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Matern_Child_Health_J-2-2-1592150

Human Immunodeficiency Virus-1 and Preconception Care

Human immunodeficiency virus (HIV) is a condition that increasingly affects women, especially women of childbearing age. Early in the HIV epidemic, HIV infection and AIDS were diagnosed in relatively few women in the United States. Today, the HIV/AIDS epidemic represents a growing and persistent health threat to women, especially young women and women of color. In 2001 and 2002, HIV infection was the leading cause of death for African American women aged 25–34 years and was among the four leading causes of death for African American women aged 20–24 and 35–44 years, as well as Hispanic women aged 35–44 years [1, 2]. Although HIV is increasing among women, the incidence in children from perinatal transmission is decreasing. With recommendations for routine screening for HIV during pregnancy and the availability of effective interventions to dramatically reduce the risk of perinatal transmission, the number of annual perinatally acquired AIDS cases from 1985–1994 has declined approximately 95% [3], representing one of the most striking success stories in the 25-year effort to reduce the impact of HIV/AIDS in the United States. HIV infection can be transmitted from mother to child in utero, during labor and delivery and through breastfeeding. In the absence of intervention and without breastfeeding, the risk of perinatal HIV transmission is approximately 25% (range 13–39%) [4]. Evidence suggests that in the absence of breastfeeding, 30% of transmission occurs before birth and 70% around the time of delivery [4]. In February 1994, the results of the Pediatric AIDS Clinical Trials Group (PACTG) Protocol 076 documented that zidovudine (ZDV) chemoprophylaxis, initiated at 14– 34 weeks gestation and continued through pregnancy, followed by intravenous ZDV during labor and oral ZDV to the infant for the first 6 weeks of life, could reduce perinatal HIV transmission by nearly 70% [5]. A U. S. Public Health Service (USPHS) task force subsequently issued recommendations for the use of ZDV for reduction of perinatal HIV transmission [6] and universal prenatal HIV counseling and HIV testing with consent for all pregnant women in the United States [7]. Epidemiologic data have further confirmed the efficacy of ZDV for reduction of perinatal transmission, even among women with advanced disease, low CD4+ T-lymphocyte counts, and prior ZDV therapy [8–13]. In addition, substantial advances have taken place in the treatment and monitoring of persons with HIV disease, which have resulted in changes in standard antiretroviral therapy for HIV infected adults. More aggressive combination drug regimens are now recommended to maximally suppress viral replication. For pregnant women, however, the benefits of antiretroviral therapy must be weighed against the risk of adverse events to the woman, fetus, and newborn; however pregnancy is generally not a reason to defer standard therapy. It should be offered with the addition of ZDV for prevention of perinatal HIV transmission to HIV infected pregnant women [4, 14]. For women with HIV infection, preconception care must focus on recommendations for safe sexual practices that prevent HIV transmission to sexual partners and protect women from acquiring potentially harmful sexually transmitted diseases or even additional strains of HIV that may be more virulent or resistant to therapy. Women also need education about perinatal transmission risks and prevention strategies, expectations for the child's future, and where desired, effective contraception until the optimal maternal health status for pregnancy is achieved, including appropriate therapy to maximally reduce viral load and optimize immune function. For HIV-infected women desiring pregnancy, specific counseling regarding available reproductive options that both prevent HIV exposure to uninfected partners, as well as preventing superinfection with resistant or more virulent virus should be discussed. The USPHS recommends that the following be included in preconception counseling for HIV-infected women: 1) selection of effective and appropriate contraceptive methods to reduce the likelihood of unintended pregnancy; 2) education and counseling about perinatal transmission risks, strategies to reduce those risks, and potential effects of HIV or treatment on pregnancy course and outcomes; 3) initiation or modification of antiretroviral therapy avoiding agents with potential toxicity for the developing fetus (e.g., efavirenz, hydroxyurea), choosing agents effective in reducing perinatal HIV transmission, attaining a stable, maximally suppressed maternal viral load, evaluating and controlling for therapy associated side effects which may adversely impact maternal-fetal health outcomes (e.g., hyperglycemia, anemia, hepatic toxicity); 4) evaluation and appropriate prophylaxis for opportunistic infections and administration of immunizations (e.g., influenza, pneumococcal, or hepatitis B vaccines) as indicated; 5) optimization of maternal nutritional status; 6) institution of the standard measures for preconception evaluation and management (e.g., assessment of reproductive and familial genetic history, screening for infectious diseases/sexually transmitted diseases, and initiation of folic acid supplementation); 7) screening for maternal psychological and substance abuse disorders, and 8) planning for perinatal consultation if desired or indicated [4]. The USPHS further notes that HIV infected women of childbearing potential receive primary healthcare services in various clinical settings, e.g., family planning, family medicine, internal medicine, obstetrics/gynecology, and stress that primary health-care providers should consider the fundamental principles of preconception counseling an integral component of comprehensive primary health care for improving maternal and child health outcomes [4]. The Infectious Disease Society of America (IDSA) recommends in-depth discussions about childbearing early in the course of HIV care for women who express a desire for a future pregnancy, who are not trying to conceive but are not using appropriate contraception consistently or who express uncertainty about reproductive plans. The goal of these discussions is to ensure informed decisions about contraception and to offer preconception counseling if pregnancy is desired. IDSA recommends that providers regularly assess women's reproductive plans and their desire to have questions answered and, that providers question patients at each visit about interval menstrual history and sexual and contraceptive practices, offering pregnancy testing as indicated [15]. The American College of Obstetricians and Gynecologists (ACOG) and the American Academy of Pediatrics (AAP) recommend that screening for HIV infection be strongly recommended for women with unknown HIV status and who are considering pregnancy [16, 17]. The Centers for Disease Control and Prevention (CDC) is considering this recommendation for its revised recommendations for HIV testing in healthcare settings planned for 2006 [3]. In addition, these organizations as well as the Institute of Medicine and the U. S. Preventive Services Task Force recommend universal prenatal screening for HIV infection [3, 17–21], so that HIV-infected pregnant women can take full advantage of the available interventions to reduce perinatal transmission as well as to receive care for themselves. These organizations support an opt-out approach to HIV screening, (i.e., patients are notified that HIV testing will be done and consent is inferred unless testing is declined) [3] to normalize HIV testing in health care settings and to reduce the barriers clinicians experience with specific HIV test counseling and written informed consent. Pre-test discussions regarding risk reduction for HIV infection are not a requirement for HIV testing and can be provided in the context of discussions of reproductive health and STD prevention. Routine HIV testing during preconception care can be integrated with other aspects of preconception care that require a blood sample and should not require substantial additional time commitments by providers. Improved implementation of preconception care will further extend the success in reducing perinatal HIV transmission in the U. S. as well as improve access to HIV testing and care to women. Because the issues accompanying HIV treatment during pregnancy and perinatal prophylaxis are rather complex, the case for preconception counseling and care for known HIV-infected women is clear.

Acta_Neuropathol_(Berl)-3-1-1868652

Silver diagnosis in neuropathology: principles, practice and revised interpretation

