic amnesia or loss of consciousness immediately after the blast) (mTBI); and (2) a matched control group without history of blast exposure or impact head injury (CON). All participants will have served in either OEF or OIF and have a diagnosis of PTSD based on DSM-IV criteria. We will measure tau biomarkers (phosphorylated tau (ptau), total tau, exosome ptau, oligomeric tau) in cerebrospinal fluid (CSF) by plate-based ELISA and conduct advanced brain imaging techniques using simultaneous PET/MRI. We will determine whether there is an association between CSF tau biomarkers, regional (dorsolateral frontal and orbital frontal) PET index metrics, mTBI metrics (number, severity, distance from blast), and neurocognitive measures of attention and executive function, Blast associated mTBI, PTSD and CTE represent significant scientific challenges in terms of determining their biological origins, their differential diagnosis and respective treatments. If not addressed immediately, the VA will be faced with daunting costs and responsibilities associated with caring for these debilitating neurological and psychiatric disorders. The development of reliable biomarkers to detect CTE will enable the diagnosis and monitoring of this condition during life and assessing the efficacy of potential therapies as they are developed. ! PUBLIC HEALTH RELEVANCE: Chronic Traumatic Encephalopathy (CTE) is a neurodegenerative disease that occurs as a consequence of mild traumatic brain injury (mTBI). CTE causes personality and behavioral changes, executive dysfunction and memory loss and is characterized by the accumulation of tau protein in the brain. OEF/OIF veterans with a history of mTBI from blast exposure may be at increased risk for CTE. We will measure tau in cerebrospinal fluid (CSF) and conduct advanced brain imaging techniques in blast-injured OEF/OIF veterans in order to develop biomarkers for CTE, as currently none exist. If research geared at determining biomarkers and treatments for blast associated mTBI and CTE is not addressed immediately, the VA will be faced with daunting costs and responsibilities associated with caring for these debilitating neurological and psychiatric disorders. !

The cytoplasmic enzyme glycerophosphate dehydrogenase (GPD) plays a pivotal role in lipid synthesis and fuel metabolism in a number of tissues. Our overall objective is to delineate the molecular processes which activate and modulate the expression of the specific gene encoding GPD in skeletal muscle. Towards this goal we have already demonstrated the responsiveness of chimeric GPD genes in myogenic cell lines, which represents the first successful regulation of appropriate GPD expression in vitro. We have now identified by deletional analysis a 1 kb intragenic region downstream from the GPD promoter which confers differentiation-dependent expression on an attached reporter gene in mouse C2-skeletal muscle cells. This DNA segment will be further dissected to localize sequences that specifically activate GPD late in the myogenic program. Our recent studies suggest that GPD regulation involves a novel myoblast silencer plus a muscle-specific enhancer, distinct from characterized muscle enhancers. To elucidate the mechanism of GPD activation, we will use in vitro binding and footprinting assays and site-directed mutagenesis of GPD regulatory sequences to define nuclear factor binding sites, their differentiation-dependent usage, and the profile of nuclear proteins that interact with them. Similarly, the DNA sequences and trans-acting factors which mediate inhibition by two catabolic agents, cyclic AMP-linked lipolytic hormones and tumor necrosis factor (the active agent of cachexia), will be characterized. Novel DNA- binding regulatory factors that control GPD expression will be isolated from cDNA expression libraries. We describe a new strategy, using chimeric c-fos/GPD constructs, to delineate the molecular basis for insulin stimulation of GPD transcription and GPD mRNA stability. Finally, we will determine the pattern of GPD expression in different muscle fibers and how disease-related states that affect insulin responsiveness in skeletal muscle (denervation, high fat feeding, genetic obesity) affect GPD expression in vivo. These experiments should yield novel insights into the mechanisms by which metabolic gene expression is regulated in skeletal muscle during development and in response to hormonal controls.