Silver-staining methods are helpful for histological identification of pathological deposits. In spite of some ambiguities regarding their mechanism and interpretation, they are widely used for histopathological diagnosis. In this review, four major silver-staining methods, modified Bielschowsky, Bodian, Gallyas (GAL) and Campbell–Switzer (CS) methods, are outlined with respect to their principles, basic protocols and interpretations, thereby providing neuropathologists, technicians and neuroscientists with a common basis for comparing findings and identifying the issues that still need to be clarified. Some consider “argyrophilia” to be a homogeneous phenomenon irrespective of the lesion and the method. Thus, they seek to explain the differences among the methods by pointing to their different sensitivities in detecting lesions (quantitative difference). Comparative studies, however, have demonstrated that argyrophilia is heterogeneous and dependent not only on the method but also on the lesion (qualitative difference). Each staining method has its own lesion-dependent specificity and, within this specificity, its own sensitivity. This “method- and lesion-dependent” nature of argyrophilia enables operational sorting of disease-specific lesions based on their silver-staining profiles, which may potentially represent some disease-specific aspects. Furthermore, comparisons between immunohistochemical and biochemical data have revealed an empirical correlation between GAL+/CS-deposits and 4-repeat (4R) tau (corticobasal degeneration, progressive supranuclear palsy and argyrophilic grains) and its complementary reversal between GAL-/CS+deposits and 3-repeat (3R) tau (Pick bodies). Deposits containing both 3R and 4R tau (neurofibrillary tangles of Alzheimer type) are GAL+/CS+. Although no molecular explanations, other than these empiric correlations, are currently available, these distinctive features, especially when combined with immunohistochemistry, are useful because silver-staining methods and immunoreactions are complementary to each other. Introduction The year 2006 was the centenary anniversary of the first clinical description of a presenile patient with progressive cognitive decline, later defined as Alzheimer’s disease (AD) [7]. It was the same year (1906) when Santiago Ramón y Cajal and Camillo Golgi were awarded the Nobel prize for their contributions on neuroanatomy achieved by using silver-staining methods, especially Golgi-silver technique. In the field of neuropathology as well, silver staining was the principal method of choice at that time, which visualized senile plaques (SPs), neurofibrillary tangles (NFTs) and Pick bodies [2]. More than 80 years have passed before biochemical compositions of these pathological structures were unveiled. During this period, technical trials to identify and localize these molecules on histological sections showed the effectiveness of immunohistochemistry and revolutionized not only research but also routine diagnosis in the field of neuropathology. Probably because of the apparent success in the molecular explanation of diseases, recent attention to silver staining has not been very enthusiastic. Although most silver-staining methods are still left with some ambiguities, neuropathologists have profited by their efficacies. Silver-staining methods have been steadily improved and one of the recent advances is undoubtedly Gallyas method [41]. In practice, it allows stable, reproducible and sensitive visualization with low background. However, it is unfortunate that their principles, technical details and interpretations, which are different according to each method, are sometimes not sufficiently shared among neuropathologists and technicians. It frequently happens that some time-honored protocols are left without possible explanations and their interpretation is often restricted to “argyrophilic or not”. This naive interpretation is based on the assumption that “argyrophilia” is a homogeneous phenomenon irrespective of the staining method and the lesion. Recent studies have demonstrated, on the contrary, that “argyrophila” is heterogeneous and dependent on the staining method and the lesion [85, 119–122]. Here, established silver-staining methods in the field of neuropathology are reconsidered with the intension of improving their use and interpretation. The first part of this review summarizes basic principles and outlines protocols about four principal silver-staining methods for formalin-fixed, paraffin-embedded samples from human autopsy brains mainly with degenerative diseases, currently in use in our laboratory and their modifications. Although other silver-staining methods have also been invented to visualize normal neuronal structures, astrocytes, microglia and experimental tissue samples, it is not possible to encompass the whole range of these techniques mainly because of the limitation of my own experience. The second part provides an overview of similarities and differences between silver-staining methods and immunohistochemistry, so that a comprehensive interpretation, including their pitfalls results. Nevertheless, this review is not intended to establish a relative grading of these methods by identifying the possible superiority of a method (for example, immunohistochemistry) over another (for example, a silver-staining method). Instead, it is designed to show how unique each staining method can be, by providing a distinct point of view, each independent of the other. If we are prepared to take these possible differences of staining methods into account, such an approach will provide a less biased, multi-faceted and fuller stereoscopic picture than can be achieved with immunohistochemistry alone. General principle of silver staining The coloration in conventional histology is based on the selective affinity of a dye to some tissue elements relative to surrounding elements and their permeability. This differential affinity to specific tissue elements is finally represented by the distinctive original color of the dye as seen with its solution. This is essentially a single step procedure, called dyeing, and does not involve chemical transformation of the dye. Subsequent washing step is also selective in dissolving out the attached dye, which is called differentiation [5]. Various combinations (for example, hematoxylin and eosin, the Klüver-Barrera method, etc.) are currently in use for neuropathological examinations. The mechanism of silver staining is, however, completely different. Because the solutions containing coloring agents used for silver-staining methods are usually colorless, simple application of these solutions to tissue sections, by itself, does not allow visualization through light microscope even if the reagent has some affinity to the tissue elements. Subsequent chemical transformation is necessary for visualization. In silver solutions, most of the silver is present as ion or complex salt. The silver ions should be first attached to the target elements as silver ions or silver salts (step 1). This attachment, however, does not allow visualization by itself. The attached silver ions (Ag+) have to be reduced (=accept electrons) to yield, in situ, metallic silver particles (Ag) now visualized only if their amount and size are sufficient for microscopic detection (step 2). Therefore, not only the differential affinity of the silver ion to some tissue elements (step 1) but also its chemical transformation for visualization (step 2) is essential. Although the attachment of silver ions to tissue elements is based on the “in situ” formation of silver salt, these silver ions, either in the solution or as salts, are reducible. They undergo reduction to form metallic silver. Gallyas demonstrated that there is a time lag between the rapid attachment of silver ions onto the section (step 1, within 15 min) and delayed formation of metallic silver particles (step 2) there [42]. Because these two steps are independently affected by pH, temperature, the concentration of silver ion and coexisting molecules [42], these steps are independent even if both take place in a single manipulation. This dual function of silver, that enables “site-directed chemical transformation”, is fundamental to various silver-staining methods. Because some tissue elements (glutathione, creatine, adrenaline etc.) exhibit an intrinsic reducing capacity, which may be strong enough to reduce silver ions into metallic silver particles in situ, these two steps (selective attachment and visualization after reduction of silver ions) may take place in a single manipulation. This is called silver reaction or “argentaffin” reaction [35, 102]. When the reducing capacity of the tissue elements is absent or not sufficient, it is necessary to provide reducing agents externally after silver ions have been attached to the tissue elements. It is also possible to facilitate or modify these steps by some reagents [40]. Anyway, this two-step procedure is called postblackening (Nachschwärzen) or “argentophilic” reaction [35, 102] and most of the current silver-staining methods for neuropathology consist, in essence, of at least these two steps. If sufficient amount of silver ions have been attached to the tissue elements but not have been reduced spontaneously after step 1, it is possible to visualize them simply by applying an appropriate reducing agent externally. This is called chemical development, during which the amount of deposited silver in the tissue section remains unchanged. However, it is not easy to increase the amount of silver deposits during step 1. For example, increasing the concentration of silver reagents or prolongation of incubation time is frequently hampered by nonspecific staining. This trade off between sensitivity and specificity, principally encountered in all kinds of staining, is especially troublesome with silver-staining methods because reactions in the silver solutions and those in reducing agents are both capricious and difficult to standardize. This is one of the reasons why more sensitive, stable and reproducible silver solutions and staining methods have been a matter of considerable endeavor for a long period, as will be discussed later. Particle size and possible determinants of selective staining It has been reported that the apparent color of silver-stained elements is correlated with the size of final silver particles. In the test tube, the smallest range in their diameter (10–20 nm) is correlated with yellow color. The largest range (>100 nm) is associated with black color. Intermediate ranges are correlated with red (>30 nm) or blue-purple (50–60 nm) [102, 103], as reported by Liesegang [70]. The emission spectra of fluorogenic semiconductor nanocrystals, recently introduced for fluorolabeling, are dependent not only on their materials but also on their particle size [20, 22]. Although the size range of semiconductor nanocrystals (2–6 nm) is much smaller than that of silver particles, it is interesting to note that these differences in their color spectra are dependent on the particle size in both silver particles and the nanocrystals. Others reported, however, that black color is correlated with a larger amount of deposits [35]. Although the deposition of silver particles is primed by an affinity of the silver ion to some tissue elements, is this affinity the only major determinant of the selective staining? According to Seki [102], some colloidal substance in the tissue may hinder formation and deposition of metallic silver particles (Schutzkolloid). In addition, packing density of tissue ultrastructures (for example, fiber density) may also be responsible, because an excessive looseness of the structure may not allow secure settlement of silver particles. It is also possible that its extreme tightness does not permit appropriate growth of silver particles. He then concluded that a small quantity of this intervening colloid (eine Armut an Schutzkolloid) and an appropriate density of the structure (eine passende Strukturdichte) of the target structure are also important for the selectivity of silver staining. This idea that some spatial constraints around silver particles, in addition to the primary affinity of the silver ion, are essential in determining the selectivity of silver staining may be related to the current concept of disease-specific “conformational status” of pathological deposits and their constituent molecules. These explanations are, however, far from complete for the majority of silver-staining methods. Furthermore, because these experiments have been performed mainly in animal tissues, in which sampling and handling of tissue materials are optimal, it remains to be clarified whether similar explanations are applicable to human samples obtained for neuropathological examinations, often containing distinct pathological lesions and suffering from suboptimal handling such as postmortem delay, suboptimal fixation and embedding in paraffin wax. Even though various methods for celloidin- or paraffin-embedded autopsy samples have been invented. Table 1 shows fundamental steps and contents of the solutions for the silver-staining methods currently in use in our laboratory. Table 1Protocols for different silver-staining methodsBielschowsky method (original) for frozen sections [8]Modified Bielschowsky method for paraffin sections [131]Bodian method [9,10]Gallyas method [41]Campbell–Switzer method [21]PretreatmentPyridine (24–48 h)––0.25% potassium permanganate (RT, 15 min), then2% oxalic acid (RT, 2 min) [114], then0.4% lanthanum nitrate/2% sodium acetate (RT, 60 min) [47]–WashingDW––DW–Silver solution I3% silver nitrate(RT, 24 h)20% silver nitrate(RT, 20 min)1% silver proteinate + metallic copperf (37°C, 24 h)Alkaline silver iodate (RT, 4 min) 0.035% silver nitrate 10% potassium iodide 4% sodium hydroxidePyridine silver (RT, 40 min) 0.52% silver nitrate 8.7% (volume) pyridine 0.39% potassium carbonateWashingDWaDWDW0.5% acetic acid 3 min × 3 times0.5% acetic acid 3 min × 3 timesSilver solution II4–5%b ammoniacal silver20%c ammoniacal silver(RT, 15 min in the dark)–Physical developerg (RT, 5–10 min) 0.1% silver nitrate 0.5% tungsto-silicic acid 0.1% ammonium nitrate 2.5% sodium carbonate 0.03–0.2% formalinPhysical developergWashingDWDW + ammoniad–Chemical developer20% formalin in tap waterDevelopere in 20% ammoniacal silver 1% hydroquinone/5% sodium sulfiteDW distilled water, RT room temperaturea20% formalin in tap water is optional [50]b5 ml of 20% silver nitrate + five drops of 40% sodium hydroxide, followed by titration with ammonia to dissolve the precipitates. DW (20 ml) is then added [8]cAmmonium hydroxide was added to 20% silver nitrate drop by drop until the precipitates turn clear. Additional two drops of ammonium hydroxide is recommended [131]dAdd three drops of ammonium hydroxide to DW [131].eAdd three drops of the developer (formalin 20 ml, DW 100 ml, concentrated nitric acid one drop, citric acid 0.5 g) to the 20% ammoniacal silver used as silver solution II [131].f4–6 g of metallic copper in 100 ml of the silver proteinate solution is optimal [9, 10]gThe physical developers reported for Gallyas and Campbell–Switzer methods are essentially identical. In our laboratory, equal amount of stock solutions A (0.5 g sodium carbonate-anhydrous, 100 ml DW) and B (0.2 g silver nitrate, 1 g tungusto-silicic acid, 0.2 g ammonium nitrate, formalin 0.73 ml, 100 ml DW) are mixed immediately before use Bielschowsky methods Improvements in silver-staining methods have often been derived from chemistry. The ammoniacal silver solution had been used for production of mirror, based on the nature of the complex “silver diamine [Ag(NH3)2]+ “ in the solution that is liable to precipitate, after being reduced, at the interface between the glass surface and the solution. This is called the silver mirror reaction and it was Fajersztajn who initially applied this phenomenon to tissue staining [34]. Later, Max Bielschowsky [8] improved this method by incubating formalin-fixed frozen sections in the silver nitrate prior to the ammoniacal silver solution (Table 1). Although the exact mechanism still remains to be clarified [100], it is plausible that this prior treatment with the silver nitrate provides active foci for subsequent silver deposition in the ammoniacal silver solution [50]. Anyway, it is reasonable to suppose that this stepwise impregnation (silver nitrate-ammoniacal silver) improved the staining probably by increasing the amount and the size of silver precipitates [66]. Early modifications of Bielschowsky method were summarized by Beech and Davenport [6]. Garven and Gairns [50] provided a comprehensive review on the possible mechanism of the so-called Bielschowsky-Gros method and its modifications. The presence of a huge number of modifications, however, indicates that this method is highly unstable and still awaits improvements. A modification for paraffin-embedded samples of normal neural tissues from various animal species was first proposed by Sevier and Munger [105]. Practically, it is probably Yamamoto–Hirano’s modification [131], which is currently accepted as the standard Bielschowsky method for neuropathological examination of paraffin-embedded autopsy samples (Table 1). This Yamamoto–Hirano’s modification is different from other conventional modifications of Bielschowsky method because the usual silver nitrate–ammoniacal silver sequence is followed by another ammoniacal silver solution containing developers. Bodian method Bodian established a new method, applicable to paraffin-embedded sections, for staining nerve fibers and nerve endings [9, 10]. This method is rather stable and reproducible and is currently in use for neuropathology. He used 1% silver proteinate (which usually contains 8% of silver) solution and placed metallic copper in it. Because the staining does not work well in the absence of metallic copper, he speculated that metallic copper might regulate the deposition of metallic silver by decreasing the concentration of silver ion [9, 10]. However, the mixture of silver nitrate and powdered egg albumin was reportedly successful even in the absence of metallic copper [92]. Nevertheless, the measured concentration of silver ion gradually decreases after addition of metallic copper, as demonstrated by two independent groups [64, 93]. Concomitant and gradual increase in the concentration of copper ion [64, 93] is accompanied by a decrease in pH [93]. These reciprocal changes between concentrations of silver and copper ions are mainly explained by a primary difference in ionization energy between copper and silver. In other words, copper is more liable to be ionized than silver. Because their concentrations are not influenced by the presence of sections in the solution, the amount of silver ion trapped in the section is practically negligible. While the addition of metallic copper up to 5 g/100 ml progressively decreases the concentration of silver ion in dose-dependent fashion (from 1,000 to 250 μg/ml), further increase in the amount of metallic copper up to 15 g/100 ml does not lead to further increase in the copper ion nor further decrease in the silver ion in the solution [64]. Indeed, this amount of copper, 5 g/100 ml, is in perfect agreement of the amount, 4–6 g/100 ml, originally recommended by Bodian [9, 10] and the amount of metallic copper more or less than this range does not give optimal results. Interestingly, not only the amount of metallic copper but also the shape of metallic copper (foil, plate, chip or granules) influences the concentration of copper and silver ions and the results of staining. The metallic copper as “foil” is correlated with the highest concentration of copper ion and the most intense staining [64, 93]. Moreover, these reactions in the silver proteinate solution occur slowly and the equilibrium is reached after 12–16 h. Figure 1 illustrates putative chronological events during incubation in the silver proteinate solution and development. In the early phase of silver impregnation, the high concentration of silver ion facilitates its attachment to the section (step 1, Fig. 1). This initial attachment is apparently not selective, because early interruption of incubation yields an overall nonselective nerve staining [93]. Gradual deposition of silver ion as metallic silver on the surface of metallic copper (step 2, Fig. 1) leads to a gradual decrease in the concentration of silver ion and a concomitant increase in the concentration of copper ion in the solution. Continued incubation in the solution with a decreasing concentration of silver ion facilitates liberation of the silver ion, once trapped in the section, back into the solution (step 3, Fig. 1). The selective staining of Bodian method is explained if this rate of liberation of silver ion back to the solution (step 3, Fig. 1) is different according to the tissue elements. This chronological change may be necessary to yield the selective staining and explains why an equivalent concentration of copper ion provided as copper sulfate instead of metallic copper does not yield similar results [64]. Some copper ions are also deposited onto the section (step 4, Fig. 1). Residual silver proteinate as colloidal fraction may release silver ions gradually throughout the incubation (step 5, Fig. 1). For practice, (1) 5 g of metallic copper as foil in 100 ml of 1% silver proteinate solution and (2) incubation for 16–24 h are recommended as optimal for Bodian method [64]. Fig. 1Mechanism of the Bodian method. Step 1: Attachment of silver ions to the section driven by high concentration of silver ions; single asterisk: a fraction of silver ions are already reduced to metallic silver even before chemical development. Step 2: Deposition of silver ions as metallic silver leads to a decrease in the concentration of silver ion and reciprocal liberation of copper ions. Step 3: Delayed liberation of silver ions from the section back to the solution with decreased concentration of silver ion after step 2. Step 4: Copper ions are now available in the solution after step 2 to be attached on the section. Step 5: Silver proteinate gradually releases silver ions during steps 1–4. Step 6: Not only silver but also copper ions/salts on the section are reduced to metallic particles. Step 7: These metallic particles are replaced with metallic gold; double asterisk: retrieval of copper ions in the gold chloride solution indicates that metallic copper has been deposited on the section (steps 4 and 6). Symbols: circle: silver; square: copper; pentagon: gold; blank symbols: reducible (ion/salts); filled symbols: metallic After being incubated in the silver proteinate solution, sections are treated with 1% hydroquinone with 5% sodium sulfite for reduction (step 6, Fig. 1) [9, 10]. It has been reported that formaldehyde, suitable for reducing the silver diamine of Bielschowsky method, is not appropriate by itself for reducing the silver ions in Bodian method. In contrast, hydroquinone suitable to reduce the silver ions in Bodian method is not appropriate for reducing the silver diamine of Bielschowsky method [35]. However, some recommended formaldehyde in combination with hydroquinone for the reducing step of Bodian method [9, 10]. Because it is usually after the incubation in the developer when the sections appear contrasted for visualization, it seems reasonable to suppose that deposited silver ions are reduced to form metallic silver only after exposure to hydroquinone. If this is the case, treatment of the silver impregnated sections with sodium sulfite, which dissolves out silver salt but not metallic silver, will diminish the staining. On the contrary, Peters [93] demonstrated the presence of sodium sulfite-resistant metallic silver and suggested that at least some fraction of silver ions have already been reduced to metallic silver during incubation in the silver proteinate solution (asterisk, step 4, Fig. 1). They may serve as nuclei that attract surrounding reducible silver ions/salts to form larger metallic silver particles during subsequent development. Indeed, Gallyas demonstrated that radiolabeled silver ions in the silver proteinate solution are deposited to the section in the form of metallic silver even before the development. This is based on the finding that the radiolabeled silver on the section is resistant to the treatment with 1% silver nitrate/10% acetic acid that washes out reducible silver ions but not metallic silver particles. Because the localization of this radiolabeled silver, now deposited as metallic silver, is in good agreement with the final image of the adjacent section stained by Bodian method, it is likely that these metallic silver deposits play a determinant role in defining final image obtained with Bodian method [44]. At any rate, it is possible to intensify the staining through subsequent gold toning by incubating in 1% gold chloride with glacial acetic acid (three drops for 100 ml). Although the mechanism of Bodian method, especially that of copper ion, is not yet completely clarified, not only silver but also copper ions are retrieved in the gold chloride solution for toning (double asterisk, step 7, Fig. 1) [64]. This indicates that the ionized copper in the silver proteinate solution has been deposited on the tissue and that the deposited copper, in addition to silver, is replaced with gold [64]. This dual replacement may explain why this gold toning is necessary and particularly effective for Bodian method. After gold toning, it is now possible and necessary to treat the sections with 5% sodium thiosulfate to dissolve out silver ions that remain unreduced in the tissue. Otherwise, these remaining ions may be reduced after exposure to light to form undesirable precipitates. Because Bodian method was originally invented to stain normal axons and neurofibrils, these normal structures are always superimposed on pathological structures such as SPs and NFTs. These intervening normal structures are one of the major hindrances of Bodian method especially when examining silver-stainable deposits overlaid on normal structures. Gallyas method Another way of improvement had been achieved based on the chemical principle of photography, “physical development”. As opposed to the chemical development, additional silver ions are provided with reducing agents to form silver particles around the reactive foci, which have been already activated after exposure to light in dose-dependent manner. This greatly increases the amount of metallic silver or oxidized silver that are now visible as photographic images. It was Liesegang who first introduced this idea to histological staining in an attempt to establish a more consistent and reproducible method of silver staining [70, 88]. The protocols with physical development were later customized successfully for neuropathology by Gallyas [40]. Because physical developer solutions contain, in general, silver ions and reducing agents that together react rapidly, protecting colloid (tungsto-silicic acid for Gallyas method) is included in order to control by retarding this reaction [43]. As with other silver-staining methods, the attachment of silver ion to specific targets determines final localization of silver particles. According to Gallyas, this specific attachment is mediated by some determinant substance, which binds to the target tissue elements or the tissue elements themselves, followed by their chemical modifications. These foci are enabled to attract silver ions to form silver salts. Otherwise, the determinant substance renders these foci capable of reducing silver ions to form submicroscopic metallic silver particles bound to the target elements. The chemical nature of the original determinant substance may determine which components (for example, fibrous astrocytes, microglia or oligodendrocytes) in the section are finally silver-stained [37–39]. For visualization of NFTs, sections are incubated with silver iodide in alkaline condition that exhibits affinity to NFTs [41] (Table 1). Although Gallyas customized protocols according to various target structures other than NFTs [37–39], this silver iodide protocol for NFTs, currently in routine use for neuropathological diagnosis, is defined in this review as “Gallyas method” for simplicity and convenience. If this silver solution is prepared with radiolabeled silver, it is possible to quantify the silver ions/particles after each step of the staining protocol. Gallyas demonstrated that most of the silver attached to the section after incubation in this silver iodide solution is reducible silver salt but not metallic silver, because it is washed out after incubation in nonlabeled 1% silver nitrate/10% acetic acid (Table 1 of Ref. [44]), a treatment that expels reducible silver ions/salts from the sections. Incubation in sodium thiosulfate or sodium cyanide, alternate ways to expel these reducible silvers, diminishes the final image, again compatible with the assumption that the attached silver after incubation in the silver iodide solution is silver ions/salts but not metallic silver [41]. Is a separate step necessary to transform these attached silver ions/salts into metallic silver particles so that they may subsequently work as nuclei for subsequent growth of silver particles in the physical developer? Because the reducing agent in the physical developer is ready to reduce these attached silver ions/salts into metallic silver nuclei, an isolated step for reduction is not necessary before the treatment in the physical developer. However, a brief exposure of the silver-impregnated sections to formalin prior to the physical development leads to an instantaneous completion of the physical development [41]. However, it is possible that silver ions contained in the physical developer potentially attaches to the tissue and are physically developed to form separate metallic silver particles. Because this may happen independently of the silver from the initial alkaline silver iodide solution [45], a prolonged incubation in the physical developer may be hampered by this unexpected staining. Therefore, pretreatments have been invented in order to minimize this background. A pretreatment initially proposed by Gallyas was 5% periodic acid for 30 min. He tried various pretreatments [46] and finally proposed an alternative pretreatment with 0.4% lanthanum nitrate/2% sodium acetate in H2O2 for 1 h, which is more effective in eliminating the background [47]. An additional pretreatment with sodium permanganate followed by oxalic acid [114] (initially intended for melanin bleaching [71]) prior to this lanthanum nitrate/sodium acetate treatment of Gallyas [47] was found highly effective in eliminating the background and is currently in use as the standard in our laboratory. The background staining is practically negligible because the expected target staining is usually obtained before long and this untoward background reaction is generally slow. Similar oxidative pretreatments are not beneficial for other silver-staining methods [68]. The subsequent gold toning is essentially similar to that for Bodian method. Compared with Bodian and Bielschowsky methods, Gallyas method is characterized by its stability, reproducibility and ability to visualize abundant deposits [15]. Moreover, normal structures are not stained in practice. This allows easy and reproducible examination especially when looking for pathological deposits such as NFTs or glial cytoplasmic inclusions (GCIs) of multiple system atrophy (MSA) [90, 119]. However, amyloid deposits are hardly visualized and it is usually neuritic components of SPs that are labeled by Gallyas method. Campbell–Switzer method The initial impregnation solution of Campbell–Switzer method contains pyridine (Table 1). Pyridine was initially used for tissue fixation. Bielschowsky [8] used pyridine for postfixation of formaldehyde-fixed frozen sections and found an enhanced selectivity. In order to avoid instability and risk of explosion of the ammoniacal silver solution, Hicks [57] added pyridine (4.4 vol%) to the silver nitrate solution (0.55%) buffered with potassium carbonate at alkaline range. Thereafter, Campbell et al. [21] combined this silver-pyridine (2.5 vol%)-carbonate solution with the physical developer invented by Gallyas and found that this staining is not only highly sensitive in detecting SPs and NFTs in AD brains but also as stable and reproducible as Gallyas method. The solution was later modified by increasing the concentration of pyridine up to 8.7% [13] and was found to be equally very powerful in detecting α-synuclein-related pathologies [101]. Gallyas demonstrated that the presence of pyridine markedly decreases the amount of silver, either metallic or reducible, attached to the section [42]. This is compatible with the observation that pyridine enhances the selectivity of Bielschowsky method [8] and that a higher concentration of pyridine is associated with reduced background [21]. Because the standard pretreatment (potassium permanganate/oxalic acid followed by lanthanium nitrate) for Gallyas method does not affect the results obtained with Campbell–Switzer method and there are essentially no differences between the physical developers used for Gallyas and Campbell–Switzer methods, the only significant difference between them is the silver solutions [86]. The reason for these differences between Gallyas and Campbell–Switzer methods remains unexplained. Enhancement of silver staining by methenamine Methenamine has been used for histological demonstration of glycogen and mucin [52]. Periodic acid-methenamine silver stain has been used for staining of the basement membrane of the glomeruli [61]. Dekura et al. [30] first described the use of the methenamine-silver solution for neurofibrils without prior periodic acid. Further modification of this methenamine-silver method was found highly sensitive in detecting SPs, which is equivalent to immunohistochemistry for amyloid β-protein (Aβ) [1, 53]. Interestingly, formic acid pretreatment, which enhances immunoreactivity for Aβ of SPs, completely diminishes argyrophilia of SPs by the methenamine-silver method or congophilia, suggesting that the methenamine-silver method detects specific structures, such as β-pleated sheets of amyloid, which are destroyed by formic acid [130]. It also stains perivascular amyloid but fails to stain Kuru plaque of Gerstmann–Sträussler–Scheinker disease [130] and renal deposits of amyloid [53]. This is in agreement with the early speculation by Seki [102] that factors other than direct affinity of silver to some tissue elements, such as the quantity of intervening colloid or packing density of tissue components are also responsible for final results of the silver staining. The paucity of normal neurofibrils with this method is helpful in delineating SPs on electron microscopy [76]. It is then possible to detect even sparse aggregations of amorphous, often ramified structures with fine granular silver deposits, suggesting that this highly sensitive method is capable of detecting the earliest stage of Aβ deposition [58]. Although the mechanism of lesion-specific enhancement remains unknown, introduction of 5% of methenamine in the silver proteinate solution of Bodian method is similarly effective in enhancing the selective affinity to SPs and NFTs [65]. It is interesting that this methenamine-silver method exhibits more affinity to Pick bodies when they are extracellular [89], while intracellular counterparts are more readily stained by conventional Bodian method even with electron microscopy [91]. Pretreatment with microwave A microwave treatment of sections in silver solution was first reported by Brinn [19]. This treatment causes an increase in molecular movements across the tissue. This presumably facilitates quicker silver impregnation. At the same time, shortened incubation time for impregnation and activated molecular movements may minimize precipitate formation. Microwave activation of silver impregnation for SPs and NFTs was first introduced in order to apply King’s method [63] (originally designed for frozen sections) to paraffin-embedded sections [74]. Microwave activation is effective with Bielschowsky [23] and the methenamine silver [36] methods in shortening the incubation time for silver impregnation and eliminating the background. Although the elevated temperature may partly explain the improvements of these silver-staining methods, it has also been reported that a lowered temperature at 5°C for initial impregnation in 20% silver nitrate for Yamamoto–Hirano’s modification of Bielschowsky method gives more reproducible results in visualizing a larger number of diffuse plaques [73]. Molecular species detected by silver staining It is interesting to identify isolated molecular species detected by silver-staining methods, but these attempts have been so far limited. Gambetti et al. used Bodian method and anti-neurofilament antibodies in parallel to stain an extract from rat spinal cord electrophoresed on the SDS-polyacrylamide gel. The typical triplet of 200, 145 and 68 kDa identified on the Bodian-stained gel is in agreement with the neurofilament triplet identified on the counterpart stained with anti-neurofilament antibodies [48]. They confirmed similar parallelism in other vertebrate and nonvertebrate species as well [94]. Additional less intense bands at a lower molecular weight range may correspond to tubulin or degradation products of neurofilament proteins [4]. Because these findings have been obtained with normal nerve tissue, further analyses based on brains containing pathological lesions are mandatory to identify components responsible for their argyrophilia on the pathological lesions. Iqbal et al. [60] used Gallyas method to stain the SDS-gel after electrophoresis of the paired helical filament (PHF) fraction extracted from AD brain. The electrophoretic mobilities of the Gallyas-positive bands correspond to those of PHF tau (intense), normal tau (less intense) and high molecular weight MAPs (weak). The absence of Gallyas-positive bands in the range of neurofilament proteins is in sharp contrast with the Bodian-positive triplet of neurofilament [48]. This difference may explain why Gallyas method is practically free from the staining of normal structures such as neuronal soma and neurites. This discrepancy suggests that molecular species detected by different silver-staining methods can be completely different, even though both of the methods label apparently identical structures such as NFTs on histological sections. Because several silver-staining methods have been modified to stain SDS gels [62, 78, 107], it may be possible to identify molecular species, either normal or specific to some pathological structures, which have a selective affinity to some silver-staining methods. Because extracted proteins electrophoresed on gel are necessarily modified or significantly degenerated, this approach may be fruitful only if the disease-specific nature, if detectable with silver-staining methods, is readily represented by a single isolated molecule even when it is modified for electrophoresis. Quantitative comparison of silver-staining methods and immunohistochemistry Even after the introduction of immunohistochemistry, modification of classic silver-staining methods or invention of new silver-staining methods have been attempted [11, 25, 26, 98]. Two major deposits targeted by these silver-staining methods are undoubtedly NFTs and SPs of AD [31, 32, 79, 82]. Different silver-staining methods [24, 27, 31, 54, 56, 67, 99, 124, 125, 127] have been compared quantitatively for their relative ability to detect NFTs or SPs, as represented in Fig. 2 [56]. In respect to detection of NFTs, these comparative studies are in agreement that Gallyas method is most powerful, followed by one of the three methods: modified Bielschowsky, Bodian or Cross methods. The methenamine-silver method is rated as least powerful for NFTs. There are two studies, each claiming a superior or equivalent number of NFTs detected with Reusche method [99] or with the nickel peroxidase method [27] compared with Gallyas method. This rating of putative sensitivity, however, is not shared when SPs are examined. SPs are well stained by modified Bielschowsky [125, 131], the methenamine-silver [1], periodic acid methenamine silver [128] or thioflavin-S [124] to similar extent. Bodian method is less powerful in detecting SPs , especially diffuse deposits [54, 56, 67, 127, 128]. Gallyas, Cross [56, 67] and Garvey [27] methods are least powerful in detecting SPs. Fig. 2Density of NFTs (a: upper panel) and that of SPs (b: lower panel) as a function of intellectual status evaluated by the Blessed Test Score. Both are dependent on the staining methods. Reproduced with permission [56] It is needless to say that a more sensitive detection is preferable in order to identify specific deposits in more detail with ease. However, if one deals with clinicopathological correlation of a disease such as AD, where symptoms and deposits are more or less quantifiable under the background of normal aging, it is possible to identify a threshold of the deposits with any of the methods that may correlate with a threshold of the cognitive decline [118]. For this purpose, stability and reproducibility of the method are preferable [31–33, 54, 79–82]. Reliable agreement between the conventional Braak staging of neurofibrillary pathology based on Gallyas method [17] and the revised protocol based on AT8 immunohistochemistry [18] is a good example. On the other hand, if one is looking for specific pathological deposits, such as Pick bodies, GCIs or astrocytic plaques not observed in normal aging [118], specificity is the primary concern for the staining method. Some of these comparative studies included immunohistochemistry for tau [31, 56, 99], Aβ [27, 54, 56, 99, 124, 127], ubiquitin [56] or neurofilament [24] for comparison with silver-staining methods. Some studies with highly sensitive Aβ immunohistochemistry demonstrated that it visualizes a larger number of deposits than with conventional silver-staining methods [1, 56]. This suggests that amorphous Aβ deposition, not identified by conventional silver-staining methods, represents the earliest stage of Aβ deposition [59, 95, 129]. On the other hand, it is noteworthy that none of the series found any quantitative superiority of immunohistochemistry in identifying NFTs over the best-possible silver-staining method. For example, it is rather frequent that pathological structures in the form of NFT do not exhibit tau-like immunoreactivity any more, while most of them are stained by some silver-staining methods or fluorochromes [116]. This is explained if some tau epitopes in NFTs are truncated out during their evolution from pretangle to extracellular NFTs [12, 116]. Furthermore, immunohistochemical detection is highly dependent on the antibody and experimental procedures. These technical uncertainties make it difficult to place immunohistochemical detection as a standard for neuropathological diagnosis, especially when the quantity of deposits is of primary importance as with the histological diagnostic criteria for AD [32, 79]. It is needless to say that the molecular specificity of immunohistochemistry is a potentially reliable and logical way in order to sort different diseases under the diagnostic flags of molecules. This highly specific detection by pinpointing a single target molecule is, however, “a very restricted, monochromic view” as pointed out by Switzer [108]”. Identification of the disease-specific molecular species and their reliable probes applicable to histological sections are desirable. Even if this kind of probes are available, immunohistochemical procedures are also difficult to standardize and much more costly than most of silver-staining methods [67]. Immunohistochemistry is highly costly especially when staining large histological samples, for example, hemispheric sections of human brain. Moreover, penetration of antibodies is sometimes not sufficient when staining thick histological sections. Silver-staining methods are more readily applicable even to large and thick histological sections [13]. It is of note as well that archival materials preserved for a long period, especially in formaldehyde, sometimes fail to exhibit immunoreactivity. It is frequently possible to visualize the lesions with silver-staining methods even in these archival materials [13, 123]. Qualitative representation by silver-staining methods As summarized in the previous section, differences in the quantity of argyrophilic AD lesions are dependent on the silver-staining method. As has been considered frequently, it may be “the sensitivity” of a staining method that governs the quantity of the argyrophilic lesions. This is probably in agreement with our general impression that “modified Bielschowsky method is more powerful than Bodian method in detecting AD lesions.” [131] This interpretation is based on the assumption that so-called “argyrophilia” is a homogeneous phenomenon regardless of the staining method and the nature of lesion, which is not the case. For example, Gallyas method is one of the most “sensitive” methods in detecting NFTs and neuropil threads in AD, while it is far less “sensitive” in detecting SPs. This empirical but evident discrepancy is not readily explained by a mere difference in the “sensitivity” of the methods and rather indicates that argyrophilic properties are dependent not only on the staining methods but also on the target lesions. Careful neuropathologists have been aware of these “method- and lesion-dependent natures” of various argyrophilic deposits in neurodegenerative diseases [16, 85, 96]. Table 2 provides a summary of the heterogeneity of argyrophilia according to the staining method and the lesion. Gallyas method is one of the most “sensitive” silver-staining method that clearly labels NFTs of AD, as well as neuronal and glial lesions of corticobasal degeneration/progressive supranuclear palsy (CBD/PSP) [113, 120]. This “sensitive” method, however, fails to stain Pick bodies, another distinct tau-positive deposit [16, 85, 96, 117, 118]. On the other hand, Campbell–Switzer method labels Pick bodies but not CBD/PSP-related lesions [120, 122]. This reversed and complementary profile with Gallyas and Campbell–Switzer methods is hardly explained by a superior sensitivity of Gallyas method over Campbell–Switzer method or vise versa. These discrepancies between Gallyas and Campbell–Switzer methods are readily demonstrable if mirror section pairs are initially fluorolabeled and one of the section pair is subsequently stained with Gallyas and the counterpart with Campbell–Switzer method, as shown in Fig. 3. It is now known that molecular species of tau are different according to the disease-specific lesions. Namely, Pick bodies are usually composed of three-repeat (3R) tau, while CBD/PSP-related tau pathologies are positive for four-repeat (4R) tau [28]. Because Pick bodies are usually positive with Campbell–Switzer method but negative with Gallyas method (Fig. 3a–d), one of the possible interpretations is that 3R tau deposits, such as Pick bodies, exhibit argyrophilia with Campbell–Switzer but not with Gallyas method. In contrast, the lesions of 4R tauopathy (CBD/PSP and argyrophilic grain disease [110, 111]) are related to the argyrophilia with Gallyas but not with Campbell–Switzer method (Fig. 3i–l) [120]. Indeed, NFTs of AD, Down syndrome, and diffuse neurofibrillary tangles with calcification, all containing both 3R and 4R isoforms, similarly exhibit argyrophilia with either method (Fig. 3e–h) [120–122]. It is interesting that Gallyas-positive Pick-like bodies were found to be positive for 4R tau but sparsely positive for 3R tau [132], which corroborates the empirical correlation between 4R tau and Gallyas method. Moreover, in one of the largest studies on sporadic and familial frontotemporal degeneration with tau-positive deposits, “Pick bodies” defined as “inclusions stained by Bielschowsky but not by Gallyas” were found in 17 cases. All exhibited the immunoreactivity with a 3R-tau specific antibody as well as 3R tau predominance in tau biochemistry except for a single case with E342V mutation in the tau gene [29]. This particular case is characterized by diffuse cytoplasmic tau staining in neurons and PHF-like electron microscopic features [72], both of which are not typical of Pick bodies. In another sporadic case, Bodian-positive inclusions positive for 4R but not 3R tau have electron microscopic features with twisted ribbon distinct from those of typical Pick bodies [83]. These apparent heterogeneities necessitate a revision of the classical definition of Pick bodies “round argyrophilic, tau-positive inclusions in neuronal cytoplasm”, because argyrophilic features and tau-species are both heterogeneous in this classical category. Among argryophilic cytoplasmic inclusions in neuron, it seems that those positive for 3R-tau [84] and those negative with Gallyas method [29] are correlated to form a cluster to be defined as Pick bodies. Argyrophilia with the Campbell–Switzer method [122] is helpful for more precise and straightforward definition of Pick body. In contrast with Gallyas and Campbell–Switzer methods, argryrophilia with Bielschowsky and Bodian methods are less dependent on the target lesions (Table 2). It is noteworthy that tau-positive cortical lesions of PSP are silver-stained with Bodian method [55], while those of CBD frequently fail to exhibit argyrophilia with Bodian method [69, 109, 115] especially in the cerebral cortices. Although possible biochemical distinctions of tau between CBD and PSP have been described [3, 104], it remains to be clarified how these biochemical differences are related to these morphological differences between CBD and PSP. Another possible limitation is related to the observations that the expression of tau-isoforms is not homogeneous throughout the brain [51, 126]. Because most of the data on tau-positive deposits summarized in Table 2 and Fig. 3 have been obtained on cerebral cortices, it remains to be determined whether these findings are similarly applicable to extracortical areas such as the brainstem. Yoshida [132] demonstrated that 3R tau immunoreactivity is represented in the brainstem even in so-called 4R tauopathies such as CBD/PSP. Because neurofibrillary pathologies related to AD or aging are sometimes found in the brainstem nuclei and usually positive for both 3R and 4R isoforms, it remains to be clarified whether the presence of 3R tau in brainstem nuclei in CBD/PSP is ascribed to tauopathies independent of normal or accelerated aging. Table 2Heterogeneity of argyrophilia according to disease and methodStaining methodsCampbell–SwitzerBodianBielschowskyGallyasPick bodies [16, 85, 96, 117, 118]++++++−AD-NFTs++++++++Down-NFTs++++++++DNTC-NFTs++++++++PSP/CBD-neurons−+a+b++PSP/CBD-glia−±c+b++Argyrophilic grains [14, 77, 112]-++++AD-SPs+++++++AD-diffuse deposits++-±-Lewy bodies [75, 119]+++++-GCIs [87, 90]++++++AD Alzheimer’s disease, NFTs neurofibrillary tangles, DNTC diffuse NFTs with calcification, PSP progressive supranuclear palsy, CBD corticobasal degeneration, SP senile plaques, GCIs glial cytoplasmic inclusions, ++: easily recognizable, +: positive, ±: questionably positive, −: negativeaPositive in PSP cases [55] but less evident in CBD cases [67, 109, 115]bNot consistent[97], positive in some cases, not appropriate for histological examinationcThe argyrophilia (Bodian) is less evident in glia than in neurons, especially in CBD casesFig. 3Silver-staining profiles of various deposits in neurodegenerative diseases compared with immunofluoresence images. (a–d Pick bodies[122]; e–h neurofibrillary tangles; i–l argyrophilic grains [121]; m–p glial cytoplasmic inclusions; q–t Lewy bodies [119]). Mirror section pairs were initially multi-fluorolabeled for PHF-tau (AT8, green for b, c, f, g, j, k) or for α-synuclein (green for n, o, r, s), with thiazin red (red, fluorochrome that labels fibrillary structure such as neurofibrillary tangles for b, c, f, g, j, k, n, o, r, s) and for ubiquitin (blue for f, g, j, k, n, o, r, s). After recording the fluorescent images from the same area of the mirror section pairs, one of the section pair was stained with Campbell–Switzer (CS: a, e, i, m, q) and the other with Gallyas (GAL: d, h, l, p, t) method. Fluorescent images and silver staining profiles of the same area from the mirror section pair were compared. Pick bodies (a–d) in the pyramidal layer of hippocampus are stained with CS (a) but not with GAL (d) [122]. Neurofibrillary tangles (NFTs, e–h) from the frontal cortex of diffuse neurofibrillary tangles with calcification are stained with both CS (e) and GAL (h) [121]. This staining profile is shared with NFTs of AD [122]. Argyrophilic grains (i–l, in the square) and tau-positive neurons are present (j, k) in the parahippocampal gyrus. The tau-positive neuron (double asterisk) and neuropil thread (arrow) stained with both CS and GAL are similar to those observed in DNTC (e–h), representing neurofibrillary pathology. Grains and pretangle neuron (single asterisk) are stained with GAL (l) but not with CS (i) in contrast. This staining profile is shared with cortical lesion of corticobasal degeneration/progressive supranuclear palsy [120]. Glial cytoplasmic inclusions (m–p) in the putamen, positive for α-synuclein (n, o), are stained with both CS (m) and GAL (p) [119]. Lewy bodies (arrowhead, q–t) and Lewy neurites (arrow, q–t) in the dorsal motor nucleus of vagus, positive for α-synuclein (r, s), are stained with CS (q) but not with GAL (t) [119]. The same blood vessel is indicated with asterisk (q–t). Bar  50 μm (a–d, m–p, q–t), bar  30 μm (e–l). Reproduced with permission. a–d [122]; e–l [121]; m–t [119] Interestingly, argyrophilic differentiation with Gallyas and Campbell–Switzer methods is not limited to tau-positive deposits. Both of the two staining methods label GCIs of MSA (Fig. 3m–p) [119]. Although GCIs are positive for some restricted tau epitopes [106], their major constituent is now considered to be α-synuclein. Indeed, Campbell–Switzer method clearly labels Lewy bodies and their neurites, another representative deposits of α-synuclein [13]. It is interesting that Gallyas method fails to label Lewy bodies and Lewy neurites (Fig. 3q–t). Therefore, these two silver-staining methods, when performed in parallel, provide staining profiles that may allow qualitative distinction between GCIs and Lewy bodies [119]. Because no biochemical markers that enable distinction between Lewy bodies and GCIs are not readily available, these differential profiles in silver staining provides an additional qualitative distinction possibly linked to underlying molecular differences. At present, these empirical distinctions are useful for diagnostic differentiation but await further explanations preferably at molecular or ultrastructural level. These distinctive features of silver-staining profiles may be explained if (1) molecular species specific to each subcategory (for example, 3R vs. 4R tau or LBs vs. GCIs) has different affinity to each silver-staining method; (2) some disease-specific ultrastructures of pathological deposit are related to each staining profile; (3) other independent molecules in these deposits play some roles in characterizing the pathological deposits so that distinctive silver-staining profiles are engendered; or (4) some disease-specific conformational changes are related to a silver-staining profile specific to a disease. Because Campbell–Switzer and Gallyas methods are useful in discriminating not only tau-positive deposits but also α-synuclein-positive deposits even with their molecular and ultrastructural diversities, it is plausible that factors such as (3) or (4) shared by tau-positive deposits and synuclein-positive deposits may play roles, at least partly, in these discriminating abilities with these silver-staining methods. Conclusions and perspective Even with my limited experience on these silver-staining methods, it is apparent that the argyrophilic profiles are dependent not only on the silver-staining method but also on the target lesion. This indicates that “argyrophilia” is highly heterogeneous. However, this heterogeneity is not capricious but rather representative of some disease-specific aspects. The apparent empirical correlations between the argyrophilic profiles and the disease-specific pathological structures are useful in sorting these neurodegenerative disorders. Combined silver-staining profiles, for example with Gallyas and Campbell–Switzer methods, are more convenient than modified Bielschowsky and Bodian method for sorting different disease-specific lesions. Although these staining profiles are discussed in this review as if disease-specific, more detailed comparison at cellular level demonstrated that they can be different from cell to cell even in the same section (intercellular differences) [117]. Furthermore, staining profiles are heterogeneous even in a single cell (intracellular differences) [49]. These silver-staining and immunohistochemical profiles are much more informative especially when combined together than when used in isolation because such combination is not a mere summation of the results obtained with different methods. It will rather provide a more stereoscopic and multi-faceted view, which may allow more precise morphological delineation of disease-specific deposits. In my view, comments such as “Silver-staining methods are unreliable, nonreproducible, nonscientific and out-dated.” are now out-dated. Awareness of their utility and reliability of silver-staining methods, different from immunohistochemistry, may facilitate diagnosis and accelerate research, which hopefully clarifies molecular basis of each silver-staining method and improves our understanding of diseases.