The Trials of Hypertension Prevention (TOHP) is a collaborative multicentered research program designed to test the feasibility and short-term effectiveness of selected nutritional and behavioral non-pharmacologic interventions in reducing or preventing an increase in diastolic blood pressure (DBP) in normotensive individuals. The information regarding the efficacy, safety and feasibility of the multiple interventions tested in Phase I would be used to assess the potential benefit of proceeding to a full-scale trial, Phase II, the objective of which would be to determine the effectiveness of the most promising of these therapies in reducing the incidence of frank hypertension. As the Coordinating Center, in conjunction with the Planning Committee, we would play a collaborative role in the design, conduct and analysis of the trial. During the 9-month planning period (Phase 1A), our major responsibility would be to coordinate the efforts of the Planning Committee in preparing for the start-up of the trial at the clinical centers. Specific activities would include the development of standardized trial materials including the study protocol, data forms, and a detailed Manual of Operations, as well as the training and certification of clinical center personnel. We would also assume responsibility for designing and implementing data processing and management programs. During the implementation period (Phase 1B), the Coordinating Center would be responsible for integrating the activities of the various collaborating units of the study (e.g., clinical centers, central laboratory, NHLBI). Major functions would include: (1) the collection, receipt, quality control, and analysis of data; (2) serving as the major interface with the clinical centers; and (3) facilitating the activities of all supervisory committees. Thus, the Coordinating Center will serve a proactive and collaborative role as an epidemiologic, statistical, clinical, and data management resource.

The yeast Pdr5 multidrug efflux pump is a member of the ABC transport family of proteins. It is similar in structure and substrate specificity to those transporters found in pathogenic fungi such as Candida albicans and Cryptococcus neoformans. A major question in the study of all ABC transporters is how signals are communicated between the drug binding sites in the transmembrane domains from which xenobiotic compounds are transported and the nucleotide-binding domains where ATP is hydrolyzed to produce the energy for efflux. Work in our lab during the present funding period has started to define the residues required for interdomain cross talk. In this application for renewal, we propose a combination of genetic and biochemical experiments that should flesh out much of this interface which appears to extend from the N-terminal nucleotide-binding domain through the intracellular loops and into transmembrane-helix 2. We will use a combination of suppressor and site-directed mutations to identify more of the interface. Residues involved in communicating signal and those specifically required for drug binding will be distinguished using several assays including a well-established iodoarylazidoprazosin-binding assay. We will also begin to explore the details of the Pdr5-specific ATPase catalytic cycle as th relationship between drug binding and hydrolysis is poorly understood in this transporter. PUBLIC HEALTH RELEVANCE: Multidrug resistance to chemotherapeutic and microbial agents constitutes a major problem in the treatment of cancer and infectious diseases. Some of this is due to the overexpression of ABC efflux pumps: proteins that use the energy from ATP to drive the transport of a very broad range of xenobiotic compounds from the cell. This proposal will investigate the relationship between drug binding, ATP hydrolysis and drug efflux in the major yeast transporter Pdr5 using bioinformatic, genetic, and biochemical approaches.

Project MATCH was a multisite clinical trial (Cooperative Agreement) that produced a rich data set on patient treatment amtching in alcoholism treatment.The objective of this contract is to support the preparation and refinement of the data collected in Project MATCH for distribution to the scientific community. Establishment of a user- friendly data tape and associated documentation, and the maintenance of a public website interface with users is required.

The long term objective of this proposal is to devise a very rapid inexpensive mutagenesis test which could be used to screen chemical compounds in industry, the environment, and food. The correlation between mutagenicity and carcinogenicity would identify these compounds as potential carcinogens/teratogens. The specific aim is to devise a system using molecular genetics, which gives a very rapid, sensitive and easily assayed response to low concentrations of mutagens. A mutagen screening test which has the same reliability as the Ames test, but is inexpensive, extremely rapid, capable of automation and consequently could be used to screen hundreds of compounds per day would have an extremely competitive commercial application.