Osteoporos_Int-4-1-2358936

Progressive vertebral deformities despite unchanged bone mineral density in patients with sarcoidosis: a 4-year follow-up study

Summary To evaluate the incidence of new and/or progressive vertebral deformities and changes in bone mineral density, we re-examined 66 patients with sarcoidosis after a follow-up period of four years. In 17 subjects (26%) new and/or progressive vertebral deformities were found, though BMD did not change significantly. Introduction Sarcoidosis is a T-cell driven chronic inflammatory disease. Although chronic inflammation has been associated with decreased bone mineral density as a result of the effects of cytokines on bone metabolism [1–4], we and others could not demonstrate changes in BMD in subjects with this condition, even if treated with glucocorticoids (GCs). In a cross-sectional study of 124 subjects with sarcoidosis, BMD values similar to an age- and sex-matched reference population were found [5]. Comparable observations were made in three small studies in untreated patients [6–8]. These studies also found a normal BMD relative to age and sex-matched controls, except for a small group of postmenopausal women in which BMD was moderately decreased at the spine in longstanding sarcoidosis only [7]. Although in our cross-sectional study normal BMD values were observed, increased levels of the bone resorption marker serum carboxy-terminal cross-linked telopeptide of type I collagen (ICTP) and the bone formation marker serum procollagen type I amino-terminal propeptide (PINP) suggestive of increased bone turnover were found [5]. ICTP levels correlated with markers of disease activity such as soluble IL-2 receptor (sIL2R) and angiotensin converting enzyme (ACE). In addition, vertebral deformities suggestive of fracture were demonstrated in 20% of the subjects studied in this series. This may imply that the fracture risk in sarcoidosis is increased due to an increased bone turnover with consequent changes in microarchitecture and decrease of bone strength which is not reflected by changes in BMD [9, 10]. If so, this may result in progressive vertebral deformities during the course of the disease. For this reason we re-examined individuals with sarcoidosis four years after the initial measurements to determine the incidence of new and/or progressive vertebral deformities and their relation with changes in BMD. Subjects and methods Subjects Sixty-six of the 124 subjects with sarcoidosis that were studied in 2002 [5] agreed to participate in the follow-up study performed in 2006. None of the 56 subjects who declined or were unable to participate had impaired mobility or a history of vertebral fractures. The mean age of this group was 45 years and did not differ with respect to gender or glucocorticoid (GC) use from the group of subjects that were re-examined in 2006. Demographic, clinical and treatment data of the subjects studied in 2002 and 2006 are summarized in Table 1. The group consisted of 22 pre-menopausal women, 11 post-menopausal women, and 33 men; median age of the total group (all Caucasian) was 43 years (20–66 y). The clinical records of all patients were reviewed. In 2002 patients were evaluated according to a standard protocol that included questionnaires, measurement of height and weight, lung function, measurement of BMD, a single energy densitometry of the spine, and laboratory evaluation [5]. In 2006 the same protocol was repeated. Informed consent was obtained from all participants and the study was approved by the medical ethics committee of our institution. Table 1Baseline and follow-up demographic, clinical, and treatment variables (n = 66)VariableBaseline (n=66)Follow-up (n=66)P*Demographic variablesFemale sex33 (50%)Postmenopausal11 (17%)14 (21%)nsAge, years43 (20–66)Body mass index, kg/m226.9 ± 5.727.2 ± 5.3nsSmoking7 (11%)Daily dietary calcium intake, mg740 (110–2360)758 (150–1340)nsClinical variablesDisease duration, years3 (1–22)7 (5–26)Chest X-ray stage (0-I-II-III-IV)24/11/12/16/327/5/12/15/7nsFEV1,% of predicted87 ± 2891 ± 26nsDLCO,% of predicted87 ± 1692 ± 18nsPhysical activity8.6 ± 3.78.1 ± 3.7nsLaboratory values (in serum)Calcium, mmol/l (2.1–2.6)2.4 ± 0.12.4 ± 0.08ns1,25(OH)2D3, nmol/l (0.040–0.200)0.14 ± 0.03ACE, U/l (9–25)22.5 ± 9.815.3 ± 7.90.001sIL-2R, kU/l (241–846)654 (188–4315)Hs-CRP, mg/l (<10)3.2 (0.2–191)2.0 (1–16)<0.05Z-score ICTP0.7 ± 1.4Z-score PINP−0.1 ± 0.9Treatment variablesGC use never31 (47)26 (39)nsGC use previous14 (21)25 (38)<0.01GC use current21 (32)15 (23)nsLifetime GC dose, mg9240 (200–48750)11187 (200–56700)<0.001Daily dose, mg12.4 ± 6.210.5 ± 3.3<0.05Started on bisphosphonates after baseline measurement6 (9)Clinical risk factors for osteoporosisFracture2 (2/24 = 8%)5 (5/28 = 18%)nsLow body weight (< 60 kg)8 (12)7 (11)nsSevere immobilization00nsLow physical activity index ≤ 518 (27)18 (27)nsMother with hip fracture6 (9)7 (11)nsData are given as mean ± SD, median (range) or number (%); *= p value between baseline and follow-up measurement. Reference parameters in bracketsAbbreviations: GC, glucocorticoid; FEV1, forced expiratory volume in one second; DLCO, diffusion capacity for carbon monoxide; 1,25(OH)2D3, 1,25 dihydroxyvitamin D; ACE, angiotensin converting enzyme; sIL-2R, soluble interleukin-2 receptor; Hs-CRP, high-sensitivity C-reactive protein; ICTP, carboxy-terminal cross-linked telopeptide of type I collagen; PINP, procollagen type I amino-terminal propeptide Pulmonary evaluation Lung function measurements, including forced expiratory volume in one second (FEV1) and forced vital capacity (FVC), were measured with a pneumotachograph. The diffusion capacity for carbon monoxide (DLCO) was measured using the single-breath method (both Masterlab, Jaeger, Würzburg, Germany). Values were expressed as a percentage of those predicted [11]. Chest radiographs were graded according to the radiographic staging of DeRemee (0 to III), adding stage IV, the end stage of lung fibrosis [12, 13]. All interpretations were made by a radiologist who was blinded to the patient’s history. Laboratory assays Serum 1,25-dihydroxyvitamin D concentration was determined by radioimmuno-assay using a commercially available kit [(IDS Ltd, Boldon, England, interassay coefficient of variation (IE-CV) 18%, intra-assay CV (IA-CV) 15%)]. High-sensitivity C-reactive protein (hs-CRP) was measured by particle-enhanced immunonephelometry on the BN Prospec (Dade Behring). The detection limit is 0.175 mg/L and the measuring range is 0.175–1100 mg/L. Soluble IL-2 receptor (sIL-2R) was determined on the IMMULITE automated analyzer, by means of a two-site chemiluminescent enzyme immunometric assay with a measuring range of 50–7500 kU/L (Diagnostic Product Corporation, Los Angeles, CA, cat no LKIP1). Serum angiotensin converting enzyme (ACE) was measured using a colorimetric method. The precision of the ACE assay was < 5.6% and the reference interval for ACE was 9–25 U/L. As a marker for bone formation, serum procollagen type I amino-terminal propeptide (PINP) was measured. As a marker for bone resorption, serum carboxy-terminal cross-linked telopeptide of type I collagen (ICTP) was assessed. Both PINP (IE-CV 3.2%, IA-CV 2.5%, lowest detectable concentration 0.4 μg/l) and ICTP (IE-CV 3.5%, IA-CV 2.3%, lowest detectable concentration <0.1 μg/l) were measured using commercial RIA kits (Orion Diagnostica Oy, Espoo, Finland). To adjust for age and gender Z-scores for these bone markers were obtained using a Dutch reference group (300 women, 150 men), checked for normal BMD of the lumbar spine and femur and normal 25-hydroxyvitamin-D levels [14, 15]. Bone mineral density and vertebral morphometry Bone mineral density (BMD) was measured by dual X-ray absorptiometry (DXA, Hologic QDR 4500). In 2002 only the BMD of the hip was measured. In 2006 the BMD of both the hip and of the lumbar spine were determined. As reference group for the hip the NHANES III database (sex- and age-matched) was used. A standard protocol as described previously was used for measurement of BMD. To adjust for age and gender, Z-scores were used. To examine changes in Z-scores between baseline and follow-up measurements a Δ Z-score was calculated reflecting the difference between the Z-score at follow-up and the Z-score at baseline. Furthermore, after bone density measurement a lateral single energy densitometry of the thoracic and lumbar spine for vertebral fracture assessment (VFA) was performed (also called Morphometric X-ray absorptiometry (MXA)) [16]. The scans obtained were analyzed twice by one trained operator (intra-observer correlation: 0.85), using the semi-quantitative method of Genant [17]. In addition we measured every vertebra quantitatively. The observer was blinded to the T-score values and to the values of the first set of measurements. After visual examination six points were placed on each vertebral body from T4 to L4. From these points three vertebral heights were measured anterior (Ha), mid (Hm) and posterior (Hp). On the basis of the average score of these morphometric measurements, ratios were calculated and a prevalent vertebral deformity was defined as a reduction of height of 20% or more (Ha/Hp; Hm/Hp and Hp/Hp below). Severity of deformities was assessed using the scoring system of Genant [17]. A score of ‘0’ was assigned to normal, non-fractured vertebra; ‘1’ for a mild deformity (20–25% reduction in anterior, middle or posterior vertebral height); ‘2’ for a moderate deformity (25–40% reduction) and ‘3’ for a severe deformity (>40% reduction). A new vertebral deformity was scored if a normal vertebra (grade 0) became deformed (grade ≥ 1) and a progressive deformity if the grade increased [17]. Questionnaires Calcium intake of all patients was scored in 2002 as well as 2006 on the basis of a detailed dietary list. Known clinical risk factors for osteoporosis (weight below 60 kg, mother with hip fracture, history of fractures after age 50, menopausal status and severe immobilization) as well as daily activities and exercise were assessed by a validated questionnaire [18], in which sports, daily and work activities are scored with a minimum of zero and a maximum of eighteen. GC therapy was evaluated by means of a patient questionnaire and verified using all the records of the patient’s pharmacist. It was scored as never, previous or current use and if subjects were currently using GCs, the daily dose was noted. Statistics Student t-tests, chi-square tests, and one-way ANOVAs were used, depending on the variables and subgroups tested. Depending on the analysis, change scores or actual scores were used. Patients with new and/or progressive vertebral deformity were clustered for the multivariate and the receiver-operating characteristics (ROC) analysis. Multivariate logistic regression analyses was performed to assess the strength of association between the incidence of new and/or progressive vertebral deformities and gender, age, weight, clinical risk factors, GC use, lifetime GC dose, daily GC dose, disease activity, bone markers, calcium intake, physical activity and BMD measurements. The variables that were entered in the multivariable analysis were those variables that appeared related (p < 0.10) to this outcome measure in univariate analyses. Odds ratio (OR) and its 95% confidence intervals (CI) were calculated by using SPSS version 12.0. ROC analysis was used to assess the ability of various levels of the T-score femoral neck to predict the incidence of a new and/or progressive vertebral deformity. The ROC curve indicates the probability of a true-positive result as a function of the probability of a false-positive result for all possible threshold values [19]. A p value < 0.05 was considered statistically significant. Results Bone mineral density and bone turnover parameters The results of BMD measurements are shown in Table 2. The BMD of the total group remained unchanged after a median follow-up of 45 months (range 35–49 months). When stratifying patients according to GC use, no decrease in each of the subgroups was found. Patients that never used GCs showed a Δ Z-score of the femoral neck (FN) of 0.03 ± 0.36 and a Δ Z-score of the trochanter of −0.08 ± 0.37. In patients with previous use of GCs these Δ Z-scores were 0.10 ± 0.36 and 0.22 ± 0.43, respectively. Even the group currently on GCs revealed no decrease of Z-score (Δ Z-score FN: 0.06 ± 0.30 and Δ Z-score trochanter: 0.00 ± 0.18) and also the subgroup of postmenopausal women (n = 11) did not show significant bone loss (Δ Z-score FN: 0.06 ± 0.45 and Δ Z-score trochanter: −0.05 ± 0.52). In the total group, bone turnover parameters at baseline showed an increased Z-score of ICTP compared to norm scores (0.7, 95% confidence interval (CI):0.4–1.1; p < 0.001). on the other hand, the marker of bone formation (Z-score PINP) did not differ from the reference population. Table 2BMD variables at baseline and follow-up for the total group (n = 66, median follow-up duration 45 months (range 35–49 months)VariableBaseline measurementFollow-up measurementP*BMD, mean ± SD gm/cm2Femoral neck0.84 ± 0.120.83 ± 0.12nsTrochanter0.74 ± 0.130.74 ± 0.12nsTotal hip0.97 ± 0.130.98 ± 0.14nsLumbar spine1.04 ± 0.14Z-score, mean ± SDFemoral neck0.17 ± 1.00.23 ± 1.1nsTrochanter0.27 ± 1.10.32 ± 1.1nsTotal hip0.18 ± 1.00.32 ± 1.00.001Lumbar spine0.03 ± 1.4T-score, mean ± SDFemoral neck−0.42 ± 1.0−0.46 ± 1.0nsTrochanter−0.02 ± 1.1−0.03 ± 1.0nsTotal hip−0.14 ± 1.0−0.10 ± 1.0nsLumbar spine−0.50 ± 1.3Abbreviations: BMD, bone mineral densityP* = p value between baseline and follow-up measurement Clinical fractures and vertebral deformities Three new non-vertebral fractures occurred during the follow-up period. These included a hip fracture (twice in the same patient), an ankle fracture and a fracture of the thumb. All these fractures were related to trauma and occurred in subjects older than 50 years. Morphometric data are summarized in Table 3. In 2002 vertebral deformities (ratio of <0.80) were found in 19 vertebrae of 13 subjects. Seventeen of these were wedge and two biconcave deformities. No crush deformities were seen. The majority of these deformities were found in the low thoracic region. At follow-up a new vertebral deformity was scored if a normal vertebra (grade 0) became deformed (grade ≥ 1) and a progressive deformity if the grade increased [17]. With this method, 36 vertebral deformities were found in 21 subjects. In one subject a vertebral deformity (ratio 0.78 of T11) found in 2002 was not found at follow-up (ratio 0.81). So, in total nine new subjects revealed one or more vertebral deformities, which means an increase of vertebral deformities from 20 to 32% of the subjects studied (p < 0.05). From the 21 subjects with a vertebral deformity in 2006, 17 subjects (26% of total group) were diagnosed with one or more new or progressive vertebral deformities and in four subjects the deformity was unchanged compared to baseline. Data on number and severity of the deformities can be found in Table 3. Six patients were started on a bisphosphonates after baseline measurement and from these six patients, two had a new or progressive vertebral deformity at follow-up. Table 3Number and grade of deformitiesBaselineFollow-upNo. of subjects with deformity13 (20%)21 (32%)*No. of deformities Mild1728 Moderate28 Severe00Total1936*P < 0.05 between number of subjects with deformity at baseline and follow-up Comparing the groups with and without new or progressive vertebral deformities at follow-up, no differences in Δ Z-scores of BMD of the trochanter or femoral neck (FN) were found (Δ Z-score trochanter −0.02 ± 0.41 and 0.08 ± 0.38 respectively and Δ Z-score FN 0.01 ± 0.32 and 0.08 ± 0.35). In addition no differences in baseline Z-scores of ICTP and PINP were seen between these groups. Multivariable logistic regression analysis, including factors that correlated in the univariate analysis, revealed that a T-score of the femoral neck at baseline (OR per 1 SD T-score reduction = 2.5 (CI: 1.0–5.9), p = 0.04), and a mother with a hip fracture (OR = 14.1 (CI:1.4–142,6), p = 0.02) were determinants of a new and/or progressive morphometric vertebral deformity at follow-up measurement. Factors such as age, gender, calcium in take, GC use, daily GC dose, lifetime GC dose, disease activity, bone markers, radiographic stage and disease duration at baseline did not predict new and/or progressive vertebral deformities. The threshold level of the T-score FN that maximized the combined specificity and sensitivity on the ROC curve (Fig. 1) was < −0.45 for predicting a new and/or progressive deformity (sensitivity 88%, specificity 51%). Fig. 1ROC curve using Femoral neck T-score to identify patients with new and/or progressive vertebral deformity. Area under the curve (AUC): 0.72. Arrow: The threshold level of T-score FN that maximized combined specificity and sensitivity was < −0.45 (sensitivity 88%, specificity 51%) Discussion In this cohort of subjects with sarcoidosis, a high prevalence of morphometric vertebral deformities suggestive of fracture was found, as well as a substantial increase in vertebral deformities during a follow-up period of four years. In 2002 20% of subjects were diagnosed with vertebral deformities according to the criteria of Genant [17], which increased to 32% of all subjects in 2006. In parallel, the total number of deformities in these subjects almost doubled. However, BMD of the trochanter and femoral neck did not change over time and BMD of the lumbar spine at follow-up measurement did not differ from the reference population. These data are suggestive of an increased risk of progressive vertebral deformities in individuals with sarcoidosis despite preservation of BMD. Although data on prevalent or incident fractures in younger healthy populations are lacking, data from other studies suggest that the incidence and prevalence of vertebral deformities in this population are indeed high. Prevalence rates of 30% asymptomatic vertebral fractures are demonstrated in elderly post-menopausal women on chronic GC therapy using the same techniques [20]. In a previous study in 60 subjects (mean age 49 ± 13 years) with differentiated thyroid carcinoma we found vertebral deformities in 7% of patients [21]. Data from the European Vertebral Osteoporosis Study (EVOS), a very large cross-sectional population based study on European subjects aged 50 to 79 years, showed a prevalence of vertebral deformities of 12% (range 6–21%) in males and females [22]. In the Rotterdam study, in which 3469 men and women aged 55 years and older were studied, the prevalence of vertebral deformity suggestive of fracture was 6.9% in men and 7.5% in women [23]. The epidemiology of vertebral fractures in women aged 50–54 years turned out to vary in different countries from 4.7% – 11.5% [24]. All these studies indicate that the fracture risk in subjects with sarcoidosis is substantial, regardless the differences in populations studied and differences in methodology. A new vertebral deformity was found in 15 subjects (23%). To identify incident deformities several approaches can be followed. Measurement of changes in vertebral heights of the same vertebral body from a baseline to a later radiograph in which a decrease in height of 15 or 20% or 4 mm is suggestive of fracture [25, 26], changes in indices of vertebral area [27] or changes in the number or presence of prevalent deformities [17, 28]. Black and coworkers evaluated these different approaches and concluded that none of these were consistently better than any other method [29]. As we aimed to assess the change of numbers of subjects with one or more vertebral deformities over time we used the last method [17], in which changes in number of prevalent deformities are scored. A comparable approach was followed in the European Prospective Osteoporosis Study (EPOS) [30], which revealed an incidence of new deformities of 3.4% after a similar follow-up period. As the mean age of subjects included in this study was substantially higher than that of our cohort, these data cannot be used as a reference, although it is likely that in younger age groups even lower incident deformities would be observed. The high prevalence of vertebral deformities at baseline, the significant increase of more than 50% of subjects after follow-up with one or more deformity and the increase of severity of prevalent deformities all imply that sarcoidosis is a relevant risk factor for vertebral deformity. What is the underlying mechanism of this predisposition to vertebral deformities in view of the lack of effects on BMD in sarcoidosis? The load bearing capacity of bone, also referred to as ‘whole bone strength’, depends on the amount of bone, the spatial distribution of the bone mass, and the intrinsic properties of the materials that comprise the bone. Thus, properties at the cellular, matrix, micro- and macro-architectural levels may all impact the mechanical properties of bone [31, 32]. Apparently, in sarcoidosis mechanisms are involved that influence bone strength without having a significant impact on bone mass. As we found in the total group an increased marker of bone resorption (Z-score ICTP) at baseline, one of the possible mechanisms could be increased bone remodelling with a negative effect on bone micro-architecture that is not reflected by a change in BMD. It is well known that chronic inflammatory diseases influence bone physiology by the production of cytokines stimulating bone turnover [1, 2, 33]. Increased bone remodelling is associated with an increased bone fragility and thus fracture risk [34, 35] and in postmenopausal women the level of bone turnover turned out to be an as strong and independent predictor of fractures as BMD [36, 37]. These data may support the hypothesis that the chronic inflammatory state in sarcoidosis results in increased bone remodelling with a negative effect on bone strength and thus an increased fracture risk. No changes in BMD in the group currently treated with GCs were found. This is unexpected as GCs are known to effect BMD via several mechanisms with consequent decrease of BMD. It may well be that this is due to intermittent GC use, as most of our patients were on intermittent glucocorticoids. Other studies have demonstrated that intermittent GC use has no major effects on BMD [38]. A recent large retrospective cohort study on clinical fracture risk among patients from the UK General Practice Research Database showed that intermittent use of high dose of oral GCs was associated with only a small increase in the risk of osteoporotic fractures [39]. Despite the on average normal BMD, we found T-score of the femoral neck and a family history of hip fractures to be predictors of a new and/or progressive vertebral deformity. This suggests that the combination of a lower BMD in combination with the increased bone turnover in sarcoidosis predisposes to progressive vertebral deformity. If so, this would mean that in these high risk individuals preventive treatment should be considered to reduce fracture risk. Controlled trials are needed, however, to substantiate this suggestion. One of the limitations of our study is the lack of an age and sex matched control population. Unfortunately data on vertebral deformities in younger populations are at present not available. The aim of this study was, however, not to compare sarcoidosis patients with healthy subjects, but to follow a cohort of these patients and to compare follow-up with baseline measurements. Another limitation is the use of morphometric X-ray absorptiometry (MXA) instead of spine radiographs. MXA is less reliable for the detection of deformities at the upper thoracic spine, where deformities are less frequent as compared to the lumbar and mid-thoracic spine. A recent study comparing MXA with lateral spine X-ray found that vertebral morphometry using MXA allowed diagnosis of vertebral fracture in the lumbar and mid thoracic spine, where vertebral fractures are most common [40]. The advantage of MXA is the low dose of radiation and the convenience of the technique for patients. The present quality of the images, with ongoing refinement of this technology, is considered sufficient to be used for the diagnosis of vertebral deformity consistent with fracture [41]. Furthermore there is a lack of a “gold standard” for VFA. We followed the method of Genant [17], which is based on a reduction of the ratios of anterior, middle or posterior heights and all measurements were performed twice to improve accuracy. This is the simplest and most practical method [42] and an association with future fracture risk is documented [43, 44]. The above mentioned EVOS study, however, applied the methodology described by McCloskey and Eastell and co-workers in which measurements are corrected for normal variations in vertebral shape [25]. Relative to the method of Genant, the method of Eastell [25] or McCloskey [28] may have resulted in lower prevalences of vertebral deformities. This does not, however, explain the differences in prevalence of vertebral deformities reported elsewhere and in this paper. The restrictions of the methodology are also the limited ability to provide a differential diagnosis for the detected deformities, a lower sensitivity for milder fractures and the inability to evaluate the uppermost thoracic levels. Other disorders that may cause changes in vertebral shape involve congenital abnormalities and conditions as severe osteoarthritis [45] and Scheuermann’s disease. We have, however, no indications that these relatively rare conditions may have interfered with our observations. In conclusion, we have shown that in subjects with sarcoidosis the number of vertebral deformities, diagnosed with morphometric assessment, increases during the course of this disease despite preservation of BMD. Although this is an uncontrolled study, it appears that subjects with sarcoidosis have an increased fracture risk, even if BMD is normal. High risk individuals can be identified by a low-normal BMD and by a family history of hip fractures. Probably these individuals will benefit from therapies that increase bone strength. A T-score FN below −0.45 may be used to identify these individuals with a high sensitivity and an acceptable specificity. Studies evaluating the effects of such therapies in individuals with sarcoidosis are however clearly needed.

Mol_Genet_Genomics-4-1-2270915

Characteristic expression of twelve rice PR1 family genes in response to pathogen infection, wounding, and defense-related signal compounds (121/180)