Here, we investigated how HCV-infection of hepatoma cells affects the response of innate immune cells, namely NK cells. As an experimental model we used hepatoma cell cultures that were either infected with the genotype 2a Japanese fulminant hepatitis-1 strain or transduced with luciferase-tagged subgenomic HCV replicons. HCV infection of hepatoma cells was found to attenuate IFN-induced expression of MHC class I. This was associated with replicating HCV RNA, and not with viral protein expression. HCV infection reduced IFN-induced synthesis of MHC class I protein and induced phosphorylation of PKR and eIF2α. IFN-induced MHC class I expression was restored by small hairpin RNA-mediated knockdown of PKR in HCV-infected cells. Co-culture of HCV-specific CD8+T cells and HCV-infected cells that expressed HLA-A2 demonstrated that HCV infection reduced the effector functions of HCV-specific CD8+ T cells; these functions were restored by small hairpin RNA-mediated knockdown of PKR. Downregulation of MHC class I expression should result in increased stimulation of NK cells. However, production of interferon (IFN)γ by natural killer (NK) cells is typically attenuated in patients with chronic HCV infection compared to those of uninfected controls. To investigate whether this is due to effects of additional cell populations, we cultured hepatoma cells that express luciferase-tagged subgenomic HCV replicons (Huh7/HCV replicon cells) or their HCV-negative counterparts (Huh7), with NK cells in the presence or absence of other populations of PBMC. Antiviral activity, cytotoxicity, and cytokine production were assessed. We found that NK cells produced greater amounts of IFNγ when exposed to PBMC co-cultured with Huh7/HCV replicon cells than with Huh7 cells. In addition, NK cells and PBMC from controls suppressed HCV replication to a greater extent than those from patients with chronic HCV infection. This antiviral effect was predominantly mediated by tumor necrosis factor (TNF)α and IFNγ. The antiviral activity of NK cells and their production of IFNγ were reduced when they were used in co-culture alone (rather than with PBMC), after depletion of CD14+ monocytes, following knockdown of the inflammasome in monocytes, or after neutralization of interleukin (IL)-18, which is regulated by the inflammasome. These findings indicate a role for monocytes in NK cell activation. Compared with control monocytes, monocytes from patients with chronic HCV infection had reduced TNFα-mediated (direct) and reduced NK-cell mediated (indirect) antiviral effects. Control monocytes increased the antiviral effects of NK cells from patients with chronic HCV infection and their production of IFNγ. Thus, patients with chronic HCV infection appear to have reduced monocyte function, which attenuates IFNγ responses of NK cells.

In the prior funding period we have introduced a new approach to genetic mapping in inbred rodent strains that exploits closely related lines differing in traits of interest. Using a 10K SNP map, we have shown that two SHR lines differ at only 13% of their genomes, but that these differences have a profound effect on susceptibility to hypertensive renal disease. The close genetic similarity has allowed us to perform fine mapping that has resulted in the identification of three highly resolved quantitative trait loci (QTL) affecting blood pressure and renal injury. Because of the similarity between the lines, each of these QTL maps to a small, isolated block where the two SHR lines have descended from different ancestors. These blocks are surrounded by extensive regions that are identical-by-descent (IBD) and thus help to narrowly define the QTL's, down to a small number of genes. In the present study we propose to identify the genes in each QTL that contribute to increased hypertensive renal disease and to understand the mechanisms by which they act. One QTL has effects on both blood pressure and renal injury. We seek to identify the causative variation and determine whether it acts first on blood pressure with secondary effects on injury or whether it lies in a pathway that produces injury that then leads to reduced renal function and increased blood pressure. Another QTL has no effect on blood pressure and appears to lead to glomerular damage directly. We have also identified the immunoglobulin heavy chain as a locus containing extensive variation across our lines. We have shown that this includes variation with important effects on IgG function including the inability to transfer IgG from mother to offspring. This variation associates with increased albuminuria. We propose to investigate whether alterations in immune function that are encoded by differences in the heavy chain of immunoglobulin contribute to the emergence of renal disease in the susceptible SHR line and whether maternal-offspring IgG transfer is involved in the transmission of risk.

Conotruncal heart defects are severe life threatening malformations whose treatment requires substantial clinical and surgical interventions throughout childhood and into adult years. But the causes of conotruncal heart defects are largely unknown. The proposed research program will focus on detecting genetic contributions to the two most common conotruncal defects, tetralogy of Fallot and d-transposition of the great arteries. The recent development of array comparative genomic hybridization (array-CGH) using mapped bacterial artificial chromosome (BAC) clones will allow us to employ this high resolution, genome-wide screening technique to detect submicroscopic chromosomal imbalances. We propose to identify de novo and familial chromosomal microdeletions among infants with conotruncal heart defects by performing array-CGH with a 32,000 clone BAC array. The microdeletions that we identify will provide us with relatively small chromosomal regions from which to identify candidate genes for conotruncal defects. We will also design and apply multiplex ligation dependent probe amplification (MLPA) assays to identify haploinsufficiency of known candidate genes for conotruncal defects. The results of this research should lead to the development of comprehensive, clinically applicable MLPA assays that will detect copy number changes of all conotruncal heart defect genes and their exons. Our 2-year research program will use data from a recently completed population-based case-control study composed of 500 California infants with tetralogy of Fallot and d-transposition of the great arteries, delivered between 1999 and 2004. This is the largest case-control study of infants with conotruncal defects and will uniquely generate population-based genotypic data on candidate genes for conotruncal defects. Overall, this research program attempts to enhance our scientific understanding of the genetic causes of conotruncal defects. Because conotruncal defects result in substantial morbidity, as well as high emotional and economic costs, expanding our understanding of their causes may lead to preventive interventions that would greatly benefit public health and society.