Pathogenesis-related (PR) proteins have been used as markers of plant defense responses, and are classified into 17 families. However, precise information on the majority members in specific PR families is still limited. We were interested in the individual characteristics of rice PR1 family genes, and selected 12 putatively active genes using rice genome databases for expressed genes. All were upregulated upon compatible and/or incompatible rice-blast fungus interactions; three were upregulated in the early infection period and four in the late infection period. Upon compatible rice–bacterial blight interaction, four genes were upregulated, six were not affected, and one was downregulated. These results are in striking contrast to those among 22 ArabidopsisPR1 genes where only one gene was pathogen-inducible. The responses of individual genes to salicylic acid, jasmonic acid, and ethylene induced defense signaling pathways in rice are likely to be different from those in dicot plants. Transcript levels in healthy leaves, roots, and flowers varied according to each gene. Analysis of the partially overlapping expression patterns of rice PR1 genes in healthy tissues and in response to pathogens and other stresses would be useful to understand their possible functions and for use as characteristic markers for defense-related studies in rice. Introduction Pathogenesis-related (PR) proteins, which are classified into 17 families, accumulate after pathogen infection or related situations in many plant species (van Loon et al. 2006). Among the PR gene family, PR1 genes have been frequently used as marker genes for systemic acquired resistance in many plant species. However, only a small proportion of PR1 genes have been studied among the many members in this family, and information on the other members is limited. For example, NtPR1a, b, and c genes for acidic proteins, and NtPRB1 (PR-1g) and NtPRB1b genes for basic proteins were reported in tobacco (Nicotiana tabacum) (van Loon and van Strien 1999), but little information is known on other PR1 members. To understand the characteristics and redundancy of the majority of PR1 family members, genome-based studies are necessary. For such studies, dicot Arabidopsis and monocot rice (Oryza sativa L.) plants have been used as the model plants. In Arabidopsis, 22 genes are listed as predicted PR1 genes that encode homologous proteins to tobacco PR1a protein, which was first reported as an acidic protein in tobacco leaves infected with Tobacco mosaic virus (TMV) (van Loon et al. 2006). Among the 22 genes, only one PR1 gene (At2g14610), which encodes a basic protein, is known to be pathogen-responsive, and the other PR1 genes reportedly did not respond to either bacterial or fungal pathogens (van Loon et al. 2006). From these results, we tend to suppose that only one PR1 gene relates to pathogen resistance in Arabidopsis and the others contribute to other functions. In rice, the induction of two PR1 genes, OsPR1a and 1b, by blast fungus infection was reported (Agrawal et al. 2001). They encode putative acidic and basic proteins, respectively, and also responded to environmental stresses and treatments with some chemicals (Agrawal et al. 2000a, b). However, for other rice PR1 gene family members, there is only limited information except for their presence and expression: (1) at least 4 signals for possible rice PR1 proteins responsive to anti-tobacco PR1a antibodies were found in an extract of blast fungus-infected rice leaves (Schweizer et al. 1997; Iwai et al. 2007), and (2) 32 predicted PR1 genes were proposed to be present in the rice genome (van Loon et al. 2006). To study the response of individual rice PR1 genes to pathogens, we selected active rice PR1 genes from the rice genome databases for expressed genes, and studied their induced expression by real time RT-PCR (qPCR). In striking contrast with the result in Arabidopsis, all 12 rice genes selected here were upregulated by blast fungus infection. The levels of constitutive expression in organs and induced expression by different treatments varied according to the gene. In addition to the data on tissue-specific PR1 expression in transgenic rice with a OsPR1 promoter::β-glucuronidase (GUS) fusion gene, we list the characteristics of the 12 rice PR1 genes. This is the first example of a comparison of the expression of the majority of members of a monocot PR family to our knowledge. This information will be useful for further studies on PR genes and on resistance mechanism in rice plants. Materials and methods Plant materials Wild-type (WT) rice (Oryza sativa cv. Nipponbare) and the near isogenic line IL7 (Ise and Horisue 1988), which contains the R gene Pi-i against blast fungus (Magnaporthe grisea) race 003 (Yamada et al. 1976), were grown for 14–16 days in soil (Bonsol No. 1, Sumitomo Chemicals L., Japan) in a greenhouse at 28°C in the day time and 25°C at night. The fourth leaves of plants at the 4-leaf stage were mainly used as the experimental material. The seeds of transformants with OsPR1::GUS were germinated on agar medium containing 30 μg ml−1 hygromycin, transferred to soil at 7 days after imbibition, and grown in the greenhouse. Five-day-old seedlings on agar medium and 2-month-old plants in the greenhouse were used for GUS-staining assays. Infection with pathogens Magneporthe grisea race 003 (isolate Kyu-89-241) was grown on oat-meal medium (Difco) for 2 weeks and conidia were induced under BLB light (FL20S BLB, Toshiba) for 2 days at 25°C. The rice seedlings of Nipponbare and IL7 plants at the 4-leaf stage were spray-inoculated with a conidia suspension (1 × 105 conidia ml−1) containing 0.05% Tween 20, and the inoculated plants were incubated under high humidity in the dark for 20 h, and then moved to the greenhouse. Under these conditions, about 100 local lesions were induced per leaf on Nipponbare and IL7. For bacterial blight infection, Nipponbare plants, which are compatible with Xanthomonas oryzae pv. oryzae (Xoo) strain T7174 (race I, MAFF 311018), were inoculated by cutting the leaf top with scissors that had been dipped in a suspension containing 108 cfu/ml of Xoo, and incubated in the greenhouse. Treatment with chemicals Plastic pots (15 × 5.5 × 10 cm) with 12 rice seedlings at the 4-leaf stage, were dipped in 500 ml solutions containing 1 mM 1-aminocyclopropane-1-carboxylic acid (ACC) or 3 mM sodium salicylate (SA) solution, and incubated for 24 h in the greenhouse. For jasmonic acid (JA) treatment, pots with 12 seedlings each was put in an air-tight clear plastic box, and a cotton pad with volatile methyl jasmonate (MeJA) dissolved in EtOH was put at the corner of the box to give a final concentration of 100 μM, and incubated for 24 h. The fourth leaves were used for RNA extraction. Quantitative real-time RT-PCR To analyze the response of OsPR1 genes to biotic and abiotic stresses, quantitative real-time RT-PCR (qPCR) was conducted using iQ SYBR Green Supermix (BioRad, Hercules, CA, USA) and an iCycler (BioRad) according to the manufacturers’ instructions. At least three independent biological samples were used with specific primers for each individual gene (Table 2). The data were normalized by the value of an actin gene (AK060893), and fold change in the expression level was calculated compared with that of healthy fourth leaves, and standard deviation (SD) values are shown. Construction of plasmids for transformation of rice The promoter region of the OsPR1.1 gene (OsPR1#074 in this work) was obtained through PCR amplification from the Nipponbare genome, using a GenomeWalkerTM kit (Invitrogen, CA, USA) in accordance with the manufacturer’s instructions (Accession no. AP008213, Kawahigashi et al. 2007). The amplified fragment was digested by NotI and BamHI and inserted into the pTH2 vector to construct the PR1T::GUS plasmid (Fig. 7a). The PR1T::GUS plasmid contained 1,919 bp of the promoter and the coding region for 16 amino acids at the N-terminus of the OsPR1 gene to express a fused GUS protein effectively (Kawahigashi et al. 2007). Transformation of rice Agrobacterium tumefaciens LB4404 was transformed with the constructed vector (Kawahigashi et al. 2007). The transformation of rice was performed by Agrobacterium infection as described by Toki et al. (2006). Histological GUS analysis Histological analysis for GUS activity was performed at 37°C essentially as described by Ohshima et al (1990) using a modified reaction mixture: 50 mM phosphate buffer (pH 7.0) containing 1 mM 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc), 5% methanol, 10 μg/ml cycloheximide, and 1 mM dithiothreitol. The reaction was stopped by the addition of ethanol. Results Characterization of the rice PR1 gene family Using the amino acid sequence of the mature tobacco acidic PR1a protein (NtPR1a; 138 amino acids, Ohshima et al. 1987) as the probe, we searched for expressed genes in rice (Oryza sativa cv. Nipponbare) encoding homologous proteins with a similar size range (within 150–200 amino acids) using a full-length cDNA database (http://www.cdna01.dna.affrc.go.jp/cDNA) and rice EST database (http://www.riceblast.dna.affrc.go.jp/). The presence and location of these genes were confirmed in the genome database of rice (http://www.rapdb.dna.affrc.go.jp/). After removing repetitive clones and confirmation of the sequences, finally 12 genes were selected as candidates for active rice PR1 genes, which encode proteins with sequences ≥41% identical to that of tobacco PR1a protein at least in the C-terminal 65 amino acids. Previously, two rice PR1 genes were reported to encode an acidic PR1a protein and a basic PR1b protein (Agrawal et al. 2000a), which were upregulated by blast fungus infection. In addition to the two, we found another 10 active genes on chromosomes 1, 2, 5, 7, 10, and 12 (Table 1). Proposed names were given to distinguish each gene to avoid confusion in this study according to the location on the chromosomes. For example, OsPR1b (AK107926, Os01g28450), located on the 5′ side of the gene for AK121108 (Os01g28500) on chromosome 1, was designated as OsPR1#011 and AK121108 was OsPR1#012. On chromosome 7, four PR1-like genes, OsPR1#071, #072, #073, and #074, were clustered in tandem from the 5′ side to the 3′ side in the sense orientation in this order. The predicted isoelectric point (pI) of each mature protein is shown in Table 1 for reference. The amino acid sequences of the predicted mature proteins for the 12 OsPR1 genes were compared with those of representative acidic and basic PR1 proteins from Arabidopsis (AtPR1-like and AtPRB1) and tobacco (NtPR1a and NtPRB1b), respectively (Fig. 1a). The sequences of the 12 OsPR1s, 2 AtPR1s, and 2 NtPR1s listed here are well conserved. As the N-termini of OsPR1#051, #052, and #121 are not conserved, these regions were eliminated for the alignment in Fig. 1a. Ten amino acid residues (closed circles) are completely conserved among the 12 rice, 2 Arabidopsis, and 2 tobacco genes, indicating they are important for the predicted roles of PR1 proteins. Table 1Classification of rice PR1 protein genesPlantAccession No. (Symbol name)Locus nameReferencesPIProposed symbolsRiceAK107926 (OsPR1b)Os01g28450Agrawal et al. (2000b)7.6OsPR1#011AK121108Os01g28500–8.7OsPR1#012AK107467Os02g54540–8.5OsPR1#021AK105575Os02g54560–5.9OsPR1#022AK071326Os05g51660–7.1OsPR1#051AK100748Os05g51680–5.5OsPR1#052AK060057Os07g03279–4.2OsPR1#071AK062949Os07g03580–4.7OsPR1#072AK063248Os07g03590–5.8OsPR1#073AU163470 (OsPR1a)Os07g03710Agrawal et al. (2000a)4.4OsPR1#074AU070895Os10g11500–10.7OsPR1#101AK100940Os12g43700–5.1OsPR1#121Arabidopsis(PR-1 like)At2g19990Metzler et al. (1991) 6.0(AtPRB1)At2g14580Santamaria et al. (2001)8.8TobaccoX06361 (NtPR1a) (X12737, X05959, X06930)Ohshima et al. (1987)4.4X66942 (NtPRB1b)Eyal et al. (1992)7.6Fig. 1Characterization of the PR1 gene family in rice plants. a Amino acid sequence alignment of 12 putative PR1 proteins from rice. The predicted mature proteins encoded by these genes were aligned by GENETICS 9.0 (Software Development Co., Tokyo). Because the N-termini of OsPR1#052, #051, and #121 are considerably different from those of the other 9 OsPR1s, these regions were eliminated from the figure and the conserved C-termini among the 12 OsPR1s were aligned. The acidic and basic PR1 proteins from Arabidopsis (AtPR1-like and AtPRB1) and tobacco (NtPR1a and NtPRB1b) were used as representatives of acidic and basic PR1 proteins, respectively. The conserved amino acid residues among the 16 proteins are marked with black circles. Identical amino acids are boxed. b Phylogenetic analysis of rice PR1 proteins based on the mature proteins using UPGMA method. The predicted isoelectric point (pI) for each mature protein is shown on the right. Acidic, basic, and neutral proteins are shown in red, blue, and green, respectively. Refer to the description in a for details Using the data of Fig. 1a, a phylogenetic tree was constructed (Fig. 1b). Among the 12 OsPR1 proteins, 7 acidic and 4 basic protein members were found (Fig. 1b). The C-terminal region of OsPR1#051 was indicated to be neutral. The PR1 proteins from Arabidopsis and tobacco grouped in one clade (framed), and OsPR1 proteins belonged to different clades. Responses of OsPR1 genes to blast-fungus infection in young rice plants Compatible interaction The response of individual OsPR1 genes to blast-fungus infection was analyzed by qPCR using a set of primers specific for each gene (Table 2). A conidia suspension of M. grisea race 003 was sprayed on the seedlings of Nipponbare at the four-leaf stage. In this system, the fungus infection results in the formation of whitish expanding lesions at 3 days post inoculation (dpi), and increasing lesion size thereafter with conidia formation. The infected plants were severely wilted and died within 7–9 days indicating a typical compatible host–parasite interaction (Sasaki et al. 2004; Iwai et al. 2006). Using triplicate cDNA samples originating from independently prepared RNA templates from inoculated fourth leaves, the expression levels of each gene were compared with that of an actin gene, which was selected as a control gene. Although expression levels of OsPR1 genes in healthy Nipponbare leaves varied according to each gene (0 dpi, white column in Fig. 2a), all OsPR1 genes tested were up-regulated by blast-fungus inoculation at 3 and/or 6 dpi (black column). When compared with mock-inoculation (gray column), the enhanced expression by the fungus was significant for each gene at 6 dpi, at which lesions were vigorously expanding. The transcripts of OsPR1#074, #011, and #012 had already accumulated to near maximal levels at 3 dpi maintaining almost the same levels at 6 dpi, indicating they are rapid response genes to blast fungus infection. However, the transcripts of OsPR1#073, #022, #101, #051, and #121 had not increased significantly at 3 dpi compared with mock-inoculated controls, indicating these are late response genes. Blast fungus infection induced around 1,000-fold transcript accumulation at 6 dpi compared with 0 dpi in 7 genes: OsPR1#074, #011, #071, #073, #052, #072, #101, and #121. Table 2Primers for qPCR of OsPR1s and actin genesNameForward primerReverse primerOsPR1#074GTATGCTATGCTACGTGTTTATGCGCAAATACGGCTGACAGTACAGOsPR1#011ACGCCTTCACGGTCCATACAAACAGAAAGAAACAGAGGGAGTACOsPR1#071CTTTAACTATGTATGGAGTATGATATAAATGTGTTATTTTCTTCTTTTATTCGAACGACAACOsPR1#012CGCTGTGTGTTTGTGTTATGTCCGTGGTTTTGTCTTTATTTCAATCCOsPR1#073TTATATATGTATGTTCGTATGTATGTATGCTGATGTACTTATTCCATCCGACACOsPR1#021CGCAGCAACCAACCAATCTTGACAGTTGTAGTACTCTTGTAACATCATCOsPR1#022CCACAGAGTTTGTCAGGATTGTCCAGATTGCACACACCTGATTCCOsPR1#052AGCTACCTGTCATTTCTTCATTTCTGCTACTCCAGAAGGAAATTAAAAGOsPR1#072AATTAATACTGGAGTAGATGCATGTACACGAATAACGTACTGTATTCTGTATGOsPR1#121ACCATCGTCGTCGTCTCATCAGCCTCTAGGGCATATCACTAACOsPR1#101TCGCTGCCGCTAGTACATTTCATTAAGATCATTACATGCTTTATTGTTCACOsPR1#051CCTGCCTGCCTTCCTCATTCAGTGAAGATTTGGTTTCCATTGTATTGActinGAGTATGATGAGTCGGGTCCAGACACCAACAATCCCAAACAGAGFig. 2Response of OsPR1 genes to pathogen infection. a Blast fungus infection. A conidia suspension of blast fungus race 003 was spray-inoculated onto Nipponbare (compatible, upper panel), and IL7 (incompatible, lower panel). The inoculated fourth leaves were harvested at 0, 3, and 6 days post inoculation (dpi) for RNA extraction. To semi-quantify the transcript level, RNA samples were subjected to qPCR, and the relative expression levels compared with that of a control actin gene are shown. The transcript levels at time 0, mock-inoculated, and blast-fungus inoculated leaves were shown as white, gray, and black columns, respectively. Bars indicate mean ± SD. The up-regulated genes by blast fungus-infection are marked with arrowheads. Gray arrowheads in the lower panel indicate early responsive genes. b Bacterial blight infection. The fourth leaves of Nipponbare rice plants were inoculated with a suspension of Xoo for bacterial blight disease, and the leaves were harvested at 0, 4, and 8 dpi for RNA extraction. The transcript level was analyzed by qPCR. The relative expression levels compared with that of the control actin gene are shown. Transcript levels at time 0, mock-inoculated, and bacteria-blight inoculated leaves are shown as white, gray, and black columns, respectively. Xoo-upregulated and -downregulated genes are marked with forward and reverse arrowheads, respectively. Bars indicate mean ± SD Incompatible interaction IL7 is a near isogenic line of Nipponbare, into which the resistance gene Pi-i for blast fungus race 003 has been introduced. In fungus-inoculated IL7, small HR lesions were induced at 2 dpi with no remarkable fungal development thereafter, indicating a typical incompatible interaction (Sasaki et al. 2004; Iwai et al. 2006). The transcript levels of OsPR1#074, #011, and #012 were maximized at 3 dpi, and slightly reduced at 6 dpi (Fig. 2a, lower part, gray arrowheads), indicating they are early responsive genes to blast fungus infection. The 3 genes had been recognized as early responsive genes in compatible interaction. The levels of OsPR1#074 and #011 transcripts increased by about 1,000-fold at 3 dpi. The transcripts of OsPR1#071, #072, #073, and #101 were higher at at 6 dpi than at 3 dpi, indicating they are late responsive genes (black arrowheads). The level of OsPR1#121 was suppressed by mock-inoculation but the level was not changed by incompatible interaction. OsPR1#051, whose transcript increased in the compatible interaction at 6 dpi, was not clearly upregulated in the incompatible interaction. Responses of OsPR1 genes to bacterial blight infection in young rice plants To study the response of the 12 OsPR1 genes to a bacterial pathogen, the fourth leaves of Nipponbare were inoculated with Xoo for leaf blight disease. In the compatible rice–bacterium interaction, the infected region was visualized as wilted necrosis, which spread from the cross cut end to the leaf base with time, and the area of necrosis grew to 2–3 cm long at 8 dpi. At 4 and 8 dpi, 5 cm long leaf pieces from the inoculated point were used to prepare RNA samples. Transcripts of OsPR1#074, #011, and #012, which are early responsive genes to blast fungus (Fig. 2a), accumulated remarkably at both 4 and 8 dpi compared with 0 dpi or a mock-inoculated control (Fig. 2b), indicating they were responsive to Xoo infection (closed forward arrowheads). Expression of OsPR1#021 and #022 were slightly suppressed after the infection compared with mock-inoculated leaves (closed reverse arrowheads). Expression of OsPR1#074, #011, #012, and #101 were upregulated by Xoo infection but other OsPR1 genes did not respond or rather suppressed, while all OsPR1 genes were upregulated in compatible blast fungus interaction (Fig. 2a). Wound-induced expression of OsPR1 genes in young rice plants To study the response of OsPR1 genes to wounding, the fourth leaves were wounded by cross cutting. Triplicate samples were extracted at 0, 1, 6, and 24 h after wounding, and used for qPCR. Among the 12 genes, OsPR1#074 was wound-inducible with peak accumulation of the transcript at 6 h after wounding, and OsPR1#051 was found to be an early wound-responsive gene (Fig. 3, forward arrowheads). The OsPR1#121 and #052 transcripts decreased in response to wounding (reverse arrowheads) (Fig. 3). Fig. 3Response of OsPR1 genes to wounding The fourth leaves were cut into 1 cm length sections for wounding, and floated on water for 0, 1, 6, and 24 h under light, and then the leaf pieces were homogenized for RNA extraction. Relative expression levels compared with that of the control actin gene are shown. The wound-upregulated and -downregulated genes are marked with forward or reverse arrowheads, respectively. Bars indicate mean ± SDFig. 4Organ-specific expression of OsPR1 genes Leaves and roots of the seedlings at the fourth leaf stage (left panel), and flowers of adult plants (right panel) were subjected to qPCR analysis. Relative expression levels compared with that of the control actin gene are shown. The genes that express at a high level in roots and flowers are marked with white and gray arrowheads, respectively. Bars indicate mean ± SD Organ specific expression of OsPR1 genes The basal transcript levels in various organs varied according to each gene; in the roots of healthy young rice plants at the 4-leaf stage, the transcript levels of the 5 genes, OsPR1#074, #071, #073, #072, and #101, were clearly higher than in the fourth leaves (Fig. 4) (open arrowheads). In flowers, the basal transcript levels of five genes, OsPR1#074, #011, #012, #121, and #051, were at a high level (gray arrowheads). These results indicate that the organ-specific expression profiles of OsPR1 genes varied considerably. The OsPR1#074, #071, #073, and #072, which are located on chromosome 7, were highly expressed in healthy roots. In healthy flowers the expression levels of five genes, OsPR#074, #011, #012, #121, and #051, were also higher. Responses of OsPR1 genes to defense signal compounds in young rice plants The responses of OsPR1 genes to defense signal compounds were studied. Intact young rice plants in soil pots were dipped in water, SA, or ACC solutions, or exposed to the vapor of MeJA. RNA from the fourth leaves was subjected to qPCR (Fig. 5). OsPR1#074 and #101 were upregulated by all three signal compounds tested, while OsPR1#071, #073, #021 and #121 were upregulated by JA but not by ACC or SA. OsPR1#074, #011, and #012, which were early responsive genes to blast fungus (Fig. 2), were SA-inducible. OsPR1#011 was upregulated by SA but suppressed by ACC. Among the six genes that were inducible by the wound signal compound JA, OsPR1#074 and #051 were also upregulated by wounding (Fig. 3). OsPR1#022 and #052 were not particularly sensitive to any of these compounds. OsPR1#011 and #072 were downregulated by ACC and SA respectively, and OsPR1#071 was downregulated by both ACC and SA. Fig. 5Response of OsPR1 genes to defense-signal compounds ACC, SA, and JA. Expression levels of each OsPR1 gene in fourth leaves at 24 h after treatment with defense-signal compounds were analyzed by qPCR. Relative expression levels compared with that of the control actin gene are shown. The ACC-, SA-, and JA-inducible genes are marked with gray, dark gray, and black arrowheads, respectively. For ACC or SA treatment, plastic pots with rice seedlings were dipped in solutions containing 1 mM ACC or 3 mM SA solution, and incubated for 24 h in the greenhouse. C1 control for ACC- and SA-treatment (leaves of water treated plant); For JA treatment, pots with seedlings each was put in an air-tight clear plastic box, and a cotton pad with volatile methyl jasmonate (MeJA) dissolved in EtOH was put at the corner of the box to give a final concentration of 100 μM, and incubated for 24 h. C2 control for JA-treatment (leaves of plant treated with 0 μM of JA), bars indicate mean ± SD Comparison of the defense-signaling pathways conferred by ACC, SA, and JA in rice with those in tobacco and Arabidopsis Using the 12 OsPR1 genes as probes, we analyzed the relationship of responsiveness to three defense signal compounds in rice. In tobacco, SA signaling was antagonistic to JA signaling on PR gene expression: Niki et al. (1998) showed that the expression of acidic PR1, 2, and 3 genes was upregulated by SA-treatment and it was suppressed in the presence of JA in a dose-dependent manner, while the expression of basic PR2, 5, and 6 genes was upregulated by JA and suppressed by SA. Thomma et al. (1998) reported that the JA-signaling pathway is different from the SA-signaling pathway in Arabidopsis. Glazebrook et al. (2003) reported that the expression patterns of some genes revealed mutual inhibition between SA- and JA-dependent signaling using global expression phenotyping analysis of mutant Arabidopsis plants. Synergistic induction by ET and JA was reported in basic tobacco PR genes (Xu et al. 1994). However, the expression patterns of OsPR1 genes in Fig. 5 showed no clear mutual inhibition between SA- and JA-dependent signaling, except for OsPR1#071. We further analyzed the relationship by means of Pearson’s correlation coefficient (Fig. 6). Correlation coefficients for SA versus ACC, ACC versus JA, and SA versus JA were 0.75, 0.61, and 0.22, respectively. The linear correlation coefficient, r, represents the strength and the direction of the linear relationship between two variables. A correlation of +1 means an increasing linear relationship and complete correlation, while −1 means a decreasing linear relationship. Cohen (1988) proposed that |r| below 0.1 is regarded as insubstantial, 0.1 ≤ |r| < 0.3 as weak, 0.3 ≤ |r| < 0.5 as medium, and 0.5 ≤ |r| < 1.0 as strong correlation. While, levels of statistical significance at 5 and 1% (n = 12) are 0.576 and 0.708, respectively. The r values in Fig. 6 suggest synergism between ACC versus SA and ACC versus JA, and low or no correlation between SA versus JA rather than antagonism. Fig. 6Relationship of the three signaling pathways in OsPR1 genes. Based on the data in Fig. 5, the response of young rice plants to ACC- , SA-, or JA-treatments was compared with other treatments, and the correlation coefficients between the responses to pairs of signals were calculated. See the text for details Tissue specific expression of a representative OsPR1 gene in rice plants Analysis of the tissue specific expression of rice PR1 gene by means of histological studies has not been performed previously. To study the tissue specific expression profiles of a representative OsPR1 gene, we selected OsPR1#074 because it clearly responded to both compatible and incompatible fungal interactions, bacterial bright infection, wounding and the 3 defense signal compounds. The promoter sequence of OsPR1#074 was obtained by PCR amplification from the Nipponbare genome. The promoter sequence, containing 1,919 bp, was inserted into the pTH2 vector (Kawahigashi et al. 2007) to construct the plasmid PR1T::GUS, which contains the promoter and the coding region for 16 amino acids of the N-terminus of the OsPR1 protein to express the GUS protein effectively as a translational fusion (Fig. 7a). The OsPR1T::GUS gene was introduced into rice plants (Oryza sativa cv. Nipponbare). Among 30 hygromycin-resistant plants regenerated, three independent lines were selected as representatives exhibiting a negligible level of GUS activity in healthy leaves and more than 30-fold higher levels after wounding or inoculation with a conidia suspension of M. grisea. The OsPR1T::GUS plants of the second or third generation were subjected to histological GUS analysis using X-gluc as the substrate. Fig. 7Histological analysis of OsPR1#074 gene expression in transgenic rice plants. a Schematic diagram showing the construct of the introduced gene. The promoter sequence of OsPR1#074 was used. b Localized GUS staining in healthy 5 day-old transgenic rice seedlings (a–e). Because similar expression profiles were obtained in three independent transgenic lines, the data from a representative line is shown. f unhulled rice. c GUS staining of 2 month-old transgenic rice plants. a Healthy roots, b roots at 2 days after wounding, c–e leaves at 2 days after wounding. d GUS staining assay of 4th leaves at 5 (a, c, d) or 11 (b) dpi. Black arrows indicate stained mesophyll tissue. Brown arrows indicate the necrotic regions Figure 7B shows the localization of GUS activity in healthy 5-day-old transgenic rice seedlings. A high level of GUS activity was detected in the primary root (arrows, Fig. 7B a–c) and the root tip (arrowheads), but it was absent or considerably reduced in the elongation zone (white arrowheads). In cross sections of the middle part of the root 80 μm in thickness (Fig. 7B e), a strong GUS activity was detected in the vascular cylinder (vc) and sclerenchyma (sc), and a weak activity in the cortex (co). GUS staining was found in the slit surface of the coleoptile (yellow arrowheads, Fig. 7B a, b), and on the surface of unhulled rice (white arrow, Fig. 7B a, f). In the anther, a weak blue staining was found in the matrix of pollen loculus (data not shown). In the vector control plants, no GUS activity was detected in roots (Fig. 7B d) or any other part analyzed under these in condition (data not shown). In 2 month-old OsPR1T::GUS plants, no GUS activity was found in healthy roots (Fig. 7C a), while high levels of GUS activity were found in healthy 5 day-old (Fig. 7Ba, b, and c) and 2 week-old (data not shown) plants. Thus, OsPR1 expression in roots is likely to be regulated developmentally. Wounding induced a high level of GUS activity in the roots of 2-month-old plants. When the aerial parts were removed by cross cut from of the plants, a strong GUS activity was induced in the roots of cut plants at 2 days after cutting (Fig. 7C b). In cross sections of the root in the middle region, GUS staining was found in all tissues, including the vascular cylinder, cortex, and sclerenchyma. No detectable GUS activity was found in healthy leaves of transgenic rice plants (data not shown); however, wounding induced a strong GUS activity in the neighbor region of the cut surface especially in the vascular systems including xylem parenchyma (xy) and phloem (ph) (Fig. 7C c–e). Next, GUS activity in rice leaves infected with blast fungus was analyzed. In the compatible cultivar (WT) lacking the Pi-iresistance gene, enlarged lesions were found 3–5 days after inoculation developing in size with time and with conidia formation on the infected leaf surface. After GUS staining, many blue spots were found at or around the lesions in the leaves 5 days after inoculation (Fig. 7D a). When the leaf of Fig. 7D a was cross cut at the marked line, and shown in Fig. 7D c, the blue staining was found to localize in the mesophyll cells (black arrows) around the necrotic regions (brown arrows, Fig. 7D d). At 11 days after inoculation, GUS activity was found around the necrotic lesions (Fig. 7D b). Discussion We studied here the response of 12 OsPR1 genes to pathogens, wounding, and defense signal compounds and their constitutive expression in organs. This is the first example of a comparative study on the characterization of a set of PR 1 family genes by qPCR, a high throughput method. The 12 genes include PR1a and b, which have been reported as pathogen-inducible genes by Agrawal et al. (2001), and ten of the 12 genes are contained in the 32 listed rice PR1-like genes including at least 3 pseudogenes whose expression profiles were not obvious (van Loon et al. 2006). We searched rice PR1-like genes from full-length cDNA (http://www.cdna01.dna.affrc.go.jp/cDNA) and EST (http://www.riceblast.dna.affrc.go.jp/). Because strongly expressed genes should be easily extracted from the databases for expressed genes, a majority of highly expressed rice PR1-like genes are thought to be contained in the 12 genes. The sequences of the 12 OsPR1 proteins were well conserved with complete matches in 10 amino acid residues in the C-terminal half (Fig. 1a). Because the 10 residues were also very well conserved in all 36 PR1-type proteins selected from 14 different plant species including Brassica napus, Hordeum vulgare, Lycopersicon esculentum, Medicago trunculata, Triticum aestivum, and Zea mays (van Loon and van Strien 1999), these regions in the C-terminus should be important for the putative functions of PR1 proteins in plants. All 12 OsPR1 genes selected here were clearly upregulated in a compatible rice-blast fungus interaction, indicating that they really are pathogen related. Notable is that the induction pattern of each OsPR1 gene by different pathogen–host interactions was characteristic (Fig. 2). Four genes, OsPR1#074, #011, #012, and #101, responded positively to blast fungus infection in both compatible and incompatible interactions, and also to Xoo infection. Among these, OsPR1#074, #011, and #012 were responsive to blast fungus in the early infection period in both interactions. Conversely, the expression of OsPR1#021 and #022 was reduced by Xoo infection compared with the control after mock-inoculation, which is a wound treatment. Assuming that Xoo infection may inhibit wound-induced expression of these genes, it is consistent that they are wound-inducible (Fig. 3). The above result that “all 12 OsPR1s are characteristically pathogen inducible” is apparently different from the data of Arabidopsis where only one PR1 gene was pathogen-inducible (AT2g14610) among 22 predicted ArabidopsisPR1 genes (van Loon et al. 2006). We studied this result using an AtGenExpress Visualization Tool from http://www.arabidopsis.org/, and confirmed that only the PR1 gene was upregulated by infection with either Pseudomonas syringae pv. tomato DC3000 or Pytophythora infestans, and the other PR1 genes were not. The reason for different responses of PR1s to pathogen infection in rice and Arabidopsis is not clear, but we could speculate that OsPR1s have evolved in the rice genome to provide resistance to a broad range of pathogens, but a limited set of ArabidopsisPR1s has evolved to respond to pathogens. In rice, the level of free SA in young healthy rice leaves was as high as about 10 μg per g fresh leaf (Silverman et al. 1995), but it was only about 20 ng (Seo et al. 2007) or 100 – 200 ng (Silverman et al. 1995) in tobacco leaves, and about 30 ng (Park et al. 2007) or 150 ng (our unpublished data) in Arabidopsis leaves. Thus, the SA content in rice leaves corresponds to 50- to 500-fold of that in tobacco or Arabidopsis leaves, which is comparable to the induced SA level after HR lesion formation by TMV-infection. Such a high SA level in rice plants could affect OsPR1 expression after pathogen infection, treatment with SA, JA, or ACC, and wounding. Actually, the responses of the OsPR1 genes to defense signal compounds indicate that the signaling pathways in rice conferred by SA and JA were synergistic as well as that by ET and JA, and SA and ET (Fig. 6). This finding is considerably different from the “antagonistic relationship of SA and JA signaling” found in tobacco (Niki et al. 1998) and Arabidopsis (Gupta et al. 2000). In tobacco, acidic PR1 proteins such as NtPR1a, b, and c were inducible by SA (Ohashi and Matsuoka 1987), which is a defense signal compound for systemic acquired resistance. The NtPRB1b (Eyal et al. 1992; Niki et al. 1998) and AtPRB1 (Santamaria et al. 2001) genes, which encode basic PR proteins in tobacco and Arabidopsis, respectively, were reported to respond positively to JA and ET, and negatively to SA. The ArabidopsisPR1 gene (At2g14610) encodes a basic protein that is inducible by SA. To compare the natures of the OsPR1 genes, the results of the expression of 12 OsPR1 genes are summarized in Fig. 8. It is again impressive that the expression upregulated by various treatments differed considerably depending on the gene, and the transcript levels constitutively found in leaf, root, and flower also varied according to the genes. When comparing the four genes OsPR1#071, #72, #073, and #074, which are clustered in a locus on chromosome 7, we found the following common characteristics: all four genes (1) encode acidic proteins, (2) are constitutively expressed in healthy roots at a high level, and (3) are upregulated by blast fungus-infection. In spite of these similarities, their expression profiles after certain treatments were significantly different from each other. For example, (1) the transcript of OsPR1#074 accumulated at an earlier time period than those of OsPR1#071, #072, or #073 in fungus-infected resistant cultivar IL7, (2) OsPR1#074 but not #071, #072, or #073 was upregulated by Xoo-infection, (3) OsPR1#074, but not OsPR1#071, #072, or #073, was wound-inducible, (4) OsPR1#074 was ACC-, SA-, and JA-inducible, OsPR1#071 was JA-inducible, OsPR1#072 was not inducible by any signal compounds used here, and OsPR1#073 appeared to be suppressed by ACC, (5) OsPR1#074 was expressed in flowers at a high level, but OsPR1#071, #072, and #073 were not. Fig. 8Comparison of expression characteristics of OsPR1 genes Induction levels of OsPR1 genes by pathogens, wounding, and defense signal compounds, and their constitutive expression levels in organs were compared. The transcript levels in healthy 4th leaves are shown in the right most column. ++ very highly inducible or expressed gene, + highly inducible or expressed gene, ± gene whose induction or expression was not clear, − not inducible or expressed gene From the expression characteristics of the 12 OsPR1 genes obtained here, the possible contribution of all OsPR1 genes for plant self-defense against pathogen attack was indicated. Although the functions of OsPR1 genes are not fully understood, PR1 proteins in some plant species have been reported to have antifungal activity including tobacco acidic PR1a overexpression that induced increased tolerance to two oomycete pathogens in tobacco (Alexander et al. 1993), and basic PR-1 proteins of tobacco and tomato, which have an antimicrobial activity against Phytophythora infestans (Niederman et al. 1995). Thus, the redundant expression of the 12 OsPR1 genes may effectively contribute to defend the attack of various pathogens. The expression levels of the 12 genes in healthy tissues and wounded leaves were also very different according to each particular gene. The data in Fig. 7 shows a representative example of tissue- and organ-specific expression of OsPR1. The level of GUS activity in transgenic rice plants carrying the OsPR1T::GUS gene was confirmed to be similar to the OsPR1#074 transcript level as shown in tobacco PR1a::GUS plants (Yamakawa et al. 1998), in which the GUS gene was expressed as a similar translational fusion gene described in Fig. 7A. GUS analysis using independent transgenic lines showed OsPR1#074 expression was developmentally regulated in roots and constitutively expressed in young rice plants, but not in 2-month old plants, in which the expression was strongly upregulated by wounding (Fig. 7A, B). The wound-induced OsPR1#074 expression in adult plants was localized mainly in the vascular system, which is important as the sensing tissue of the water pressure change after wounding and as the site of bacterial pathogen propagation. The information on the 12 PR1 genes obtained here will be useful for further studies on self-defense mechanisms in rice plants, and for the usage of each PR1 gene as a characteristic marker gene.

Diabetologia-4-1-2362135

Reconstituted HDL infusion restores endothelial function in patients with type 2 diabetes mellitus

To the Editor: HDL-cholesterol is inversely correlated with cardiovascular events in all major epidemiological studies [1]. HDL-increasing strategies have demonstrated that HDL-cholesterol increase is associated with decreased cardiovascular risk in high-risk individuals such as patients with type 2 diabetes [2]. Endothelial dysfunction, a hallmark of type 2 diabetes patients, has been shown to predict future cardiovascular events [3]. Therefore, we investigated the effect of reconstituted HDL (rHDL) on endothelial function measured both acutely (4 h after infusion) and 7 days after infusion in type 2 diabetes patients. Control volunteers were measured only at baseline and 4 h after infusion. Seven non-smoking patients with type 2 diabetes (four men and three women, BMI 24.4 ± 1.6 kg/m2) and seven matched control volunteers (four men and three women, BMI 22.9 ± 1.8 kg/m2) were enrolled. Inclusion criteria for type 2 diabetes patients were: (1) fasting glucose >7.0 mmol/l; (2) no insulin therapy; and (3) triacylglycerol and LDL-cholesterol levels <2.0 and <3.5 mmol/l, respectively. Matched control individuals were volunteers who were recruited via advertisements. The presence of macrovascular disease (ECG abnormalities, abnormal ankle–brachial index or a history of cardiovascular events) served as exclusion criteria. Female participants were postmenopausal and not using hormone replacement therapy. The study protocol was performed at least 4 weeks after discontinuation of vasoactive medication, including ACE inhibitors, angiotensin receptor blockers and non-steroidal anti-inflammatory drugs. None of the patients or control volunteers used lipid-lowering medication. The Internal Review Board of the Academic Medical Center approved the study and all individuals gave written informed consent. Vascular function was assessed using venous occlusion strain-gauge plethysmography (EC-4; Hokanson, Washington, DC, USA) [4]. Forearm blood flow (FBF), expressed as ml min−1 100 ml−1 forearm tissue volume (FAV), was measured simultaneously in both arms. FBF responses to cumulative doses of the endothelium-dependent vasodilator serotonin (Sigma, Poole, UK; 0.6, 1.8 and 6 ng 100 ml−1 FAV min−1), the endothelium-independent vasodilator sodium nitroprusside (SNP; Spruyt Hillen, IJsselstein, the Netherlands; 6, 60, 180 and 600 ng 100 ml−1 FAV min−1), and the competitive inhibitor of endothelial nitric oxide (NO) synthase NG-monomethyl-l-arginine (l-NMMA; Kordia, Leiden, the Netherlands; 50, 100, 200 and 400 μg 100 ml−1 FAV min−1) were measured. Agents were administered intra-arterially for 6, 4 and 8 min at each dose, respectively. Average FBF values of the measurement (cannulated) and control arm were obtained from the last six measurements of each measurement period. The three different infusion blocks proceeded after a 15 min rest period or until FBF had returned to baseline. The ratio of flow in the infused measurement (M) and non-infused control (C) arm was calculated for each recording (M/C ratio). The average value of the M/C ratio was calculated from these four to six M/C ratios, thus providing an internal control by excluding systemic factors from influencing the results [5]. Subsequently, a venous catheter was inserted in the contralateral arm for administration of rHDL (CSL-111; CSL Bioplasma, Parkville, VIC, Australia) at a dose of 80 mg/kg body weight over a period of 4 h. Subsequently, the infusion blocks were repeated. Blood samples were drawn from the individuals after a 12 h overnight fast, and at 4 h and 7 days after rHDL infusion. Descriptive statistics between the two groups were compared by two-tailed independent Student’s t tests or non-parametric tests, depending on skewedness of the data. Analysis of measurements for individuals between baseline and 4 h as well as baseline and 7 days after rHDL infusion was performed by two-way ANOVA for repeated measures with Bonferroni correction. Infusion of rHDL was well tolerated and no adverse events were recorded. Characteristics of type 2 diabetes patients and control volunteers during each measurement are listed in Table 1. Baseline FBFs were not significantly different between type 2 diabetes patients and control individuals (Table 1). Intra-arterial infusion of serotonin increased FBF in a dose-dependent manner in both groups (see Fig. 1a). At baseline, the FBF response to serotonin was attenuated in type 2 diabetes compared with control volunteers (M/C ratio in type 2 diabetes: 1.5 ± 0.2 vs controls: 2.5 ± 0.3; p < 0.05). Four hours after rHDL infusion in type 2 diabetes, FBF response to serotonin increased significantly (M/C ratio to 1.9 ± 0.2; p < 0.05 compared with baseline). rHDL infusion had no significant effect on serotonin-induced vasodilation in control volunteers. In type 2 diabetes, 7 days after rHDL infusion serotonin responses had returned to baseline values (7 days, 1.5 ± 0.2). At baseline, the maximal vasoconstrictor response to L-NMMA was blunted in type 2 diabetes compared with control volunteers (M/C ratio controls 0.6 ± 0.1 vs type 2 diabetes 1.0 ± 0.2; p < 0.05, Fig. 1b). After rHDL infusion, the L-NMMA response improved in type 2 diabetes compared with baseline (0.7 ± 0.1; p < 0.05). Although not significant, 7 days after rHDL infusion there still was a tendency towards improvement in type 2 diabetes (M/C ratio 0.8 ± 0.1 compared with baseline). rHDL infusion had no effect on L-NMMA response in control individuals. Finally, SNP responses were lower in type 2 diabetes vs control individuals at baseline (M/C ratio in type 2 diabetes, 3.0 ± 0.6 vs control individuals, 5.3 ± 0.7; p < 0.05, Fig. 1c) and rHDL infusion had no effect on SNP responses. Fig. 1Change in M/C ratio after stimulation with the endothelium-dependent vasodilator serotonin (a), NO inhibitor L-NMMA (b) and the endothelium-independent vasodilator SNP (c) before (white circles) and 4 h (black circles) and 7 days (diamonds) after rHDL infusion in type 2 diabetes patients as well as in control volunteers (before, white triangle; after 4 h, black triangle). bp < 0.05 for type 2 diabetes patients compared with control individuals and ap < 0.05 for changes after rHDL within type 2 diabetes patients. Data are means ± SEMTable 1Clinical characteristics of type 2 diabetes patients and control volunteers Type 2 diabetes patients (n = 7)Control volunteers (n = 7)Baseline4 h7 daysBaseline4 hWaist circumference (cm)101 ± 5  90 ± 6† Metabolic syndrome6/7  0/7 Systolic BP (mmHg)148 ± 12146 ± 11143 ± 7135 ± 16138 ± 12Diastolic BP (mmHg)78 ± 1379 ± 1382 ± 883 ± 985 ± 8Heart rate (beats per min)65 ± 967 ± 966 ± 1261 ± 964 ± 8Total cholesterol (mmol/l)5.6 ± 0.46.5 ± 1.45.4 ± 1.35.3 ± 0.46.7 ± 1.3LDL-cholesterol (mmol/l)2.9 ± 0.63.3 ± 1.13.1 ± 1.03.0 ± 0.73.9 ± 1.2HDL-cholesterol (mmol/l)1.1 ± 0.22.7 ± 0.7*1.6 ± 0.61.2 ± 0.32.5 ± 0.4*ApoA-I (g/l)1.2 ± 0.12.8 ± 0.4*1.5 ± 0.31.2 ± 0.22.7 ± 0.4*Triacylglycerol (mmol/l)1.5 ± 0.41.6 ± 0.61.6 ± 0.50.8 ± 0.3†1.8 ± 1.3*Glucose (mmol/l)8.3 ± 1.26.8 ± 1.77.3 ± 1.35.2 ± 0.4†4.9 ± 0.2hsCRP (mg/l)3.5 ± 1.64.4 ± 1.73.6 ± 0.81.0 ± 0.9†1.8 ± 1.1ASAT (U/l)22.5 ± 2.520.4 ± 4.022.4 ± 4.320.2 ± 1.821.4 ± 2.7ALAT (U/l)31.2 ± 6.926.0 ± 9.727.8 ± 8.714.9 ± 1.7†15.6 ± 3.2Basal FBF (ml 100 ml−1 FAV min−1)4.1 ± 2.03.7 ± 0.83.9 ± 1.32.6 ± 0.92.8 ± 0.7Values are means ± SD*p < 0.05 compared with baseline within one group; †p < 0.05 between type 2 diabetes and control volunteers ALT, alanine aminotransferase; ASAT, aspartate aminotransferase; hsCRP, high-sensitivity C-reactive protein In conclusion, the present study confirms that basal and stimulated NO bio-availability is reduced in type 2 diabetes patients compared with control volunteers. Besides hyperglycaemia-induced reactive oxygen radical formation, other components of type 2 diabetes-associated dyslipidaemia such as small dense LDL are known to influence endothelial function in type 2 diabetes [3, 6]. Moreover, the high prevalence of metabolic syndrome in our patients is in line with previously published data [7]. rHDL resulted in a significant improvement of endothelial function within several hours in type 2 diabetes patients. More importantly, there was still a tendency towards improved NO availability 7 days after infusion, at a time when apolipoprotein A-I (ApoA-I) increase had largely disappeared. Acute HDL-increasing strategies are actively being pursued for further reducing cardiovascular burden [8], and thus far the lack of selective and potent HDL-increasing drugs has limited the success of the HDL-cholesterol increase concept. Our reported beneficial effects of ApoA-I infusion may lend further support to the development of ApoA-I-increasing strategies, also for patients with type 2 diabetes.

Psychopharmacologia-3-1-1915592

Effects of the cannabinoid CB1 receptor antagonist rimonabant on distinct measures of impulsive behavior in rats

Rationale Pathological impulsivity is a prominent feature in several psychiatric disorders, but detailed understanding of the specific neuronal processes underlying impulsive behavior is as yet lacking. Introduction Despite its relative recent discovery, the central endocannabinoid system has been implicated in a variety of behaviors such as food intake (for review, see Di Marzo and Matias 2005) and nociception (for review, see Cravatt and Lichtman 2004), and in mediating the reinforcing properties of drugs of abuse (for review, see De Vries and Schoffelmeer 2005). In addition, the endocannabinoid system has been shown to play an important role in various cognitive processes. In this respect, memory encoding, retrieval, and extinction processes have received most interest (e.g., Marsicano et al. 2002; Takahashi et al. 2005; Varvel et al. 2005; for review, see Lichtman et al. 2002), presumably due to the high expression level of CB1 receptors in the hippocampal formation (Egertova and Elphick 2000; Tsou et al. 1998). High densities of CB1 receptors are also present in frontal cortical and striatal regions (Egertova and Elphick 2000; Tsou et al. 1998), suggesting involvement of the endocannabinoid system in executive functions that appear to be largely controlled by frontal corticostriatal systems (for review, see Miller and Cohen 2001). Indeed, several clinical and preclinical observations have demonstrated that Δ9-tetrahydrocannabinol (THC), the principle active cannabinoid of Cannabis sativa and other synthetic cannabimimetics impair selective attention (Arguello and Jentsch 2004; Solowij et al. 1995; Verrico et al. 2004) and behavioral flexibility (Egerton et al. 2005; Hill et al. 2006), alter time estimation (Han and Robinson 2001, McDonald et al. 2003), and impair working memory (Ilan et al. 2004; Jentsch et al. 1997). Nonetheless, to date, little is known about the involvement of the endocannabinoid system in other executive functions such as inhibitory control processes subserving impulsivity. Pathological levels of impulsive behavior are important features in attention-deficit/hyperactivity disorder, substance-related disorders, bipolar disorders and personality disorders (American Psychiatric Association 2000). Further elucidating the neurobiological basis of impulsivity may therefore enhance our understanding of these psychiatric disorders. It is becoming increasingly clear, however, that the concept impulsivity is multifaceted and covers various distinct and independent measures. These measures range from poor inhibitory control (impulsive action) to probability and delay aversion or impulsive choice (Barkley 1997; Evenden 1999; Moeller et al. 2001). Recent studies have implicated CB1 receptors in some of these measures of impulsivity. For instance, it has been shown that acute THC impairs response inhibition in healthy volunteers, whereas time estimation and impulsive choice were not affected (McDonald et al. 2003). On the other hand, it has been demonstrated more recently that marijuana acutely increases risk taking in volunteers (Lane et al. 2005). Collectively, these data suggest a role of the cannabinoid system in impulsivity, although its precise role therein is still unclear. The present experiments were aimed at further elucidating the importance of cannabinoid CB1 receptor activation on distinct measures of impulsivity. To this end, we tested the effects of the potent and selective CB1 receptor antagonist rimonabant (SR141716A; Rinaldi-Carmona et al. 1994) and agonist WIN55,212-2 (D’Ambra et al. 1992) on impulsive behavior in various operant paradigms measuring different and presumably independent aspects of impulsivity (for review, see Winstanley et al. 2006), namely, (1) the five-choice serial reaction time task to measure inhibitory control; (2) the delayed reward paradigm to measure impulsive choice, and (3) the stop-signal paradigm to measure response inhibition. Materials and methods Subjects In total, 48 male Wistar rats were obtained from Harlan CPB (Horst, The Netherlands). At the start of the experiments, animals were 12 weeks old, weighed approximately 250 g, and were housed in pairs in macrolon cages (42.5 × 26.6 × 18.5 cm; l × w × h) under a reversed 12 h light/dark cycle (lights on at 7:00 p.m.) at controlled room temperature (21 ± 2°C) and relative humidity of 60 ± 15%. Animals were maintained at approximately 90% of their free-feeding weight, starting 1 week before the beginning of the experiments by restricting the amount of standard rodent food pellets (Harlan Teklad Global Diet, Blackthorn, UK). Water was available ad libitum throughout the entire experiment. All experiments were conducted with the approval of the animal ethical committee of the Vrije Universiteit, Amsterdam, The Netherlands. Apparatus Experiments were conducted in 12 identical rat five-hole nose poke operant chambers with stainless steel grid floors (MED-NPW-5L, Med Associates, St. Albans, VT, USA) housed in sound-insulating and ventilated cubicles. Set in the curved wall of each box was an array of five circular holes, 2.54 cm in diameter, 2.2 cm deep, and 2.25 cm above floor level. Each hole was equipped with an infrared detector located across each nose poke unit 1.0 cm from the front, and a yellow LED stimulus light (6.4 mm in diameter). Rodent food pellets (45 mg, Formula P, Research Diets, New Brunswick, NJ, USA) could be delivered at the opposite wall via a dispenser. In addition, the chamber could be illuminated by a white houselight, and sound stimuli were generated using a programmable audio generator (ANL-926, Med Associates). A computer equipped with MED-PC version 1.17 (Med Associates) controlled experimental sessions and recorded data. Animals were tested once daily from Monday until Friday, during the dark phase of the light/dark cycle. Behavioral procedures Separate groups of n = 16 animals were trained for each different paradigm, and for all procedures, a similar habituation and magazine training protocol was used. This protocol consisted of a habituation exposure to the boxes for 20 min with the houselight on and the food cup containing three food pellets for two consecutive sessions. Subsequently, in the next two sessions, in total, 100 pellets were delivered with an average delay of 15 s, to allow the animals to associate the sound of pellet delivery with reward. Five-choice serial reaction time task A more detailed description of training in the 5-CSRTT in our laboratory has been reported previously (Van Gaalen et al. 2006a). In short, rats were trained 5 days per week to detect and respond to a brief visual stimulus in one of five holes to obtain a food reward. Each session terminated after 100 trials or 30 min, whichever occurred first. Initially the duration of this stimulus was 32 s and was gradually decreased to 1 s over sessions until animals reached stable baseline performance (accuracy >80% correct choice and <20% errors of omission). Responding during stimulus presentation or within the limited hold (LH) period of 2 s was counted as a correct response. Incorrect, premature responses and errors of omission (no responses or a response after the LH) did not lead to the delivery of a food reward and resulted in a 5-s time-out period during which the houselight was extinguished, whereas perseverative responses, i.e., repeated responding during the presentation of the stimulus, were measured but did not have any programmed consequences. Two different measures of inhibitory control were measured, namely, (1) the number of premature responses before the onset of the visual stimulus, reflecting aspects of loss of inhibitory control and (2) the number of perseverative responses into the stimulus hole after correct choice, presumably measuring aspects of compulsive behavior. In addition, the following other behavioral parameters were measured that reflect task performance, namely, (3) accurate choice, i.e., percentage correct responses calculated as [number correct trials / (correct + incorrect trials)] × 100; (4) latency to make a correct choice, i.e., the mean time between stimulus onset and nose poke in the illuminated hole; (5) omission errors, i.e., the number of omitted trials during a session; and (6) feeder latency, i.e., the latency to collect a pellet following correct choice. Delayed-reward paradigm In addition to the 5-CSRTT, the delayed reward paradigm, as employed in our laboratory, has also been described more elaborately (Van Gaalen et al. 2006b). Briefly, rats were trained 5 days per week in this paradigm. In the final stages of training and during drug testing, a session was divided into 5 blocks of 12 trials, each block starting with 2 forced trials during which, after initiating the trial through a nose poke into the central hole, either the left hole or the right hole was illuminated in a counterbalanced fashion. In the next ten trials, the animals had a free choice, and both the left and right hole were illuminated. Poking into one position resulted in the immediate delivery of a small reinforcer (one food pellet), whereas a nose poke into the other position resulted in the delivery of a large, but delayed, reinforcer (four food pellets). If an animal did not make a response during this choice phase within 10 s, an intertrial interval was initiated, and the trial was counted as an omission. The position associated with the small and large reinforcer was always the same for each individual and counterbalanced for the group. The delay for the large reinforcer progressively increased within a session per block of 12 trials as follows: 0, 10, 20, 40, and 60 s. Responding into non-illuminated holes during the test was recorded, but had no programmed consequences. The behavioral measure to assess task performance, i.e., the percentage preference for the large reinforcer as a function of delay, was calculated as: the number of choices for the large reinforcer / (number choices large + small reinforcers) × 100. In addition, we calculated the total number of omitted choice trials within a session. Stop-signal paradigm Shaping During initial shaping for two consecutive sessions, both the middle nose poke hole and the hole immediately adjacent to the right or left were illuminated (counterbalanced for all subjects). A nose poke into either one of the two active holes extinguished the visual stimuli in both holes and resulted in delivery of a pellet. After an intertrial interval of 10 s, the next trial started. Nose poking within this intertrial interval period did not have any programmed consequences. A session ended when the rat had earned 100 pellets or after 30 min, whichever occurred first. Shaping: go trials During the next phase, only the stimulus light in the middle nose poke hole was illuminated (start stimulus). A response into the active middle hole switched off the stimulus light and was followed by the illumination of the stimulus light (go stimulus) in the hole immediately adjacent to the left or right. A nose poke into this illuminated hole switched off the stimulus light and resulted in the delivery of a pellet. After an intertrial interval of 10 s, the next trial started. Responding in the start stimulus hole during presentation of the go stimulus was counted as perseverative start pokes, whereas prestimulus responses into the go stimulus hole resulted in a timeout period of 5 s. Subsequently, the response requirements into the start stimulus hole before onset of a go stimulus were varied into a variable ratio 2 schedule (VR2, i.e., either FR1, FR2, or FR3) to avoid the development of a prepotent response pattern from start stimulus to go stimulus hole and to ensure that animals waited until the appearance of a go stimulus. During this phase, rats were trained until they reliably completed 200 successful go trials. Following this phase, a LH period was introduced for the go stimulus and only during this period was the go stimulus present. Initially, the LH was set at 5 s, and in subsequent sessions, was individually titrated to meet performance criterion of 80% successful hits and <20% prestimulus responses. Omissions of a go stimulus response within the LH resulted in a 5-s time-out period, during which both the houselight and stimulus light were turned off. From initial shaping to criterion performance in this phase, 26 sessions were required. Shaping: introduction stop signal During the final training phase, a stop signal was introduced in 25% of all trials. Initially, this stop signal (duration, 50 ms; frequency, 4,500 Hz; intensity 80 dB) was contingent with the appearance of the go signal. Responding during the onset of the stop signal or during the LH immediately extinguished the go stimulus and houselight, turned off the stop signal, and was followed by a 5-s time-out. In contrast, if the animal successfully refrained from responding during a stop trial, a pellet was delivered. Initially, the LH during stop and go trials were equal; however, when performance during stop trials was below 80% successfully inhibited stop trials, the LH during stop trials was lowered over sessions in steps of 100–200 ms until animals improved performance. Subsequently, the LH was then gradually increased in these individuals over sessions until the LH during both go and stop trials were equal. As soon as animals reached the criterion of approximately 90% successfully inhibited stop trials, delays for the onset of the stop signal were introduced. The stop-signal delays (SSD) were presented in a pseudorandom order, and to compensate for differences between rats, SSDs were based on each individual rats’ mean reaction time on go trials in the preceding drug-free training session. SSDs were calculated as follows: mRT minus either 25, 50, 100, 200, or 400 ms. In addition, an equal amount of zero delays were presented during sessions. Drug testing commenced upon stable baseline performance for at least five consecutive sessions, i.e., 80% accuracy during go trials and a significant SSD-dependent decrease in correctly inhibited stop trials. It took 30 sessions before animals reached stable baseline performance after the introduction of the stop signal; therefore, from initial shaping to stable baseline performance in the stop-signal paradigm, in total, 56 sessions were required. Stop-signal paradigm: estimation stop-signal reaction time and correction for omissions during go trials Calculations to estimate the stop-signal reaction times (SSRT) and a correction for omission errors were adapted from Logan (1994) and Solanto et al. (2001). For estimating the SSRT, data of the three SSDs of 200, 100, and 50 ms were used, as the probability of correct inhibition on these intervals was within the range of 0.2 < p < 0.8, and thus, most informative for estimating SSRT (Band et al. 2003). For each of the three intervals, the probability of responding was calculated including a correction for nonresponses based on the number of omissions during the go trials, the latter, as omissions cannot be distinguished from successful inhibitions during stop trials. The following formula, adapted from Solanto et al. (2001) was used for these calculations: where x is the number of stop-signal trials at each delay interval; correct inhibitions are the number of correctly inhibited trials, and y is the probability of omissions during the go trials within the entire session. To calculate SSRTs, reaction times on all go trials were rank ordered. From this list with RTs, the “nth” RT was taken, where “n“ was obtained by multiplying the total number of go trials by the probability of responding for a particular SSD. This RT value approximates the latency between onset of the go stimulus and completion of the stopping process. The SSRT for each interval is then obtained by subtracting the SSD interval from this RT. The average estimated SSRT that is used for the analyses in the present study is calculated by taking the mean of each SSRT at the three SSDs (200, 100, and 50 ms). Drugs SR141716A was generated and kindly donated by Solvay Pharmaceuticals (Weesp, The Netherlands), whereas WIN55,212-2 was purchased from Tocris Biosciences (Bristol, United Kingdom). Both SR141716A and WIN55,212-2 were dissolved as described previously in a mixture of ethanol, Tween80, and sterile saline (ratio 1:1:18; cf. De Vries et al. 2001). In all experiments, SR141716A was injected 30 min before testing, whereas WIN55,212-2 was injected 20 min before testing. In all paradigms, the order of testing the drugs was (1) SR141716A and (2) WIN55,212-2. The drug combination was only performed in the five-choice serial reaction time task and followed the studies with SR141716A and WIN55,212-2. Drugs were freshly prepared each day before testing and intraperitoneally injected in a volume of 1 ml/kg bodyweight according to a Latin square design for both the dose–response studies and the drug combination on Tuesdays and Fridays, with baseline training sessions on the other weekdays. Statistical analyses Data were subjected to repeated measures analysis of variance (ANOVA) with drug dose (all paradigms), delay to large reinforcer (delayed reward paradigm), and stop-signal delay (stop-signal paradigm) as within subjects variables using the Statistical Package for the Social Sciences version 11 (SPSS, Chicago, IL, USA). The homogeneity of variance across groups was determined using Mauchly’s tests for equal variances, and in case of violation of homogeneity, corrected, and therefore, more conservative Huynh–Feldt probability values were used for subsequent analyses. In the stop-signal paradigm, some further exploratory analyses were performed between mean go reaction times and the limited hold period using a bivariate Pearson correlation. In addition, the estimated SSRT data were also subjected to a median split analysis to assess whether SR141716A or WIN55,212-2 had differential effects on response inhibition in individuals with relatively “fast” or “slow” stopping abilities. In case of statistically significant main effects, further post hoc comparisons were conducted using Student–Newman–Keuls Tests. The level of probability for statistically significant effects was set at 0.05. Results Effects of SR141716A and WIN55,212-2 on measures of inhibitory response control in the five-choice serial reaction time task The number of premature responses, a measure of inhibitory control reflecting impulsive behavior, was dose dependently decreased by SR141716A [Fig. 1a; F3,45 = 12.24, p < 0.001], and further post hoc analyses revealed that all doses significantly lowered premature responding compared with vehicle, whereas the highest dose (3.0 mg/kg) even further lowered the number of premature responses compared with 1.0 mg/kg SR141716A. In contrast, perseverative responding after correct choice, a different measure of inhibitory control reflecting compulsive behavior, was not affected at any dose [Fig. 1b; F3,45 = 1.84, p = 0.15]. Attentional function was improved by SR141716A, and further analyses revealed that only 0.3 mg/kg SR141716A significantly increased the percentageof accurate choice from approximately 77% under vehicle conditions to 82% as shown in Table 1 [F3,45 = 3.43, p = 0.025]. In addition, a marginal, but significant, increase in the latency to make a correct choice was also detected, and further comparisons showed that only the high dose of 3.0 mg/kg significantly slowed correct response reaction time [Table 1; F3,45 = 4.53, p = 0.007]. The latency to collect a food reward and errors of omission, however, were not affected at any dose [Table 1; feeder latency: F3,45 = 0.39, p = 0.65 and omissions: F3,45 = 1.30, p = 0.29]. Fig. 1Effects of SR141716A (a, b) and WIN55,212-2 (c, d) on different measures of inhibitory control in the 5-CSRTT. Data depict mean (±SEM) numbers of premature responses (a, c) and perseverative responses after correct choice (b, d). *p < 0.05 and **p < 0.005 vs vehicleTable 1Effects of SR141716A and WIN55,212-2 on measures of attentional function and motivation in the 5-CSRTT. Data depict mean ± SEMSubstances Accuracy (%)Response latency (ms)Omissions (%)Feeder latency (ms)SR141716AVehicle77.2 ± 2.4336 ± 95.4 ± 1.1954 ± 480.3 mg/kg82.0 ± 1.9*338 ± 105.6 ± 1.0954 ± 441.0 mg/kg80.5 ± 2.5347 ± 145.1 ± 0.7937 ± 433.0 mg/kg80.2 ± 2.3374 ± 14*7.3 ± 1.0939 ± 37WIN55,212-2Vehicle 77.7 ± 2.5324 ± 73.9 ± 0.7956 ± 530.3 mg/kg79.6 ± 2.3333 ± 103.4 ± 0.7910 ± 411.0 mg/kg77.2 ± 2.3380 ± 23*14.4 ± 4.3*943 ± 443.0 mg/kg76.8 ± 2.7399 ± 17**20.2 ± 4.1**1212 ± 138*p < 0.05**p < 0.005 vs vehicle WIN55,212-2 did neither change the number of premature responses nor the number of perseverative responses after correct choice [Fig. 1c and d; premature responses: F3,45 = 1.02, p = 0.39 and perseverative responses: F3,45 = 1.64, p = 0.21; respectively]. Furthermore, as indicated in Table 1, WIN55,212-2 did not change accurate choice or the latency to collect a pellet after correct choice [accurate choice: F3,45 = 0.72, p = 0.54 and feeder latency: F3,45 = 3.19, p = 0.052]. In contrast, errors of omission were increased, and latencies to make a correct choice were lengthened by WIN55,212-2 [omissions: F3,45 = 7.44, p = 0.002 and correct response latency: F3,45 = 6.59, p = 0.004]. As shown in Fig. 2, the decrements in premature responding by 3.0 mg/kg SR141716A were prevented in the presence of WIN55,212-2 at a dose of 1.0 mg/kg [F3,45 = 7.33, p < 0.001]. In addition, this selected dose of WIN55,212-2, by itself, did not affect inhibitory control as indicated by the absence of an effect of this dose on the number of premature responses. Fig. 2Coadministration of WIN55,212-2 at 1.0 mg/kg (WIN1) prevents the effects of 3.0 mg/kg SR14716A (SR3) on inhibitory control in the 5-CSRTT. All data are depicted as mean (±SEM). *p < 0.05 and **p < 0.005 SR141716A and WIN55,212-2 do not affect decision making in the delayed reward paradigm Stable baseline performance on the delayed reward paradigm occurred after approximately 30 training sessions on a full delay range (0, 10, 20, 40, and 60 s), and therefore, we commenced drug testing from session 35 onwards. A clear, highly significant delay-dependent decrement in the percentage preference for the large reinforcer was observed [Fig. 3; delay: F4,60=270.22, p < 0.001]. Nonetheless, SR141716A did not shift the preference for a large reinforcer over delays at any of the tested doses [dose: F3,45 = 1.56, p = 0.21 and dose × delay: F12,180 = 1.24, p = 0.29]. In addition, SR141716A also did not change the total numbers of omitted choice trials, i.e., the failures to start a trial during the choice phase [dose: F3,45 = 3.10, p = 0.052]. Fig. 3Effects of SR141716A (a) and WIN55,212-2 (b) on the mean (±SEM) percentage preference for the large reinforcer in the delayed reward paradigm In the WIN55,212-2 experiments, at the highest dose, in total, five animals omitted all choice trials of some delays, and therefore, were excluded from all analyses of the delay discounting data. Similar to SR141716A, increasing the delay highly significantly shifted the preference from large to the small reinforcer [Fig. 4; delay: F4,40 = 179.06, p < 0.001]. Although there was no overall effect of WIN55,212-2 on decision making [dose: F3,30 = 0.68, p = 0.57], there was a dose by delay interaction effect suggesting a shift in preference for the large reinforcer over delays [dose × delay: F12,120 = 2.61, p = 0.011]. Nonetheless, further post hoc comparisons revealed no differences between vehicle and any of the other doses. The number of omitted choice trials was not affected by WIN55,212-2 [dose: F3,30 = 1.61, p = 0.21]. Fig. 4Effects of SR141716A on response inhibition as measured in the stop-signal paradigm. Data are depicted as mean (±SEM) percentage of correctly inhibited stop trials with varying SSDs before the mean go RT (a), go reaction times (b) and estimated stop signal reaction times from SSDs 200, 100, and 50 ms (c). **p < 0.005 vs vehicle Effects of SR141716A and WIN55,212-2 on response inhibition in the stop-signal paradigm Under baseline conditions in the stop-signal paradigm, there was stable individual variation in mean reaction times during go trials over consecutive sessions ranging from 290 to 470 ms (mean: 365 ms; standard deviation: 58 ms). Likewise, the limited hold periods during go and stop trials also stably varied and ranged from 400 to 1,100 ms (mean: 594 ms; SD: 167 ms). A highly significant positive correlation indicated that individuals with “shorter” limited hold periods also displayed shorter mean go reaction times compared to individuals with “longer” limited hold periods that were slower in go reaction speed [r = 0.85, p < 0.001]. Response inhibition, as displayed in the inhibition function curve in Fig. 4a, was not affected by SR141716A at any of the tested doses [dose: F3,45 = 1.28, p = 0.29 and dose × SSD: F12,180 = 1.25, p = 0.26]; however, the percentage of correctly inhibited stop trials significantly declined with decreasing stop-signal delays [SSD: F4,60 = 39.24, p < 0.001]. SR141716A did slow mean reaction times during go trials by approximately 30 ms [Fig. 4b; dose: F3,45 = 7.93, p = 0.001], and further analyses indicated that both the 1.0 and 3.0 mg/kg dose significantly slowed reaction times compared with vehicle. Nonetheless, the number of omissions during go trials were not significantly affected [mean ± SEM: vehicle = 16.2 ± 3.0; 0.3 mg/kg = 18.1 ± 1.2; 1.0 mg/kg = 17.7 ± 1.7 and 3.0 mg/kg = 23.3 ± 2.6; dose: F3,45 = 1.76, p = 0.17]. Lastly, the average estimated SSRT across three stop-signal delays, putatively reflecting response inhibition, was not significantly changed by SR141716A at any dose [Fig. 4c; dose: F3,45 = 1.47, p = 0.24]. Furthermore, a median split analysis (median SSRT: 238 ms) did not reveal differential effects of SR141716A on the estimated SSRT in individuals with relatively “fast” vs “slow” stopping abilities [slow vs fast stopper: F1,14 = 9.54, p = 0.008; dose: F3,42 = 1.04, p = 0.38 and dose × slow vs fast stopper: F3,42 = 2.48, p = 0.074]. In the WIN55,212-2 experiments, at the highest dose, in total, four animals did not start any go trials and were therefore excluded from all stop-signal data analyses. Response inhibition, as displayed in the inhibition function curve in Fig. 5a, was affected by WIN55,212-2 [SSD: F4,44 = 23.51, p < 0.001; dose: F3,33 = 5.12, p = 0.005 and dose × SSD: F12,132 = 1.11, p = 0.36], and further comparisons revealed that 0.3 mg/kg WIN55,212-2 significantly deteriorated the percentage of correct inhibition compared to vehicle. Mean reaction times during go trials were slowed by WIN55,212-2 [Fig. 5b; dose: F3,33 = 15.07, p < 0.001], and further analyses indicated that 1.0 and 3.0 mg/kg WIN55,212-2 significantly slowed mean reaction times compared to vehicle. Nonetheless, the number of omissions during go trials were not significantly affected [mean ± SEM: vehicle = 14.8 ± 1.9; 0.3 mg/kg = 23.2 ± 4.8; 1.0 mg/kg = 26.1 ± 5.4 and 3.0 mg/kg = 34.7 ± 7.0; dose: F3,33 = 3.06, p = 0.073]. Lastly, the average estimated SSRT across three stop-signal delays, putatively reflecting response inhibition, was not significantly changed by WIN55,212-2 at any dose [Fig. 5c; dose: F3,33 = 2.06, p = 0.13]. Furthermore, a median split analysis (median SSRT: 227 ms) did not reveal differential effects of WIN55,212-2 on the estimated SSRT in individuals with relatively “fast” vs “slow” stopping abilities [slow vs fast stopper: F1,10 = 9.57, p = 0.011; dose: F3,30 = 2.12, p = 0.12 and dose × slow vs fast stopper: F3,30 = 1.30, p = 0.29]. Fig. 5Effects of WIN55,212-2 on response inhibition as measured in the stop-signal paradigm. Data are depicted as mean (±SEM) percentage of correctly inhibited stop trials with varying SSDs before the mean go reaction times (a), go RTs (b) and estimated stop signal reaction times from SSDs 200, 100, and 50 ms (c). *p < 0.05 and **p < 0.005 vs vehicle Discussion To our knowledge, this is the first report demonstrating behavioral effects of a CB1 receptor agonist and antagonist on independent measures of impulsivity. Thus far, evidence pointing towards cannabinoid involvement in impulsivity mainly originates from studies in which effects of THC or marijuana were tested in human volunteers (Lane et al. 2005; McDonald et al. 2003). Our data obtained in the 5-CSRTT strongly suggest that inhibitory control is modulated by an endogenous cannabinoid tone, as premature responding was dose dependently decreased by SR141716A. This notion is further supported by the observation that in the presence of WIN55,212-2, the effects of 3.0 mg/kg SR141716A on premature responding were no longer observed. Furthermore, the finding that the CB1 receptor agonist WIN55,212-2, by itself, did not impair inhibitory control by increasing the number of premature responses is in keeping with previous findings (Arguello and Jentsch 2004). It should be noted though, that in the study by Arguello and Jentsch (2004), SR141716A (dose range: 0.1–1.0 mg/kg), by itself, did not change premature responding in a lateralized reaction time task, thereby, contrasting our findings. Nonetheless, differences in baseline performance may have contributed to the discrepancy in findings, as premature responding in well-trained rats in the task by Arguello and Jentsch (2004) was low (approximately 3 per session) compared to premature responding in well-trained rats in the present study (approximately 22 per session). Presumably, a floor effect may have masked the effects of SR141716A on premature responding in the lateralized reaction time task. Together, our data indicate that performance in the 5-CSRTT is associated with profound occupation of CB1 receptors by endogenously released cannabinoids. As might then be expected, the endocannabinoid uptake inhibitor AM404 did not affect inhibitory response control in the 5-CSRTT (unpublished data). In contrast to premature responding, perseverative responding after correct choice, a different measure of inhibitory control that putatively reflects compulsive behavior (Robbins 2002), was neither altered upon activation nor blockade of CB1 receptors. This finding further underscores the dissociation between premature and perseverative responding in the 5-CSRTT that has been shown to have a neuroanatomical basis as well (Chudasama et al. 2003). The endocannabinoid system interacts with many other central neurotransmitters systems including the cholinergic, GABAergic, glutamatergic, and opioid systems (Schlicker and Kathmann 2001; Schoffelmeer et al. 2006). Moreover, cannabimimetics have been shown to indirectly modulate the release of dopamine and glutamate in corticostriatal regions (e.g., Cheer et al. 2004; Szabo et al. 1999; Tanda et al. 1997; Xi et al. 2006), most notably in the nucleus accumbens, and particularly, these effects have been linked to the involvement of the cannabinoid system in addiction (for review, see De Vries and Schoffelmeer 2005). Although it is beyond the scope of the present study and additional experiments are required to substantiate this, one might speculate that the present findings are explained by these modulatory effects of the cannabinoid system on mesolimbic dopamine release, as inhibitory control processes in the 5-CSRTT have been shown to depend upon dopamine receptor activation within the nucleus accumbens (Cole and Robbins 1987; Pattij et al. 2007). On the other hand, explanations for the beneficial effects of SR141716A on inhibitory control may be due to the role of the endocannabinoid system in feeding behavior (for review, see Di Marzo and Matias 2005). Accordingly, it has been demonstrated that SR141716A reduces intake of normal and palatable food (e.g., Arnone et al. 1997; Freedland et al. 2000; Thornton-Jones et al. 2005) and the motivation to obtain food (Solinas and Goldberg 2005). Nonetheless, despite these reported effects of SR141716A on food intake, anorexic effects of this compound seem unlikely to explain the current data, as both primary indices of food-motivated behavior in the 5-CSRTT, namely, errors of omission and feeder response latencies, were not changed. It is interesting to note that we also observed a moderate beneficial effect of SR141716A on visuospatial attention, as it increased the level of accurate choice at 0.3 mg/kg. Likewise, it has been shown that SR141716A improves social recognition and spatial and aversive memory at comparable dose ranges between 0.3 and 3.0 mg/kg (Lichtman 2000; Takahashi et al. 2005; Terranova et al. 1996; Wolff and Leander 2003). Although the mechanisms responsible for the beneficial effects of disrupting endocannabinoid signaling on mnemonic and attentional processes need to be elucidated further, it is interesting to note that SR141716A increases, among others, cholinergic neurotransmission in the medial prefrontal cortex in vivo (Tzavara et al. 2003). This finding may be particularly relevant for attentional function, as performance in the 5-CSRTT has been shown to be accompanied by increments in acetylcholine release in the medial prefrontal cortex (Passetti et al. 2000), whereas decrements in acetylcholine release in this brain region have been shown to correlate with poor visuospatial attention (McGaughy et al. 2002). A different measure of impulsivity we studied was impulsive choice in the delayed reward paradigm. Conceptually, impulsive choice differs from inhibitory control in the 5-CSRTT and rather reflects a cognitive decision-making process, as rats have to weigh the immediate vs delayed outcomes of their behavior. Impulsive choice then is reflected in insensitivity towards the delayed larger reward and a preference for the small immediate reward. In the present experiments, we observed that neither SR141716A nor WIN55,212-2 did have any effects on impulsive choice. In accordance with these findings, it has been shown recently that THC did not change delay and probability discounting in human volunteers (McDonald et al. 2003), and together, these data suggest that the (endo)cannabinoid system is not critically involved in impulsive choice. Our observations do contrast with previous findings indicating that marijuana elevates risk-taking behavior in volunteers (Lane et al. 2005). Risk taking, however, differs from delay and probability aversion in that it is most likely to occur when the behavioral options may result in losses or have aversive consequences (Rachlin et al. 1986). In the delayed reward paradigm and the paradigm used by McDonald et al. (2003), subjects could only win and not lose reward (food or money, respectively), whereas only the magnitude of the reward depended upon choice. It is therefore possible, that these procedural and conceptual differences explain the discrepancy in findings, and furthermore, that delay aversion and risk-taking processes are differentially regulated by the (endo)cannabinoid system. While response inhibition has been shown to be impaired in human volunteers after THC administration (McDonald et al. 2003), neither disruption of endocannabinoid signaling nor administration of a CB1 receptor agonist had clear observable behavioral effects on stop-signal task performance. In agreement with the assumption of a “race model” between “go” and “stop” processes (Logan 1994) and previous stop-signal data in rats (Eagle and Robbins 2003a, b), the probability of successfully inhibiting a response during a stop trial decreased in rats when the onset of the stop signal was delayed in time. Although SR141716A did not shift this inhibition curve as a function of increasing stop-signal delays, the low dose WIN55,212-2 (0.3 mg/kg) significantly deteriorated the ability to inhibit responding with increasing stop-signal delays. However, the primary parameter in this paradigm, the average estimated stop-signal reaction time, or simply put, speed of stopping, was not changed by any dose of SR141716A or WIN55,212-2. In addition, median split analyses revealed no differential drug effects in individuals with “slow” vs “fast” stopping abilities as has been reported previously for the effects of d-amphetamine on stopping speed in both rats and humans (De Wit et al. 2002; Feola et al. 2000). Collectively, our findings suggest that response inhibition processes, as measured in the stop-signal paradigm, are not under control of CB1 receptors. In the present study, the speed of stopping was estimated from separate SSRTs obtained from three stop-signal delays (200, 100, and 50 ms before mean go RT), and consistent with this estimation method, there was some variability in the SSRTs depending on the delay (Band et al. 2003; Logan 1994), with shorter stop-signal delays resulting in SSRTs of approximately 140 ms and longer delays resulting in SSRTs of approximately 300 ms. However, the variability in SSRTs over delays was similar across all doses of both drugs (data not shown), thus, ruling out the possibility that this variability may have masked effects of SR141716A or WIN55,212-2 on response inhibition. In contrast to the measures of response inhibition, reaction times during performance on go trials were slowed by both compounds in line with their effects in the 5-CSRTT. With regard to these effects of SR141716A, in the absence of changes on omission errors in both the 5-CSRTT and the stop signal paradigm, they cannot be solely interpreted in terms of motor effects. Rather, the minor but significant increase in response latencies of approximately 30 ms in both paradigms (Table 1 and Fig. 4b) may indicate changes in information processing speed induced by blockade of endocannabinoid signaling. However, this notion is not confirmed by previous sensorimotor gating data (Mansbach et al. 1996; Martin et al. 2003). In contrast, the effects of 1.0 and 3.0 mg/kg WIN55,212-2 on reaction times in the stop-signal paradigm may in part be secondary to changes in locomotor activity, as these doses also increased the number of omissions in the 5-CSRTT. Remarkably, although both the stop-signal paradigm and 5-CSRTT measure aspects of impulsive action, only inhibitory control in the 5-CSRTT was improved by SR141716A. Impulsive action in the latter paradigm is mainly measured as the inability to inhibit inappropriate (premature) responses, whereas in the stop-signal paradigm impulsive action is reflected in the inability to inhibit ongoing behavior, i.e., the inability to stop a behavioral response that has just been initiated. Our observations support the notion of different and separable forms of inhibitory control, and moreover, suggest a differential role for the endocannabinoid system therein. Likewise, lesion studies have suggested that different brain regions are involved in inhibitory control measured in either the 5-CSRTT or stop-signal paradigm, as for instance, lesions of the nucleus accumbens or subregions of the medial prefrontal cortex have been shown to impair inhibitory control in the 5-CSRTT (Christakou et al. 2004; Chudasama et al. 2003; Muir et al. 1996) and not in the stop-signal paradigm (Eagle and Robbins 2003b). In summary, the present study provides evidence for a differential involvement of the endocannabinoid system in independent measures of impulsivity, as SR141716A primarily affected inhibitory control, and neither impulsive choice nor response inhibition, whereas WIN55,212-2 only slightly affected response inhibition. In this regard, the present data add to existing clinical and preclinical evidence demonstrating that distinct measures of impulsivity can be dissociated at a pharmacological and neuroanatomical level (e.g., Chudasama et al. 2003; De Wit et al. 2002; McDonald et al. 2003; Winstanley et al. 2004). Our findings may be of particular interest with respect to the heterogeneity observed in attention-deficit/hyperactivity disorder (Sonuga-Barke 2002) and suggest that possible novel pharmacotherapies targeted at the cannabinoid system may benefit the subtype resulting from poor inhibitory control, but not the motivational style, or delay aversion, subtype.