We propose to elucidate certain conformational aspects of gonadotropins (e.g., luteinizing hormone, chorionic gonadotropin, pregnant mare serum gonadotropin, and follicle-stimulating-hormone), growth hormone, prolactin, adrenocorticotropin, and placental lactogen. This will include measurements of circular dichroism, fluorescence, electron-spin-resonance, ultra-centrifugation, and viscosity. For the gonadotropins particular attention will be devoted to the nature of the conformational changes occuring upon subunit association-dissociation. For growth hormone the studies will be concerned mainly with membrane interactions as monitored by adiabatic rapid passage electron-spin- resonance. We propose to investigate the molecular mechanisms involved in the metabolism (i.e., plasma clearance, urinary excretion, tissue uptake, intracellular catabolism, and inactivation) of the various pituitary hormones. The data will be analyzed using a compartment model with an intravascular pool and several extravascular pools. Structure- function relationships of gonadotropins will be examined by determining the effects of specific amino-acid modifications on binding to Leydig cells and on the stimulation of steroidogenesis and cyclic-AMP in Leydig cells. The effects of chemical and enzymic modification of growth hormone will be investigated in an effort to define active-site peptides and residues essential for somatomedin induction. These studies are aimed at elucidating on a molecular level the relationships between hormone structure and function, biological actions, and metabolism.

The purpose of this research program is to gain new insights into cellular and molecular mechanisms that regulate expression of ion channels in heart. Although our understanding of mechanisms regulating contractile protein gene expression has advanced rapidly, we know relatively little about regulation of expression of another structure crucial to control of cardiac contractile performance: the L-type calcium channel. A goal of this project is to learn how neural, ionic and paracrine factors determine how many functional copies of the L-type calcium channel are present in the sarcolemma of heart cells. Recent experiments from this laboratory demonstrate that sympathetic innervation in vitro increases calcium channel expression determined by ligand binding and patch clamp studies. In a cultured rat ventricular myocyte model, we will determine by what mechanisms sympathetic innervation and specific neuroeffectors regulate dihydropyridine (DHP) receptor gene expression and the relation between abundance of message, DHP receptor protein, and channel function. A myocyte-sympathetic ganglion co-culture model will be utilized that permits studies on direct myocyte innervation and denervation. Then, in cultures of myocytes alone, three candidate neuroeffectors will be studied: norepinephrine, nerve growth factor and calcitonin gene-related peptide. The role of protein kinases C and A in mediating expression of DHP message and protein will be assessed. There is evidence suggesting that alterations in cytosolic calcium concentration may regulate expression of calcium channels. Accordingly, we will determine whether, and by what means, a decrease or increase in cytosolic calcium concentration alters the abundance of DHP receptors in heart. Furthermore, immunofluorescence and immunocolloidal gold labeling studies will be conducted to determine where in cardiac myocytes DHP receptors are localized. The topology of the DHP receptor in the sarcolemmal membrane will be examined with recently developed site-directed antibodies. Novel probes and powerful contemporary techniques will be used to examine the integrated cellular biological response to altered DHP receptor gene expression. Abnormalities of calcium channel expression have been implicated in. pathophysiological conditions, including hypertrophy, arrhythmias, and cardiomyopathy. Gaining further understanding of mechanisms that regulate calcium channel expression is of fundamental biological importance and will have therapeutic implications.