Neuroimage-2-1-2278232

Phonological processing in deaf signers and the impact of age of first language acquisition

Just as words can rhyme, the signs of a signed language can share structural properties, such as location. Linguistic description at this level is termed phonology. We report that a left-lateralised fronto-parietal network is engaged during phonological similarity judgements made in both English (rhyme) and British Sign Language (BSL; location). Since these languages operate in different modalities, these data suggest that the neural network supporting phonological processing is, to some extent, supramodal. Activation within this network was however modulated by language (BSL/English), hearing status (deaf/hearing), and age of BSL acquisition (native/non-native). The influence of language and hearing status suggests an important role for the posterior portion of the left inferior frontal gyrus in speech-based phonological processing in deaf people. This, we suggest, is due to increased reliance on the articulatory component of speech when the auditory component is absent. With regard to age of first language acquisition, non-native signers activated the left inferior frontal gyrus more than native signers during the BSL task, and also during the task performed in English, which both groups acquired late. This is the first neuroimaging demonstration that age of first language acquisition has implications not only for the neural systems supporting the first language, but also for networks supporting languages learned subsequently. Introduction Phonology describes the level of analysis at which meaningless, contrastive units of language combine to form meaningful units. In spoken languages these are auditory/articulatory elements. Substitution of a single element creates a new lexical item, e.g., in English /pin/–/bin/. The same level of analysis has been applied to signed languages, where phonology is visual, with handshapes, movements and locations combined to form signs (Stokoe, 1960; Brentari, 1999; Sandler and Lillo-Martin, 2006). As with words, the substitution of just one element can create a new sign. For example, the BSL sign NAME is located at the forehead while AFTERNOON differs only in that it is located at the chin (see Fig. 1). The primary aim of the current study was to examine whether the application of the term ‘phonology’ to signed languages has neurological as well as linguistic and psycholinguistic validity. We address, for the first time, whether similar neural processing is involved in phonological analysis of both signed and spoken languages. We asked participants to judge whether spoken word labels for pictures rhymed or not. Of the phonological parameters of signs, location is one of the primary factors determining whether signs are judged to be similar (Hildebrandt and Corina, 2002). Therefore, we also asked deaf participants to judge if BSL signs for pictured items shared the same location. Studies of written rhyme judgement by hearing people report reliable activation of the posterior portion of the left inferior frontal gyrus (IFG) and the ventral premotor cortex (Broca's area; Sergent et al., 1992; Poldrack et al., 1999; Kareken et al., 2000; Lurito et al., 2000; Xu et al., 2001; Seghier et al., 2004; Burton et al., 2005). Temporary disruption of this region using transcranial magnetic stimulation (TMS) impairs phonological processing (Gough et al., 2005). Given the well-established role of the posterior IFG in speech production (Ojemann and Mateer, 1979), the contribution of the left posterior IFG and ventral premotor cortex to phonological processing is often attributed to articulatory processes or representations (Démonet et al., 1996). In addition to the left IFG, the left parietal lobe is also recruited during rhyme judgement tasks using written stimuli (e.g., Pugh et al., 1996; Lurito et al., 2000; Xu et al., 2001; Seghier et al., 2004). However, the precise role of this region in phonological processing remains unclear (see Eden et al., 2004) and will be addressed further in the Discussion section. If similar processing is required to make phonological similarity judgements about BSL and English, left inferior frontal and left parietal regions should be recruited during both tasks. An important feature of the current study was the use of picture stimuli (see Fig. 2). This enabled the same stimuli and response requirements to be used in both the BSL and English tasks. This is not possible in studies contrasting comprehension (e.g., MacSweeney et al., 2002b) or production (e.g., Braun et al., 2001) of speech and sign. Picture stimuli also allowed us to tap individuals' own phonological representations of words and signs. Previous studies of rhyme judgement with hearing people have used auditory or written stimuli, which directly or indirectly provide the phonology of the item. To determine whether rhyme judgements in response to pictures elicit the pattern of activation reported in studies using written words, hearing participants were also tested. Including this group also allowed us to directly contrast activation patterns observed in deaf and hearing participants to determine the effect of hearing status on the neural systems supporting phonological processing. A second aim of this study was to determine if the neural systems supporting language are influenced by the age of first language acquisition. To address this, deaf native and non-native signers were contrasted. Approximately 5% of deaf people are born to deaf, signing parents (Mitchell and Karchmer, 2004). Typically, these children learn a signed language as their native language, reaching acquisition milestones along the same timescale as hearing children acquiring speech (Morgan and Woll, 2002). However, for nearly all of the 95% of deaf children who are born to hearing parents (non-native signers), exposure to a signed language is delayed. Deaf native and non-native signers should differ on sign-related tasks since this is the language acquired early or late. In addition, Mayberry et al. have shown that deaf native signers perform better than deaf non-native signers on grammaticality judgements of written English (Mayberry et al., 2002; Mayberry and Lock, 2003). In these studies, both groups encountered written English at the same age. However, what did differ between these groups was their early language experience. Native signers have a well-established first language which can facilitate the acquisition of a later learned language; non-native signers do not. For all deaf people, both native and non-native signers, exposure to spoken language is late and incomplete. This is because speechreading cannot provide full access to speech since many of the articulators of speech are invisible. From this perspective, Mayberry and Lock (2003) have argued that deaf non-native signers can be considered to have ‘no early language’. This is a very different situation to that of hearing people learning a signed language late since they already have a native spoken language. Studies contrasting early and late acquisition of signed language in hearing participants (cf., Newman et al., 2002) cannot therefore be generalised to the deaf population. We examined how the incomplete acquisition of a language early in life is reflected in the brain. Consistent with the behavioural findings of Mayberry et al., we predicted differences between deaf native and non-native signers, on both the BSL and English tasks, despite these groups' similar experience of English. In summary, in the current study profoundly deaf and hearing adults made phonological similarity judgements in response to picture pairs (see Fig. 2). Participants judged if the spoken English labels rhymed or whether the BSL labels shared the same location (deaf group only). Activation during these experimental conditions was contrasted with a ‘same picture?’ control task. The following research questions were addressed: are the same neural networks recruited during phonological decisions based on signed and spoken language? What is the impact of age of signed language acquisition and hearing status on this network? Materials and methods Participants Twenty-three deaf adults and 24 hearing adults were scanned. All were right-handed and had normal or corrected to normal vision. Of the deaf participants, one was excluded because of excessive movement in the scanner and another two were excluded because they did not complete both the sign and speech tasks. Therefore, 20 deaf and 24 hearing participants, matched on age and non-verbal IQ (Block Design, WAIS-R: p > 0.1), were included in the analyses (see Table 1 for participant characteristics). The deaf participants included in the fMRI study were selected from a larger sample of volunteers. To enhance the likelihood that they would perform the phonological judgement tasks well, those selected were good readers and had performed well on a test of rhyme ability in a previous test session outside the scanner. Therefore, the deaf participants included in the fMRI study were good readers (mean reading age = 15years, 6months) in comparison to the population mean for deaf people, generally considered to be approximately 9 or 10years (see Conrad, 1979; Allen, 1986; Holt, 1993). The deaf participants were also skilled speechreaders, outperforming the hearing participants on the Test of Adult Speechreading (Mohammed et al., 2003; t = 5.1, (34), p < 0.0005). Nevertheless, the hearing group were significantly better readers (Vernon-Warden, 1996: t = − 3.9, (42), p < 0.0005) and had a higher English vocabulary score (shortened version of the Boston Naming Test, Kaplan et al., 1983: t = − 4.1, (42), p < 0.0005) than deaf participants. These differences are accounted for in the fMRI data analyses. All deaf participants reported being born profoundly deaf and audiograms obtained at the time of testing confirmed that all had a mean hearing loss greater than 92dB in the better ear over four octaves, spanning 500–4000Hz. All deaf participants encountered written English upon entering primary school, aged 4/5. Twelve of the deaf participants were native signers, having acquired BSL from their deaf, signing parents. The remaining eight deaf participants (non-native signers) had hearing parents. One native signer and one non-native signer reported attending schools which used a total communication approach, in which signs are used to support spoken English. The remaining 18 of the 20 deaf participants had attended ‘oral’ schools in which spoken English was the main form of communication. This educational approach was the norm for this generation of deaf adults in the UK, even for those who used BSL as their native language. Of the eight non-native signers, five learned BSL after leaving secondary school, aged 17 to 21. One participant learned BSL at their total communication primary school aged 4/5. Two other participants who attended oral schools reported learning BSL at school; one aged 4/5, the other aged 11. These participants will have been exposed to BSL by their deaf native signing classmates. The deaf subgroups were well matched. There were no significant differences between deaf native and deaf non-native signers in age (p = 0.06), non-verbal IQ (p > 0.1), reading age (p > 0.1), English vocabulary (p > 0.1) or speechreading skill (p > 0.1). All participants gave informed, written consent to participate in the study, which was approved by the Institute of Psychiatry/South London and Maudsley NHS Trust Research Ethics Committee. Stimuli The same pictures were presented in both the rhyme and location judgement tasks (n = 60). All pictures represented highly familiar/frequent, monosyllabic English words. Fifty-eight of the pictures were black and white line drawings, taken predominantly from the Snodgrass and Vanderwart (1980) standardised picture set and other language assessments. Two colour pictures were also included, to represent ‘red’ and ‘blue’. Rhyme task (shared English phonology pairs) Thirty pictures were combined as 15 rhyming pairs. Orthography was inconsistent so that rhyme decisions could not be based on spelling, (e.g., chair–bear, tail–whale; see Fig. 2A). Location task (shared BSL phonology pairs) The remaining thirty pictures were combined as 15 pairs sharing the same location when signed in BSL, but differing in handshape and movement (e.g., pig–witch; hat–cow, see Fig. 2B). Only signs touching the body or occurring in close proximity to it were considered to have a defined location. Participants were told that any pairs articulated in neutral space in front of the signer's body (see Stokoe, 1960) should receive a ‘no’ response. The two sets of experimental pictures (rhyme and location pairs) were matched on familiarity (Coltheart, 1981) (t = 0.64, 53, p > 0.1) and concreteness (Coltheart, 1981) (t = − 1.1, 53, p > 0.1) and the English labels were matched on frequency (Kucera and Francis, 1967) (t = 0.92, 56, p > 0.1) and length (t = 1.5, 58, p > 0.1). The ‘no’ (non-shared phonology) trials were established by re-pairing the pictures from the complementary task. Rhyming pictures were re-paired such that there was no overlap in signed or spoken phonology to form the ‘no’ trials in the location task (e.g., chair–whale). Likewise, location pairs were re-paired to form the ‘no’ trials in the rhyme task (e.g., hat–pig). Thus, the same stimuli were presented in both tasks. The stimuli used in the experimental conditions doubled as their own controls in the ‘same picture?’ control task. Fifteen of the pictures were presented as identical pairs (e.g., chair–chair, see Fig. 2C). Another 30 pictures were re-paired to form different picture trials. The labels for these items did not share any phonological features in either English or BSL. Thus, of the 60 pictures seen in both experimental conditions, 45 were also presented in the ‘same picture?’ control condition. Whether an item was first seen in the experimental or control condition was counterbalanced such that any repetition effects were balanced across conditions. All participants performed a picture naming pre-test before the scan session. If an unexpected label was generated, the desired English word or BSL sign (deaf participants only) was supplied. Correct naming of these items was checked again at the end of the pre-test session. Design Deaf participants performed the rhyme and location similarity judgement tasks in separate, counterbalanced runs. Hearing participants performed only the rhyme task. Each run consisted of six 30-s blocks of the experimental task (rhyme or location), alternating with six 30-s blocks of the ‘same picture?’ control task. Each run lasted 6 min. In the English phonology task, participants were required to decide whether the English labels for two pictures rhymed. Deaf participants had already been involved in a behavioural study of rhyme awareness as part of a wider project. They were reminded of the concept of rhyme and were given examples and practice trials prior to the start of the experiment in the scanner. In the sign phonology task, signing participants were required to decide if the BSL labels for the two pictures shared the same location. The control condition was interleaved between the same phonology? task blocks. This consisted of deciding if two pictures were the same. The trials in each condition were half ‘yes’ trials and half ‘no’ trials. Subjects indicated their response using a two-choice button box. A one-syllable task prompt appeared at the bottom of the screen, without a pair of pictures, for 2000ms at the beginning of each block (‘Rhyme?’—rhyme task; ‘Place?’—location task; ‘Same?’—picture matching task). The prompt remained on the screen throughout the block. Each pair of pictures was presented for 5s. This relatively long presentation duration was selected on the basis of pilot studies in which deaf people made self-paced rhyme decisions in response to pictures. The inter-stimulus interval was 500ms. Each 30-s block was a mixture of five ‘yes’ and ‘no’ trials (see Fig. 2). fMRI parameters Gradient echo echoplanar MRI data were acquired using a 1.5-T GE NVi MR system (General Electric, Milwaukee, WI, USA) using a standard quadrature head coil. Head movement was minimised by positioning the participant's head between cushioned supports. One hundred and twenty T2⁎-weighted images depicting BOLD contrast were acquired during one experimental session at each of 38 near-axial 3mm thick planes parallel to the intercommissural (AC–PC) line: 0.3mm interslice gap; TR = 3s, TE = 40ms; flip angle = 90°). The field of view for the fMRI runs was 240mm, and the matrix size was 64 × 64, with a resultant in-plane voxel size of 3.75mm. An inversion recovery EPI dataset was also acquired to facilitate registration of individual fMRI datasets to Talairach space (Talairach and Tournoux, 1988). This comprised 43 near-axial 3mm slices (0.3mm interslice gap), which were acquired parallel to the AC–PC line (TR = 16s; TE = 80ms; TI = 180ms; flip angle = 90°). The field of view for the EPI dataset was 240mm, and the matrix size was 128 × 128, with a resultant in-plane voxel size of 1.875mm. fMRI data analysis The fMRI data were analysed using an in-house non-parametric software package (XBAM_v3.2) which uses standard preprocessing steps: realignment, normalisation, baseline correction, spatial smoothing, and GLM parameter estimation using a combination of gamma variate basis functions (for details see Brammer et al., 1997; Bullmore et al., 1999, 2001; Suckling and Bullmore, 2004). The data were realigned to minimise artefacts due to subject motion. First, a template was computed by averaging the image intensity over all time points at each voxel. The 3D volumes at each time point for each participant were then realigned to the template by computing the rigid body motion parameters (3 rotations, 3 translations) that maximised the correlation between each volume and the template. Normalisation was conducted using an affine transform and by computing the parameter set that maximised the correlation between the template image (in standard space—Talairach and Tournoux) and the image to be normalised. The data were then smoothed using a Gaussian filter (FWHM 7.2mm). Experimental responses were then analysed by convolving the experimental design with two gamma variate functions (peak responses four and eight seconds) with delays chosen to span the likely range of BOLD delays and computing the least squares fit of the resulting convolution to the time series at each voxel. A goodness of fit statistic was derived by calculating the ratio between the sum of squares due to the model fit and the residual sum of squares (SSQ ratio). The value of this statistic was then tested for significance using the wavelet-based time series permutation method (Bullmore et al., 2001; Suckling and Bullmore, 2004). Group analysis Data were transformed into standard space (Talairach and Tournoux, 1988). Voxel size in standard space was 3.3 × 3.3 × 3.3mm. Significant activations were identified using data-driven significance testing of the median activations at each voxel over all members of the group (Brammer et al., 1997). Median statistics were used to minimise outlier effects in the group sizes normally used in fMRI studies. Analysis was extended to the cluster level with the clusterwise false positive threshold set to less than one across the whole brain (Bullmore et al., 1999). Since the XBAM analysis method takes into account first level as well as second level variance, it resembles what Thirion et al. (2007) have called a “pseudo mixed effects analysis”. Group differences Differences in activation between groups and conditions were assessed by fitting the following linear model to the data at each voxel, Y = a + bX + e, where Y is the vector of BOLD effect sizes for each individual, X is the contrast matrix for the particular inter-condition/group contrasts required, a is the mean effect across all individuals in the various conditions/groups, b is the computed group/condition difference, and e is a vector of residual errors. The model was fitted by minimising the sum of absolute deviations to reduce outlier effects. The null distribution of b was computed by permuting data between conditions (assuming the null hypothesis of no effect of experimental condition) and refitting the above model. Group difference maps were computed as described above at voxel or cluster level by appropriate thresholding of the null distribution of b. Conjunction analysis Conjunction analyses were carried out to identify brain regions in which there was consistent activation across tasks. First, the minimum SSQ ratio (effect/error) at each voxel across conditions was determined. This measure was then tested (at appropriate voxelwise and clusterwise p-values), as described above under Group analysis, to determine whether it was significantly different from zero. Brain areas showing significant levels of activation were considered to show significant conjunctions of brain activation. Results Behavioural data See Table 2 for accuracy and reaction time data. Deaf participants only A mixed-model ANOVA was conducted on the accuracy data (Task (rhyme/location/control) × Group (native/non-native signers)). A main effect of Task indicated that the control task was performed better than both experimental tasks (F(2,36) = 25.5, p < 0.0005). There was no significant effect of Group and no interaction. Excluding the control task from the ANOVA yielded no significant main effects and no interaction. Thus, deaf native and non-native signers were equally accurate on both the rhyme and location tasks. The same mixed-model ANOVAs were applied to the reaction time data. Deaf participants were faster on the control than experimental tasks (F(2,36) = 211.7, p < 0.0005). There were no further significant effects. When the control task was omitted from the model, a main effect of Task (F(1,18) = 9.7, p < 0.01) indicated faster reaction times to the rhyme than location task. All deaf versus all hearing participants performing the rhyme task A mixed-model ANOVA was conducted on the accuracy data (Task (rhyme/control) × Group (deaf/hearing)). A main effect of Task indicated better performance in the control than rhyme task (F(1,42) = 52.5, p < 0.0005). A main effect of Group indicated better performance by hearing than deaf participants (F(1,42) = 13.4, p < 0.002). This was qualified by a significant interaction (F(1,42) = 11.4, p < 0.005) indicating that the hearing group performed better than the deaf group on the rhyme task, with no difference on the control task. With regard to the reaction time data, a main effect of Task (F(1,42) = 744.7, p < 0.0005) indicated faster responses on the control than rhyme task. A significant interaction (F(1,42) = 11.5, p < 0.005) indicated slower performance by deaf than hearing participants to the rhyme task, but no group difference on the control task. fMRI data Rhyme and location similarity judgements in deaf participants only To identify neural systems involved in phonological processing of sign and speech in the deaf group, data from all deaf participants were combined. Analysis of the rhyme (English) and location (BSL) tasks separately, relative to the control task (voxelwise p = 0.025; clusterwise p = 0.01), resulted in remarkably similar patterns of activation (see Figs. 3A and B/Table 3). A conjunction analysis was performed to clarify the overlap in activation between the two phonological tasks (English rhyme and BSL location) in deaf participants (voxelwise p = 0.05; clusterwise p = 0.025). As in the individual task analyses, a network consisting of three regions was identified. The most extensive activation was in the left frontal cortex (19.41cm3 volume; X = − 40, Y = 30, Z = 17; these Talairach and Tournoux coordinates, and those reported in the text to follow, represent local maxima). This extended from the insula, through the inferior and middle frontal gyri into the ventral precentral gyrus. The second significant activation extended from the superior portion of the supramarginal gyrus (SMG) into the superior parietal lobule (SPL) and the precuneus (11.28cm3 volume; X = −29, Y = −67, Z = 40). Finally, significant activation was identified in the medial portion of the superior frontal gyrus (SFG), incorporating the anterior cingulate (7.37cm3 volume; X = 0, Y = 1, Z = 50). These data suggest that a left-lateralised network of three regions is engaged by deaf participants performing a phonological similarity judgement task, regardless of whether the task is performed in English or BSL. Differences between the rhyme and location tasks and the effect of age of BSL acquisition in deaf participants To determine the differences between the networks recruited during the rhyme and location judgement tasks and to examine the effect of age of signed language acquisition, a mixed-model ANOVA was conducted. This included Task (rhyme/location) as a within subjects factor and Age of BSL Acquisition (native/non-native) as a between subjects factor (voxelwise p = 0.025; clusterwise p = 0.005). The main effect of Task showed that two regions were recruited to a greater extent for the rhyme than location task. These were the left dorsal IFG, extending into the precentral gyrus (4.13cm3 volume; X = − 51, Y = − 4, Z = 40; BA 6), and the medial portion of the SFG, at the junction with the anterior cingulate (2.30cm3 volume; X = 0, Y = − 11, Z = 53; BA 6). In contrast, a region in the left parietal lobe was recruited to a greater extent for the location than rhyme task (see Fig. 4). This extended from the superior portion of the SMG, into the SPL and medially to include the precuneus (5.10cm3 volume; X = − 4, Y = − 70, Z = 43; BA 7). No regions were recruited to a greater extent by native than non-native signers. In contrast, non-native signers recruited the left inferior frontal cortex to a greater extent than native signers (see Fig. 5A; 5.32cm3 volume; X = − 40, Y = 19, Z = 30). This activation extended from the IFG (BA 44/45), into the middle frontal gyrus and precentral gyrus. Follow-up analyses were conducted comparing native and non-native signers on the rhyme and location tasks separately (voxelwise p = 0.025; clusterwise p = 0.005). These analyses confirmed that non-native signers engaged the posterior IFG more than native signers, during both the location task (3.45cm3 volume; X = − 40, Y = 15, Z = 30) and the rhyme task (3.74cm3 volume; X = − 40, Y = 7, Z = 23). A significant Group × Task interaction was also identified, the focus of which was at the junction of the left IFG (BA 44), the precentral gyrus (BA 6) and the middle frontal gyrus (BA 9; see Fig. 5B; 2.44cm3 volume; X = − 43, Y = 19, Z = 30). Follow-up analyses (voxelwise p = 0.025; clusterwise p = 0.005) demonstrated that there was no significant difference in the extent to which non-native signers recruited this region during the rhyme and location tasks. In contrast, native signers engaged this region more during the rhyme task, performed in English which they learned late, than the location task, performed in their native language (2.87cm3 volume; X = −47, Y = 0, Z = 36, precentral gyrus (BA 6)). The medial portion of the SFG, at the junction with the anterior cingulate, also demonstrated the same effect (2.16cm3 volume; X = 0, Y = 11, Z = 53). Rhyme similarity judgements in hearing participants (voxelwise p = 0.025; clusterwise p = 0.01) Hearing participants performing the rhyme task engaged four regions (see Table 3): [1] the left prefrontal cortex extending from the insula, through inferior and middle frontal gyri and into the precentral gyrus, [2] the superior portion of the SMG, extending into the SPL and medially into the precuneus, [3] the anterior medial portion of the SFG and superior portions of the anterior cingulate, and [4] the right inferior occipital gyrus extending into the fusiform gyrus and incorporating superior parts of the cerebellum. The pattern observed in hearing participants performing the rhyme task was similar to that observed in deaf people performing the rhyme and location tasks. To clarify the overlap in regions recruited during phonological similarity judgements a conjunction analysis was conducted on the data from deaf and hearing participants performing the rhyme task and deaf participants performing the location task (voxelwise p = 0.05; clusterwise p = 0.01). Not surprisingly, given the individual group patterns, this analysis identified three regions as being significantly activated across all tasks/groups: the dorsal portion of the left IFG, extending into the middle frontal gyrus (14.81cm3 volume; X = −40, Y = 7, Z = 33 BA 44/9); the left SPL (5.82cm3 volume; X = −29, Y = −63, Z = 46 BA 7) and the medial portion of the SFG (5.61cm3 volume; X = 0, Y = 11, Z = 50 BA 6). These regions appear to make up a core network involved in phonological processing of both signed and spoken language, recruited by both deaf and hearing participants. Deaf versus hearing participants performing rhyme task, matched for performance To determine the effect of hearing status on the neural system supporting phonological processing, we contrasted activation patterns in deaf and hearing participants performing the rhyme task. Since group performance on the rhyme task was poorer in deaf than hearing participants, subsets of 12 participants were selected from each group who were matched on accuracy and reaction time on the rhyme task performed in the scanner. To further control for differences between deaf and hearing participants the subgroups were also matched for age, non-verbal IQ, reading age, and accuracy and reaction time on a more extensive test of rhyme awareness, run prior to the scan session. There were no significant differences between the two groups on any of these variables (all p-values > 0.1; see Table 4). A between subjects ANOVA (voxelwise p = 0.05; clusterwise p = 0.005) indicated no regions in which hearing participants showed greater activation than deaf participants. The deaf subgroup showed greater activation than the hearing subgroup in the left IFG, extending into the middle frontal and the precentral gyri (7.01cm3 volume; X = −40, Y = 0, Z = 33; BA 44/6) and in a small portion of the SFG, at the junction with the anterior cingulate (1.29cm3 volume; X = − 4, Y = 4, Z = 50; BA 6/32). Further analyses confirmed that the same pattern was observed when deaf native signers and deaf non-native signers were compared separately to matched hearing participants. This suggests that combining native and non-native signers, in order to carefully match subgroups of deaf and hearing participants, did not influence the outcome of this analysis. Discussion Our results demonstrate that a very similar neural network supports phonological similarity judgements made in both English and British Sign Language (BSL). Given that these languages operate in such different modalities, these data suggest that this phonological processing network is multimodal or possibly to some extent supramodal: that is, involving representations that in some way ‘transcend’ the sensory modalities (see Fowler, 2004 for discussion). This network, which was also recruited by hearing people making rhyme judgements, consists of the medial portion of the superior frontal gyrus (SFG), the left superior parietal lobule (SPL) incorporating the superior portion of the supramarginal gyrus (SMG), and, most extensively, the left posterior inferior frontal gyrus (IFG) extending into the ventral precentral gyrus. We do not argue that these regions are dedicated to phonological processing. Rather we argue that they act together as a network to support phonological similarity judgements and other linguistic and, it is likely, non-linguistic processes (see Corina and Knapp, 2006). Nevertheless, our data are consistent with prior demonstrations, concerning semantic and syntactic processing, that modality has relatively little influence on the neural systems that support language (Neville et al., 1998; Petitto et al., 2000; Braun et al., 2001; Emmorey et al., 2002; MacSweeney et al., 2002b; Corina et al., 2003; MacSweeney et al., 2006). Demonstrating this in the context of phonological processing is even more striking since awareness of phonology is more directly linked to sensory input (which differs for sign and speech) than either semantic or syntactic processing. Although the observed network is recruited by both signed and spoken language, we demonstrate that it does not perform identically across languages or groups. Recruitment of different parts of this network is modulated by age of first language acquisition, language modality and hearing status. The impact of age of first language acquisition was explored by comparing deaf native and non-native signers. Non-native signers engaged the left posterior inferior frontal cortex to a greater extent than native signers. This was the case not only during the task performed in BSL, of which both groups had different language experience, but also during the task performed in English, of which both groups had similar experience. The differential recruitment of the left posterior IFG is even more striking given that native and non-native signers were matched on non-verbal IQ, English vocabulary score and reading age and that there were no significant group differences in accuracy or reaction time on the two phonological tasks. These are the first neuroimaging data to demonstrate the impact of age of acquisition of a first language in the brain. Lack of exposure to a fully accessible language early in life has implications for the neural systems supporting not only that language, but also for languages learned subsequently, whether signed or spoken. In conjunction with the behavioural data of Mayberry and colleagues (Mayberry et al., 2002; Mayberry and Lock, 2003) these data highlight the importance of early exposure to an accessible language for those born profoundly deaf. Even when signed, early language leads to the normal establishment of language systems that may then be used to facilitate a later learned language. Enhanced recruitment of the left posterior IFG has previously been reported during grammaticality judgements performed by late in contrast to early learners of German (Wartenburger et al., 2003) and during semantic judgement tasks in low- in contrast to high-proficiency late language learners (Chee et al., 2001; Wartenburger et al., 2003). Regions within the left IFG are differentially modulated not only by age of language acquisition and proficiency level, but also by extent of language use (Perani et al., 2003), age at time of testing, and task demands (Tatsuno and Sakai, 2005). Thompson-Schill et al. (2005) argue that the left IFG is increasingly engaged as selection demands increase. In particular, it is argued that this region is involved in regulating the cognitive control necessary to resolve competition between incompatible responses (Snyder et al., 2007). It is possible that selection demands increase for bilinguals because responses from both first and second languages are available. This situation applies to native signers (first language: BSL; second language: English), but may apply to a greater extent to non-native signers. Deaf non-native signers have knowledge of both BSL and English; however, both languages are acquired late. Despite equivalent behavioural proficiency on our tasks, both languages are likely to be poorly established in non-native signers, leading to greater conflict between potential responses, possibly resulting in enhanced recruitment of the left IFG. An alternative, phonology-specific argument can also be made for the role of the posterior IFG. It has been argued that different parts of the left IFG may show preferential engagement in different aspects of language processing: phonological (the dorsal region: BA 44/6), syntactic (the more anterior region: BA 45), and semantic (the ventral portion: BA 47) (Fiez, 1997; Price et al., 1997, Poldrack et al., 1999; Bookheimer, 2002; Devlin et al., 2003). While the baseline task used in the current study did not require picture name retrieval, given the relatively long presentation time (5s) it is likely that participants did name these stimuli (cf. Meyer and Damian, 2007). Furthermore, the network we identify in the current study has been reported in numerous previous studies of phonological processing involving written words (e.g., Lurito et al., 2000; Xu et al., 2001; Seghier et al., 2004). The most parsimonious interpretation of the current data is thus in terms of phonological processing. With regard to the left frontal cortex, as in previous studies, it was specifically the posterior portion of the IFG, extending into the ventral premotor cortex within precentral gyrus, that was the focus of activation involved in the phonological similarity matching tasks reported here. Moreover, this region was engaged to a greater extent by deaf participants during the rhyme than location task and more by deaf than hearing participants performing the rhyme task (see also Aparicio et al., 2007), even when behavioural performance was taken into account. To account for these findings, we suggest that when the auditory component of speech is absent, the articulatory/motoric component makes a greater contribution to speech-based phonological processes. A similar explanation may account for the observed increased involvement of the left IFG during reading in children with developmental dyslexia following phonological remediation (e.g., Temple et al., 2003). Further studies with deaf participants are underway to test this hypothesis. We also found that the posterior portion of the left IFG was engaged more by non-native than native signers during tasks performed in both languages. Broca's area, in left posterior IFG, is engaged in sign production, just as it is in speech production (Braun et al., 2001; Corina et al., 2003; Emmorey et al., 2007). One possibility that may account for the effect of age of first language acquisition in this region is that the articulatory component of both speech and sign is less established in deaf non-native than native signers, leading to enhanced recruitment of this region during both tasks. From our data, it is not possible to distinguish between phonology-specific and cognitive control/selection demands accounts of the differential engagement of the left IFG by native and non-native signers. Indeed both accounts may apply since the area showing differential activation in all of the contrasts reported here involved both the posterior portion of the left IFG and the ventral premotor cortex, in the precentral gyrus. Snyder et al. (2007) propose that the left IFG is involved in cognitive control, while the ventral premotor cortex demonstrates phonology specific properties. The relative role of these regions in language processing, and phonological processing in particular, will be greatly informed by future studies examining different domains of language (phonology, syntax, semantics) within the same group of deaf late language learners, while manipulating age of acquisition and proficiency. The left parietal lobe was also recruited during both the location task (deaf participants) and the rhyme task (deaf and hearing participants). In all groups and tasks this activation included the superior portion of the supramarginal gyrus (SMG), extending into the superior parietal lobule (SPL) and medially into the precuneus. Previous studies of rhyme judgement of written words by hearing adults also report activation of this area (Lurito et al., 2000; Xu et al., 2001; Seghier et al., 2004; Snyder et al., 2007). Nevertheless, the exact role of this region in phonological processing remains unclear. It has been proposed that this multimodal integration region may be recruited during mapping between orthographic and phonemic representations (Booth et al., 2002; Eden et al., 2004). The fact that the stimuli in the current study were pictures, not written words, does not necessarily pose a problem for this account (but see Katzir et al., 2005). Hearing adults have been shown to engage this region more than children during auditory rhyme decisions (Booth et al., 2004). In addition, deaf people are more accurate and faster to make rhyme judgements when the labels for picture stimuli share orthography, e.g., cat–mat, than when they do not, e.g., chair–bear (Sterne and Goswami, 2000). Both of these lines of evidence suggest that orthographic representations may be activated in both deaf and hearing participants when making spoken language phonological decisions in response to pictures. Further studies are needed to examine this hypothesis and the role of the left parietal lobe in phonological processing. Whatever the functional role of the left parietal lobe in spoken language phonological processing, a growing number of studies suggest that this area may play a particularly important role in signed language processing (see Corina and Knapp, 2006). Perception of BSL sentences that involve spatial description engage the left inferior parietal lobule (IPL) and SPL to a greater extent than ‘non-spatial’ sentences in deaf native signers (MacSweeney et al., 2002a). Direct stimulation of the IPL induces phonological errors during sign production (Corina et al., 1999). Emmorey et al. (2007) have reported that sign production engages the left IPL (X = −60, Y = −25, Z = 27) and the left SPL (X = −26, Y = −51, Z = 54) more than speech production. Emmorey et al. (2007) propose that the left IPL may be involved in phonological processing while the left SPL may be involved in proprioceptive monitoring of motor output. In the current study, the observed greater activation in deaf signers during the location task than the rhyme task was located between the two foci reported by Emmorey et al. (2007) and incorporated the superior portion of the SMG in the IPL, extending into the SPL (X = −4, Y = −70, Z = 43). Given the nature of the current task, a phonological account of this activation seems more likely than an account based on proprioceptive monitoring. Corina and Knapp (2006) have argued that the left parietal lobe plays a greater role in signed than spoken language processing because signed language can build on the “...prior existence of a general human system for manual action observation and production” (p. 537). Portions of the parietal lobe are particularly attuned to the location of the hands/body in space (Gerardin et al., 2000; Hermsdorfer et al., 2001). Accordingly, this region may be particularly engaged in tasks that focus on these spatial relationships, including those involving sign phonology. Whether this activation is specifically related to linguistic processing or to more general processing of body-related information requires further investigation, using linguistic tasks exploring both comprehension and production, and non-linguistic tasks. Examining other parameters of sign phonology, e.g., movement and handshape, is also required. It is not possible to claim a straightforward parallel between rhyme in spoken language and any of the parameters of sign phonology. It has been suggested that when any sign parameter is shared, this is more analogous to alliteration than to rhyme (Sutton-Spence, 2004). Location was chosen in the current study because it has been demonstrated to be important in judging sign similarity (Hildebrandt and Corina, 2002) and because, among the phonological parameters of signed languages, it appears to have the smallest inventory, as do vowels, which are the nucleus of the syllable and crucial for rhyme in spoken languages. However, sign phonologists may argue that movement is more analogous to vowels with respect to syllable structure because a sign is ill-formed without movement, just as a syllable is ill-formed without a vowel (Brentari, 1999; Sandler and Lillo-Martin, 2006). Whether different patterns of activation are observed when different sign parameters are examined or whether any form of sublexical analysis of signs elicits activation in the network reported here remains to be seen. In summary, these data suggest that phonological processing, at least in the context of a phonological similarity judgement task, is to some degree supramodal. We show that a similar neural network supports phonological processing of both signed and spoken language in signed language users born profoundly deaf. Furthermore, this network was also engaged by hearing non-signers performing the similarity judgement task in English. However, different parts of this network were differentially weighted depending upon language modality, hearing status and, most importantly, age of first language acquisition. These data highlight the importance of learning a language, whether signed or spoken, early in life. Early acquisition of a first language is critical not only for processing that language, but also appears to form a base on which subsequently learned languages can successfully build.

Int_J_Parasitol-1-5-1906861

Altered phenotype and gene transcription in endothelial cells, induced by Plasmodium falciparum-infected red blood cells: Pathogenic or protective?