DESCRIPTION (adapted from the application): Inflammation of the airway epithelium is often required for effective innate defense against microbes, and epithelial cells provide critical biochemical signals that regulate this response. One major mechanism that epithelial cells in the airway use to participate in the inflammatory response is through regulation of leukocyte trafficking and/or activation by expression of intercellular adhesion molecule-1 (ICAM-1). ICAM-1 serves as a ligand for leukocyte beta2-integrins and thereby mediates epithelial-leukocyte interactions that may allow for "appropriate" inflammatory responses (e.g., to a respiratory bacterial infection) or "inappropriate" responses (e.g., airway inflammation in cystic fibrosis). This proposal focuses on Haemophilus influenzae, which frequently colonizes human respiratory mucosa and often produces respiratory tract disease, particularly in patients with chronic bronchitis, bronchiectasis, and cystic fibrosis. The specific aims of this proposal are based on four observations regarding airway epithelial cell ICAM-1 expression in response to H. influenzae: 1) H. influenzae induces airway epithelial cell ICAM-1 expression in vivo and in vitro; 2) ICAM-1 expression is required for efficient bacterial clearance in a murine model of airway infection with H. influenzae; 3) increased ICAM-1 expression can be initiated by epithelial cell interaction with a constitutive molecule on the bacterial cell surface; and 4) airway epithelial cell interaction with H. influezae results in generation of soluble ICAM-1 inducing activity containing a novel mediator(s) of ICAM-1 expression. Based on these observations, they hypothesize that direct induction of specific epithelial genes (such as ICAM-l) allow for rapid targeting and/or activation of neutrophils and other leukocytes at sites of H. influenzae infection, resulting in efficient innate defense in the airway. Accordingly, there are two specific aims. 1) Define mechanisms for induction of epithelial cell ICAM-1 expression by H. influenzae. This aim will take advantage of in vitro coculture models of epithelial cell interaction with bacteria. Definition of mechanisms for ICAM-1 gene activation in response to H influenzae will be accomplished by analysis of ICAM-1 promoter function and identification of mediator molecules. This latter refers to the observation that airway epithelial cells challenged with H. influenzae release a novel soluble factor into the medium capable of eliciting ICAM-1 in naive epithelial cells. 2) Determine functions of ICAM-1 in defense against H. influenzae infection. This aim will take advantage of in vivo murine models of airway infection by bacteria. The functions of ICAM-1 will be determined by examining ICAM-1 expression, leukocyte recruitment and function, and bacteria clearance under conditions that allow for manipulation of airway defense factors.

This study is designed to describe the spectrum of clinical course from "silent" IDDM to fasting hyperglycemia and clinical symptoms, and to determine if factors which are known to regulate glucose homeostasis are associated with further clinical deterioration.

Toxoplasma gondii is a serious pathogen of humans and livestock in the U.S.A. and world-wide. In addition to its well-known pathogenesis in the developing fetus, in recent years this protozoan parasite has ncreased its notoriety through the fatal disease it can cause in AIDS patients. Currently, there is no vaccine for Toxoplasma that is designed to impact human health and no drug capable of eliminating the persistent, chronic infection. Disease in AIDS patients is thought to largely result from the reactivation of a chronic infection that persists through the ability of the parasite to differentiate from the actively dividing tachyzoite stage to an encysted bradyzoite stage. The resulting tissue destruction, particularly in the brain and lungs can lead to severe disease or even death. Our goal is to identify and characterize parasite genes (and their respective products) that are critical for the differentiation of tachyzoites to bradyzoites. To identify these genes, we will use a combination of genetic and molecular techniques. For the molecular analysis, we will use microarrays to monitor changes in gene expression over a time-course of differentiation. A novel method developed by us will be used to determine whether these differences are the result of changes in mRNA synthesis or stability. A combination of forward and reverse genetics will then be used to determine the cis-elements mediating these changes. To identify genes with a central role in the differentiation process, we will select mutants that are not able to differentiate from tachyzoite to bradyzoite. This will be done by selecting for parasites that fail to express bradyzoite-specific surface markers. The genes will be identified and the role of their protein products will be determined. In this way, we will learn what pathways are key to differentiation and growth of bradyzoites. This work will lay the foundation for generation of an animal vaccine that produces a self-limiting infection that cannot be transmitted to other animals or humans. Such a vaccine would break the zoonotic cycle of animal to human transmission.

"Accurate diagnosis of solid pediatric tumors requires a combination of diagnostic techniques including reverse transcription polymerase chain reaction (RT-PCR). Many pediatric solid tumors exhibit fundamental cytogenetic abnormalities that have implications in their pathogenesis. The Ewing's sarcoma family of tumors (ESFT) and alveolar rhabdomyosarcoma (RMS) are characterized by consistent chromosomal translocations which result in the fusion of genes and subsequent formation of novel chimeric genes. These molecular markers can be detected by RT-PCR or fluorescence in situ hybridization (FISH) and can be used not only to establish the diagnosis in difficult cases, but also to understand the pathogenesis of these tumors. Recently, the products of these fusion genes have become the target of vaccine therapies in newly established protocols in the Pediatric Oncology Branch (POB) at the NCI. The objective of this project is: (1) to provide state of the art diagnosis of solid pediatric tumors (2) to assist in the evaluation of pediatric tumor specimens for the presence or absence of specific fusion transcripts and (3) to evaluate the significance of molecular markers in the diagnosis, classification and pathogenesis of pediatric sarcomas. The following accomplishments have been made in the last year: (1) A total of 120 Pediatric Pathology reports were issued. (2) A total of 60 RT-PCR reports were issued. (3) A series of 50 pediatric sarcomas were evaluated by RT-PCR and conventional methods. The study showed that RT-PCR is needed for diagnosis in selected cases and should be interpreted in conjuction with the morphlogic findings (4) FISH is a very useful technique to detect fusion products in paraffin sections (5) Olfactory neuroblastoma is negative for the EWS/Fli-1 fusion transcripts by FISH, in contrast to the ESFT. (6) Microsatellite markers at the tyrosine hydroxylase locus in chromosome 11p are helpful in the distinction of RMS from other spindle cell sarcomas, but not in the distinction of alveolar from embryonal RMS."

Phycobilisomes are major light harvesting complexes in cyanobacteria and some eukaryotic algae. The complex includes both pigmented proteins (phycobiliproteins) and nonpigmented proteins (linker proteins). The phycobiliproteins in eukaryotic algae are synthesized in the chloroplast while the nonpigmented linker proteins are synthesized in the cytoplasm (and must be imported into the organelle). Since the subunits among the different phycobiliproteins are related, they are thought to represent a gene family localized to the plastid DNA. Recently, a gene encoding the Beta subunit of phycocyanin has been placed on the small single copy region (this region encodes at least one additional phycobiliprotein subunit) of the plastid genome of Cyanophora paradoxa. We propose to locate the other phycobiliprotein subunits (Alpha phycocyanin and Alpha and Beta allophycocyanin) on the plastid genome, determine whether these genes are transcribed separately or as a unit, and generate specific probes for identifying analogous genes in cyanobacteria. The synthesis of several phycobiliprotein subunits in cyanobacteria is regulated by light (complementary chromatic adaptation), and this regulation may be at the transcriptional level. The probes generated from the plastid genome of C. paradoxa will enable us to identify these light-regulated phycobiliprotein genes and analyze the molecular events involved in complementary chromatic adaptation. Experiments involving differential hybridizations of RNA isolated from cyanobacteria grown in different light qualities to clone banks of cyanobacterial DNA, and immunological techniques which would enable us to identify clones synthesizing phycobilisome polypeptides, would also aid in the isolation of light-regulated phycobilisome genes. Once these genes are obtained, we will examine their arrangement on the cyanobacterial genome (clustered or dispersed), characterize their transcription (especially in response to different light qualities) and sequence their 5' ends to establish features which might be essential for light-regulated transcription.

NovaScreen Biosciences Corp. has successfully completed a Phase I SBIR project (#1 R43 AA014542-01, entitled "Novel Molecules to Treat Alcoholism") and is submitting this Phase II SBIR application in response to RFA-AA-04-002, "Medications Development to Treat Alcoholism." The specific objective of this Phase II application is to discover and to advance at least one novel, optimized, new chemical entity (NCE) up to the stage of preclinical development. That NCE will be designed to be concurrently active at multiple (two) molecular targets, each a validated drug target for treating alcoholism. Compounds active at multiple targets may have greater therapeutic efficacy than agents acting at single targets, and display fewer side effect liabilities than cocktails of two or more different drugs each active at single targets. Our Phase I SBIR research has produced a promising lead compound and an array of structure-activity relationship (SAR/QSAR) models of small molecules that concurrently modulate the serotonin 5HT3 receptor and the mu-opioid receptor. Additionally, we also produced SAR/QSAR models for compounds that display dual activity at 5HT3 and at other opioid receptor subtypes (i.e., 5HT3-delta opioid and 5HT3-kappa opioid). Our emphasis in this Phase II builds on Phase I results and employs these SAR/QSAR models to continue optimization and development of the identified lead compound to produce a new generation of therapeutic candidates for the treatment of alcohol dependency. [unreadable] [unreadable]