Severe malaria is associated with sequestration of Plasmodium falciparum-infected red blood cells (PRBC) in the microvasculature and elevation of intercellular adhesion molecule-1 (ICAM-1) and TNF. In vitro co-culture of human umbilical vein endothelial cells (HUVEC), with either PRBC or uninfected RBC, required the presence of low level TNF (5 pg/ml) for significant up-regulation of ICAM-1, which may contribute to increased cytoadhesion in vivo. These effects were independent of P. falciparum erythrocyte membrane protein-1 (PfEMP-1)-mediated adhesion but critically dependent on cell–cell contact. Further changes included increases in IL8 release and soluble TNF receptor shedding. Microarray analysis of HUVEC transcriptome following co-culture, using a human Affymetrix microarray chip, showed significant differential regulation of genes which defined gene ontologies such as cell communication, cell adhesion, signal transduction and immune response. Our data demonstrate that endothelial cells have the ability to mobilise immune and pro-adhesive responses when exposed to both PRBC and TNF. In addition, there is also a previously un-described positive regulation by RBC and TNF and a concurrent negative regulation of a range of genes involved in inflammation and cell-death, by PRBC and TNF. We propose that the balance between positive and negative regulation demonstrated in our study will determine endothelial pathology during a malaria infection. 1 Introduction Pathogenesis of severe malaria has stirred considerable interest over the years, however there are many questions at the mechanistic level that remain unanswered. Severe malaria is characterised, in part, by sequestration of Plasmodium falciparum-infected red blood cells (PRBC) at microvascular sites in the brain tissue, leading ultimately to coma and subsequent death. There is evidence that the sequestration of PRBC on the endothelial surface can result in loss of integrity, thus compromising blood–brain barrier function either directly (Brown et al., 1999a,b, 2001a), by indirect mechanisms including binding via platelets (Wassmer et al., 2004; Combes et al., 2006) or by the production of microparticles from endothelial cells (EC) activated by adhering PRBC (Combes et al., 2005, 2006), and also cytokine-driven modulation of endothelial cell metabolism, but that this is usually a small, transient effect in vivo. One question is the mechanism underpinning the accumulation of PRBC leading to micro-vascular occlusion as seen in post mortem cerebral malaria (CM) brain tissue, which is believed to be a progressive phenomenon of PRBC sequestration. We suggest that the initial sequestration of PRBC, if maintained for a prolonged period of time, has the ability to activate the endothelium to promote sequestration, leading to deleterious effects on the host. Our studies investigate the direct effects on the endothelium of PRBC retention for prolonged periods of time. First, sustained exposure of the endothelium to PRBC can result in modulation of the endothelium, thus increasing its responsiveness to low levels of TNF (TNFlow). This may contribute to promoting further cytoadhesion in microvasculature containing sequestered PRBC. This hypothesis is based on previous evidence of endothelial cell activation by abnormal red blood cells in various conditions, including sickle cell disease, diabetes and also malaria (Shiu and McIntire, 2003). Sickle cell RBC (sRBC), diabetic RBC (dRBC) and also PRBCs can all induce expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) and E-selectin (Brown et al., 2001b; Shiu and McIntire, 2003; Viebig et al., 2005; Tripathi et al., 2006). In addition, recent co-culture studies have demonstrated PRBC-induced increases in the activity of pro-apoptotic genes involved in the caspase pathway (Pino et al., 2003). Other changes induced on EC by sRBCs include induction of endothelin and prostacyclin production (Shiu et al., 2002) and by PRBC include increased expression of pro-inflammatory genes (Pino et al., 2003) and release of IL6 (Viebig et al., 2005). Serum levels of IL8 were elevated in severe malaria compared with healthy volunteers (Lyke et al., 2004), correlated with parasite count and severity of disease at the time of admission (Burgmann et al., 1995) and there were no apparent differences between febrile and a-febrile volunteers (Hermsen et al., 2003). IL8 is an endothelial-derived acute response activation marker. Prestored IL8 is exocytosed at a basal level from Wiebel palade bodies in ECs and is rapidly released in response to various stimuli (Wolff et al., 1998; Utgaard et al., 1998; Oynebraten et al., 2004). Exposure of ECs to sRBCs in vitro resulted in increased sensitivity to the pro-inflammatory cytokine IL1β (Shiu et al., 2000). We propose that local increases in TNF levels occur in the microvasculature in early stages of infection, during shizogony (Bisser et al., 2006). In a similar fashion to that observed with sRBCs, contact between PRBCs and the endothelium could potentially sensitise the ECs to TNF. There is evidence for an important role for TNF receptors (TNFR) in CM pathology, however, most of this comes from studies on animal models. Lucas et al. (1997a) showed a close correlation between absence of TNFR II expression and protection from CM-associated brain damage in TNFR II knock-out mice, while TNFR II was up-regulated on brain endothelium in CM-susceptible mice (Lucas et al., 1997b). Up-regulation of the soluble TNFR I, which is the predominant type in human serum, was reported in the serum of patients with acute P. falciparum infection (Wenisch et al., 1994). Interestingly, EC from CM-susceptible mice had a greater sensitivity to TNF than that of CM-resistant mice. TNF induced up-regulation of TNFR I and II together with an associated increase in IL6, ICAM-1 and VCAM-1 to a greater extent in CM-susceptible mice than in CM-resistant mice (Lou et al., 1998). In addition, there was an absence of ICAM-1 up-regulation in TNFR II knock-out mice, suggesting a link between TNFRs and ICAM-1 up-regulation during malaria infection (Lucas et al., 1997a). Our aim was to investigate the ability of PRBC to modulate the endothelium in the presence and absence of the inflammatory cytokine, TNF, in a co-culture system. Firstly, functional markers of EC modulation included endothelial expression of ICAM-1, which has been attributed a critical role in parasite adhesion, and the release of IL8. Changes in levels of TNFR I and II, were also investigated as a potential mechanism for any changes in the sensitivity of EC to TNF. Second, we sought to assess the global transcriptional changes in ECs and elucidate the regulation of cellular processes following co-culture under the same conditions, using a human genome Affymetrix (Affymetrix, Santa Clara, CA, USA; http://www.affymetrix.com) chip. Our results have led us to propose a novel mechanism for the modulation of the endothelium during malaria infection that is dependent on low level TNF and involves a pro-inflammatory component but also a concurrent down-modulation of RBC-induced inflammation due to the presence of the parasite within the infected cell. 2 Materials and methods 2.1 Malarial parasites Plasmodium falciparum ItG strain was derived from the Brazilian line IT4/25/5 (Ockenhouse et al., 1992). This strain was used for the PRBCs in these studies. The ItG strain is a strong ICAM-1 binder and also binds to CD36 (Gray et al., 2003). The PRBCs were cultured in RPMI-1640 supplemented with 2 mM l-glutamine, 37.5 mM N-2-hydroxyehtylpiperazine-N′-2-ethanesulfonic acid (Hepes), 10 mM glucose, 25 μg/ml gentamicin and 10% human serum, at pH of 7.2 (Trager and Jensen, 1976). All reagents were obtained from Sigma, UK. Human serum was isolated from whole blood obtained from the Royal Liverpool Hospital and was approved by the Liverpool School of Tropical Medicine Ethical Committee. 2.2 Endothelial cells Pooled human umbilical vein endothelial cells (HUVEC) were obtained from Promocell (Heidelberg, Germany), HUVEC from different batches were used for each experiment, at passages three to five. HUVEC were grown to confluence on 1% gelatin (Sigma, UK) coated flasks and plates. All co-culture experiments were performed in serum-depleted basal HUVEC medium (quiescing medium) which consisted of M199 (Invitrogen, UK) containing 1% FCS. These conditions were designed to increase the signal window, while maintaining the integrity of the HUVEC monolayer (i.e. there was no indication of cell apoptosis or necrosis during the experiments). For all co-culture studies, ECs were co-cultured with PRBCs and uninfected RBCs in the absence and presence of TNFlow. The sub-optimal dose was verified in separate TNF dose response studies; 5 pg/ml was 1/100th of the dose of TNF used as standard for optimal induction of ICAM-1 on HUVEC in our laboratory (0.5 ng/ml), and had no significant effect on ICAM-1 expression (data not shown). For the positive control a high dose of TNF (TNFhigh) was used (10 ng/ml) to stimulate the HUVEC, while medium alone served as a negative control. All parasite and EC cultures were regularly monitored for mycoplasma using the Takara PCR mycoplasma detection kit (Cambrex Biosciences). 2.3 PRBC-EC co-culture conditions HUVEC grown to confluence in either 24-well plates or 25 cm2 flasks, for functional studies and microarray studies, respectively, were co-cultured with PRBCs and uninfected RBCs in the absence and presence of TNFlow at 37 °C. The RBC suspension was adjusted to 1% haematocrit. PRBC at the trophozoite stage (20–28 h post-invasion) were used in all co-culture studies; for the functional studies the parasites were synchronised using sorbitol, while for the microarray studies the trophozoites were additionally enriched using plasmagel flotation. In selected experiments, the parasites were retrieved after the 20-h, stained with giemsa and examined by light microscopy. There was no apparent rupture of RBC or change in the parasite stage over this period. For all functional studies the PRBCs were at a parasitaemia of 3% and co-cultured with HUVEC for 20 h. For microarray studies, the PRBCs were enriched on Plasmagel to parasitaemia ranging between 50% and 60% and co-cultured for 6 h. The uninfected RBCs were from the same batches used for parasite culture and were maintained under the same conditions as PRBCs, in separate flasks. 2.4 RNA expression: microarray analysis Following incubation, the supernatant was aspirated, HUVEC was washed with cold RPMI-1640 and then with 0.02 M EDTA to remove the adherent RBCs and the cells harvested using Trizol (Invitrogen, UK). RNA integrity was evaluated by electrophoresis on a 1% agarose gel and by spectrophotometry using the absorbance ratio at 260/280 nm. Five micrograms of each complementary biotin-labelled RNA was hybridised on the Affymetrix GeneChip, Human Genome version 2, HGU133plus2.0 (Santa Clara, CA), according to the manufacturer’s instructions. The signal intensity for each feature on the array was determined using the 70th percentile method provided by GCOS software (Affymetrix). Hybridisations were performed for four replicates of each of the seven conditions (control, RBC, PRBC, RBC + TNFlow, PRBC + TNFlow, TNFlow and TNFhigh). Data analysis was performed using Bioconductor (Gentleman et al., 2004). The microarrays were pre-processed using a Robust Multiarray Averaging program (Irizarry et al., 2003). The expression values were calculated using the R statistical computing environment (http://www.r-project.org/) with the “affy” package (Gautier et al., 2004). The differential expression was assessed and variability estimated by fitting a linear model to the data that fully models the systematic part of each gene, using the “limma” package. From the “limma” output, log-fold changes from one condition compared with another, P-values corrected for a 5% false discovery rate (Hochberg and Benjamini, 1990), together with B-statistic (log-odds that a gene is differentially expressed) can be obtained and used to determine up- and down-regulation of genes. Due to the complexity of the microarray design (i.e. four replicates, seven conditions); for non-hierarchical clustering, the 47,000 transcripts present on the human chip were filtered down to 8105, based on their moderated F-statistic at P-value <0.001. These were then clustered by similarity. The unsupervised clustering program enabled us to discriminate biological clusters (ArrayMiner, OptimalDesign). For each cluster Gene Ontology (GO; http://www.geneontology.org) assignments were extracted using the GOstats package (http://bioconductor.org/packages/1.9/bioc/html/GOstats.html). The gene list of each cluster was used to identify all the unique GO terms in order to determine whether genes that comprise a given cluster have a common function, process or location in the cell. For each GO term, GOstats analysed whether that term was being significantly over-represented in the data, using a P-value cut-off of P ⩽ 0.05. 2.5 ICAM-1 protein expression HUVEC grown to confluence in 24-well plates were co-cultured with PRBC and uninfected RBC at a haematocrit of 1% and a parasitaemia of 3% and suspended in M199 supplemented with 1% FCS in the absence and presence of 5 pg/ml TNF (TNFlow) for 20 h at 37 °C. Following the incubation period, the supernatant was removed, centrifuged at 300g for 3 min to remove any RBCs and stored at −80 °C. The HUVECs were washed once with cold RPMI-1640 and then with 0.02 M EDTA to remove the adherent RBCs and subsequently harvested by trypsinisation for analysis by flow cytometry. FACS ICAM-1 expression on HUVEC was determined by staining the cells using a fluorescein isothiocyanate (FITC)-conjugated mouse anti-human ICAM-1 antibody (MCA1615F; Serotec) using standard staining protocols and the cells fixed in 2% paraformaldehyde and analysed by flow cytometry. ICAM-1 expression was expressed as geometric mean of the fluorescence intensity. 2.6 IL-8 and TNF receptor expression The supernatants stored from the co-culture studies were analysed using a standard sandwich ELISA kit (IDS), using a horse-radish peroxidise based colorimetric detection system, to quantify IL8 released from ECs. IL8 production was expressed as a concentration in pg per ml. Similarly, soluble TNFR I, sTNFR I (p55) and soluble TNFR II, sTNFR II (p75), were detected using sTNFR I (KAC1761) and sTNFR II (KAC1771) ELISA kits (Biosource). TNFR level was expressed as TNFR concentration in ng per ml. In order to understand the kinetics of TNFR expression on the surface of ECs in response to co-culture with PRBCs, the ECs were co-cultured with PRBCs and uninfected RBCs for 0.5, 1, 2 and 3 h. Following the incubation period, HUVECs were harvested and dual stained for surface TNFRs with monoclonal anti-human RII-FITC (FAB226F) and monoclonal anti-human RI-PE (FAB226F) antibodies (R&D Systems Europe). The receptor expression was expressed as the geometric mean of the fluorescence intensity. 2.7 Trypsin digestion of RBC The ability of PRBCs to induce changes in surface ICAM-1 levels following trypsinisation was determined. PRBCs and uninfected RBCs were washed twice with PBS and incubated with 0.1 mg/ml trypsin in PBS for 15 min at room temperature with gentle mixing (modified from Chaiyaroj et al., 1994). After incubation the trypsin was inhibited with FCS at a final concentration of 10%. Conditions for trypsinisation were optimised to prevent cell lysis and the cells were stained with giemsa before and after trypsinisation to confirm RBC integrity was maintained. The RBCs were washed three times in PBS and resuspended in M199 supplemented with 1% FCS and co-cultured with HUVEC, as described. We also analysed adhesion of PRBCs to microslides coated with 50 μg/ml Fc-tagged recombinant ICAM-1, ICAM-1-Fc (Gray et al., 2003), under laminar flow conditions at shear stress of 0.05 Pa, before and after trypsinisation, in order to evaluate PfEMP-1 mediated cytoadhesion. 2.8 Transwell experiments Confluent HUVECs were co-cultured with PRBCs separated using a 0.4 μm transwell filter (Falcon), which prevents contact between HUVECs and RBCs but allows soluble factors to diffuse through. This set-up allowed us to determine whether contact between RBCs and ECs was necessary for induction of ICAM-1 expression on the ECs. 3 Results 3.1 Up-regulation of ICAM-1 expression following co-culture with infected and uninfected RBC ICAM-1 is expressed constitutively at low levels on the surface of HUVEC in culture. Neither PRBCs nor uninfected RBCs was capable of inducing appreciable ICAM-1 expression over 20 h (Fig. 1). However, exposure to both PRBCs and TNFlow simultaneously, resulted in significant up-regulation of ICAM-1 expression (P < 0.05). TNFlow alone was incapable of inducing ICAM-1 expression (Fig. 1). 3.2 Up-regulation of IL-8 release following co-culture with infected and uninfected RBC Following incubation with either PRBCs or uninfected RBCs for 20 h, IL8 levels in the supernatant were increased over basal levels (P < 0.01) (Fig. 2). When the HUVECs were concurrently exposed to PRBCs and TNFlow, IL8 release was further amplified over basal levels (P < 0.0002). Uninfected RBCs in combination with TNFlow induced similar levels of IL8 release (P < 0.001) (Fig. 2). 3.3 Up-regulation of soluble TNF receptor release and down-regulation of surface expression following co-culture with infected and uninfected RBC Soluble forms of both TNFR I (p55) and TNFR II (p75) were significantly increased by 35% (P < 0.05) following treatment with either PRBCs or uninfected RBCs for 20 h. Thus, the phenomenon of soluble receptor release from the surface of ECs, when co-cultured with RBCs, appeared to be independent of the parasite (Fig. 3a and b). Surface levels of the TNFRs were monitored over 3 h to understand the dynamics of TNFR expression. Both TNFR I and TNFR II were significantly reduced in response to both PRBCs and uninfected RBCs by 70% (P < 0.05) and 50% (P < 0.01), respectively, within 30 min (Fig. 4a). Interestingly, the levels of the TNFR I, but not TNFR II, fell over time under basal conditions, albeit at a slower rate than co-culture with RBCs. This could be a result of serum depletion when complete medium was replaced with M199/1% FCS for the duration of the co-culture experiment. In the case of both receptors, the observed down-regulation in response to RBCs was significantly greater than that seen under basal conditions. 3.4 Loss of ICAM-1-mediated adhesion phenotype following trypsinisation of PRBC Following trypsinisation, PRBCs did not lose the ability to up-regulate ICAM-1 expression on co-culture with ECs (Fig. 5a). As before, both PRBCs and uninfected RBCs induced an increase in surface levels of ICAM-1 in the presence of TNFlow. ICAM-1 mediated adhesion under laminar flow, however, was abolished following trypsin treatment of PRBCs (Fig. 5b). In additional experiments, when the PRBCs were separated from the ECs in 24-well plates using 0.4 μm transwell inserts, there was no stimulation of ICAM-1 expression (data not shown). Thus, the EC activation observed in our system was critically dependent on cell–cell contact, although it did not involve specific PfEMP-1-mediated cytoadherence. 3.5 Modulation of the endothelial cell transcriptome following co-culture with infected and uninfected RBC Using ArrayMiner software (OptimalDesign), seven non-hierarchical clusters were identified (Fig. 6a). As illustrated on the resulting heatmap, there is minimal change in the gene expression profiles with either PRBCs or RBCs compared with controls (Fig. 6b). However, when HUVECs were exposed to either PRBCs or RBCs in combination with TNFlow, differential expression was visible in three out of the seven clusters, compared with control or TNFlow alone (Fig. 6a and b). A comprehensive analysis of the microarray data was performed and has been submitted to Array Express (Accession No. E-SGRP-3). Our analysis focuses on genes that were differentially and significantly expressed at P < 0.001, compared with control levels. We have concentrated on over-represented gene ontologies and cited genes that best exemplify these GO terms. In cluster 3, both PRBCs and uninfected RBCs, in combination with TNFlow, produced over-expression (Fig. 6a and b). TNFlow alone, however, produced a significant under-expression. Genes in this cluster included the inflammatory protease, caspase 1, the acute response cytokine, IL8, inflammatory cytokines IFN-γ and lymphotoxin β, adhesion molecules ICAM-1, E-selectin and chemokines, CXCL2, CXCL3, CX3C1 and CXCL6. In clusters 1 and 2, opposing effects were induced when HUVECs were exposed to either PRBCs or RBCs in combination with TNFlow. Over-expression of genes was observed in response to uninfected RBCs and TNFlow and conversely, PRBCs and TNFlow resulted in under-expression of genes in these clusters (Fig. 6a and b). In cluster 1, TNFlow on its own had no effect compared with controls, while in cluster 2 TNFlow alone produced a small reduction in gene expression (Fig. 6b). Genes in cluster 1 included the protease calpain 13, toll-like receptor-6 and the junction protein desmocollin 2, while genes in cluster 2 included the junction protein decorin, MHC class II, transferrin, endothelin 3 and endothelin receptor type A. While it is difficult to extrapolate roles for these genes from this in vitro analysis of the transcriptome, we can make some speculations based on recent studies. For instance, transferrin induction may be a mechanism for iron homeostasis to prevent accumulation of iron in endothelial cells which can lead to oxidative damage of the cells (Nanami et al., 2005). Endothelin is a known vasoconstrictor, in vivo; down-regulation of endothelin and its receptor may be a mechanism for maintaining vascular tone and integrity during malaria infection (Basilico et al., 2004). Down-regulation of MHC class II may represent suppression of the host immune response by the parasite. The genes in each cluster were further analysed and categorised by GO) terms using the GOstats package of R/Bioconductor to understand their basic biological functions. The over-represented GO terms, defining genes in each cluster, in terms of biological process, cellular component and molecular function, showing ⩾2% of the total number of genes being annotated with that term, were evaluated. Fig. 7 illustrates distribution of the over-represented gene ontologies with respect to biological process and cellular component, in clusters 1 and 2, which reflect inflammatory and protective functions in the endothelial cells. The full analysis for all three clusters is detailed in Supplementary Fig. S1(see on-line supplementary data). Whilst some GO terms fell below the 2% cut-off, it is important to note that they were nonetheless significant and have been tabulated for further information (Supplementary Tables 1, 2 and 3; see on-line supplementary data). A wide range of GO terms were over-represented in each cluster within categories of biological process, cellular component and molecular function, with a number of common and parallel themes, including cell communication, signal transduction, cell adhesion, organismal physiological process, ion transport, response to external stimuli and immune response. Interestingly, the genes in each of these clusters were primarily cell membrane-associated, or related to the extracellular region and the extracellular matrix. The molecular functions of the genes in each cluster were wide ranging, although receptor activity, signal transducer activity, ion channel and protein binding activities were parallel themes in all three clusters. The fold changes for the 8105 genes were calculated for each of the conditions for visualizing the changes in expression, compared with control levels. Expression of ICAM-1 and IL8 RNA was up-regulated, reaching 3.7-fold and 7-fold, respectively, over control levels (P = 0.001), when HUVECs were incubated with a combination of PRBCs and TNFlow. Interestingly, ICAM-1 transcription reached similar levels in the presence of a combination of uninfected RBCs and TNFlow. Table 1 illustrates the changes in ICAM-1 and IL8 expression under the different co-culture conditions. Table 2 shows the changes in expression of selected genes which illustrate specific biological processes of ECs that may have a role in mediating either pathology or protection, and their respective fold changes in the presence of PRBCs, uninfected RBCs and TNFlow compared with controls. Although the magnitudes of change are relatively small, they are highly reproducible and biologically significant at P < 0.001, over four separate replicates. The chemokines, CXCL2 (Gro-β), CXCL3 (Gro-γ), CXCL6 and E-selectin were all over-expressed in the presence of PRBCs and TNFlow, with variable increases in expression of CXCL2 and CXCL6 in the presence of uninfected RBCs and TNFlow (Table 2). Gro-β has been shown to have a role in the adhesion of monocytes to ECs (Schwartz et al., 1994) while CXCL6 has similar properties to IL8 and can be co-induced with IL8 (Gijsbers et al., 2005). E-selectin was also over-expressed in response to PRBCs, uninfected RBCs and TNFlow, reaching up to 7-fold over controls with PRBCs and TNFlow. Molecules involved in cell adhesion such as ICAM-1 and CX3C1 were both over-expressed in PRBCs, uninfected RBCs, and TNFlow, with a greater effect in PRBCs (Table 2). CX3C1 (fractalkine) has recently been implicated in mediating adhesion of PRBCs to ECs (Hatabu et al., 2003). Other adhesion receptors such as desmocollin 2 and integrin β1 were both under-expressed with PRBCs and TNF, and over-expressed with uninfected RBCs and TNF (Table 2). A number of inflammatory genes such as MHC class I, superoxide dismutase 2 (SOD 2) and lymphotoxin-β were all over-expressed in the presence of PRBCs and TNFlow (Table 2). Whilst both MHC class I and SOD 2 were also over-expressed with uninfected RBCs and TNFlow, lymphotoxin β was under-expressed. Table 2 illustrates differential expression of molecules such as TNFR-associated factor 1 (TRAF 1), Fas associated factor 1 (FAF-1), prostaglandin receptor 3 and TGF-β3, which all have a role in signal transduction. A number of cell apoptosis-related genes were differentially regulated in our co-culture system (Table 3). Both caspase 3 and caspase 9, which are believed to be inducers of apoptosis (Ho and Hawkins, 2005), were under-expressed in the presence of PRBCs and TNFlow. In addition, caspase 3 expression was markedly increased in the presence of uninfected RBCs and TNF. Calpains, which are inducers of cell death via a caspase-independent pathway (Utz and Anderson, 2000; Perrin and Huttenlocher, 2002), were also regulated in our system. We looked at the expression of two calpains, 9 and 13. Calpain 9 was under-expressed and there was a small increase in calpain 13 expression in the presence of PRBCs and TNFlow (Fig. 8). Thus, our data does not suggest induction of apoptosis; in fact, there appears to be a trend towards protection from apoptosis and this was supported by our inability to detect any positive staining with annexin V and the absence of any morphological changes in the ECs after co-culture. 4 Discussion Sequestration of PRBCs at vascular sites is a critical event in the pathogenesis of severe malaria. Here we have investigated the effect of prolonged exposure to PRBCs on the endothelium. We have demonstrated a phenomenon whereby ECs are differentially modulated when co-cultured with PRBCs in the presence of TNFlow, which could occur at microvascular sites early in infection, for instance during schizogony. Unlike previously published work, in our system there was no apparent induction of ICAM-1 or modulation of RNA transcription by PRBCs alone. Significant de novo ICAM-1 induction and differential regulation of gene transcription were dependent on the presence of low levels of TNF. PRBCs alone did modulate certain EC functions such as IL8 release and TNFR shedding from the cell surface, but IL8 release was further enhanced in the presence of TNFlow. Up-regulation of ICAM-1 was maintained following removal of surface expressed PfEMP-1 by trypsin digestion, but was abolished when cell–cell contact was prevented. This demonstrated that while cell-to-cell contact was crucial for modulating EC function, there was no requirement for adhesion via specific receptors in this model. Despite the differences in the reported effects of PRBCs on ECs, the unifying theme in all the co-culture studies is this critical requirement for close apposition of the two cell types in the co-culture system, in the induction of EC activation. The transwell system, whilst preventing cell-to-cell contact, allowed the movement of parasite-derived soluble factors. Therefore we could exclude the role of soluble parasite-derived factors in EC activation. In all cases, when the cell–cell contact was compromised using a filter, EC activation was abolished (Randolph and Furie, 1996; Pino et al., 2003; Viebig et al., 2005). Thus the variable observations may reflect differences in the respective co-culture conditions, for instance, studies by Viebig et al. (2005) were performed in complete HUVEC culture medium and studies by Pino et al. (2003) were performed in parasite culture media, while our studies were performed in a serum-depleted basal HUVEC medium in order to enhance the signal window for the EC measurements, whilst maintaining EC integrity. It is clear from our work and that of others, that care will need to be taken in interpreting the results of model co-culture systems and that further work will be required to develop ex vivo models of severe disease. It is possible that no single model will be able to reflect the variable pathology represented in malaria infection. Our study is unique in the observation that concomitant presence of low level TNF is necessary for PRBCs to modulate the endothelium, namely HUVECs. Indeed, a recent study also suggested that PRBCs alone do not have the ability to induce ICAM-1 in HUVECs, and that this may be a phenomenon specific to brain-derived ECs (Tripathi et al., 2006). Using HUVEC as an in vitro model for studying PRBC-EC interactions in vivo, we have demonstrated that close apposition or cell–cell contact of the PRBCs with ECs is a critical factor in mediating this activation. Whilst uninfected RBCs also had an effect on ECs in the presence of low level TNF, it is noteworthy that in vivo, apposition of uninfected RBCs on ECs only occurs secondary to cytoadhesion or as a result of vessel occlusion. The ability of normal RBCs to stick to and modulate ECs in vitro, however, is not a novel observation. Indeed, normal RBCs can bind to ECs mediated by plasma factors such as fibrinogen and fibronectin (Wautier et al., 1983) and the binding can induce low-grade ICAM-1 expression in vitro (Brown et al., 2001b). RBCs express a wide range of molecules on their surface which have been implicated in mediating adhesion to endothelial cells under different conditions. These include: (i) VLA-4 (alpha4beta1) which can bind to endothelial VCAM-1 (Walmet et al., 2003); (ii) the blood group Lutheran molecule (LU) over-expressed on sRBCs can bind to laminin present on cells or in the intercellular space (Eyler and Telen, 2006); (iii) advanced glycation end products (AGEs) present on RBCs bind to their receptor (RAGE) on endothelium (Wautier and Schmidt, 2004), activating endothelial cells; and (iv) a molecule related to blood group Rhesus, ICAM-4 (Hermand et al., 2003) binds to integrins present on leukocytes (CD11–CD18) and on platelets (alpha2beta4) offering a surface which can be involved in thrombosis (reviewed by Wautier and Wautier, 2004). Thus, although PfEMP-1 does not play a role in the induction of ICAM-1 expression seen by us, it would normally be required to bring the PRBCs and ECs together in vivo. Contrary to our hypothesis that up-regulation of TNFRs in response to PRBCs contributed to the increased sensitivity to TNF, unexpectedly, we observed significant down-regulation of surface TNFR expression and increased receptor shedding. It is possible that the enhancement of the effects of RBCs is at the level of intracellular signal transduction. Rapid TNFR shedding by cleavage of the extracellular region of the receptors as observed in our studies is a common response to various stimuli (Aderka et al., 1998) and may be a mechanism for down-modulation of both TNF receptors. This rapid release of TNFRs in response to an insult to the endothelium or physical cell contact with the endothelium is believed to be a non-specific pro-inflammatory response and up-regulation of soluble TNFR I has been correlated with parasitaemia in the serum of patients with acute P. falciparum infection (Wenisch et al., 1994). The TNFR shedding seen in response to PRBCs or uninfected PRBCs alone, may be part of a non-specific inflammatory response of ECs to prolonged cell–cell contact. Similarly, IL8 release in our system may also represent a non-specific inflammatory response to cell–cell contact with RBCs, as was demonstrated in a monocyte/EC co-culture model and was further shown to be independent of ICAM-1 mediated binding (Lukacs et al., 1995). Similarly to our direct measurements of ICAM-1, there was no significant change in EC gene transcription in response to PRBCs or RBCs alone but in combination with TNFlow, significant regulation of the EC transcriptome was induced. Analysis of the transcriptome revealed a wide diversity of gene ontologies representing genes that were significantly regulated, however, care must be taken in interpreting the data since there is a high degree of redundancy across GO terms, which was not accounted for in this analysis. IL8, for instance, has diverse biological roles including cell communication, cell adhesion, signal transduction and immune response. In spite of such deficiencies, the GO terms that were over-represented, including cell communication, cell adhesion, signal transduction, ion transport and immune response, fit well with our hypothesis that the infected RBC is able to modulate the host endothelial response. From previous work, we had expected to see a pro-inflammatory response induced by PRBCs (cluster 3, Fig. 6b). Less predictable was the down-regulation of genes in the presence of PRBCs and TNFlow (clusters 1 and 2, Fig. 6b), which may represent a parasite-specific adaptive response that favours parasite survival and development by protecting the integrity of the host endothelium. Up-regulation of these genes under the same conditions may result in the induction of a pro-adhesive effect that can potentially feed back to reinforce pathological sequestration that is typically seen in the later stages of severe malaria. In addition, induction of an immune response, as in cluster 3, may confer a protective effect on the host. Thus, we propose that the balance between these converse effects of PRBCs and TNF on the endothelium may be the critical factor in determining the response by endothelium and the clinical outcome of a malaria infection in vivo. The over-expression of genes in response to uninfected RBCs and TNFlow in two of the clusters, whilst unexpected, is a common phenomenon in static in vitro co-culture studies. For instance, normal RBCs can interact with ECs to induce the expression of a variety of pro-inflammatory molecules including ICAM-1 (Brown et al., 2001b), VCAM-1 (Brown et al., 2001b; Wautier et al., 2001) and activation of NADPH oxidase, which mediates the generation of reactive oxygen intermediates (Wautier et al., 2001). This is a potential mechanism to induce expression of genes to counteract the response to PRBCs and may reflect a partially protective host response during a malaria infection, balanced against inducing a pro-adhesive environment, particularly early in infection when systemic inflammation is unlikely to be a major contributor to pathogenesis. Modulation of host cells has been demonstrated in other co-culture models, for instance tyrosine phosphorylation of scyncitiotrophoblasts in a model of placental malaria (Lucchi et al., 2006) and ecto-phosphorylation of CD36 following the initial PRBC attachment which is believed to further enhance CD36-mediated cytoadhesion (Yipp et al., 2003). While co-culture models give useful insights into mechanisms of action of PRBCs on host cells in malaria pathogenesis, care must be taken in the interpretation and extrapolation of this data to pathogenesis in vivo, since these mechanisms may not be reflected in the in vivo situation. In summary, our co-culture model identifies a novel mechanism whereby PRBCs, whilst mobilizing a pro-inflammatory and a seemingly pro-adhesive effect on the host endothelium, have the additional ability to suppress gene expression pathways involved in signal transduction, apoptosis and immune response as part of a mechanism to support parasite survival through maintaining the overall integrity of the host EC. This fits well with histo-pathological observations in severe malaria where transient dysfunction of the blood–brain barrier is observed (Brown et al., 1999a) but the overall health of the endothelium seems to be maintained despite the presence of many adherent PRBCs (Medana and Turner, 2006). Our suggestion is that the host response to sequestration is complex and involves a balance between pro- and anti-inflammatory pathways, both mediated by apposition of the PRBC, and interacting with other mechanisms of host response to malaria infection variably throughout an infection. This balance is critical to the health of an EC and the resulting pathology associated with severe malaria.

Mol_Syst_Biol-3-_-1800356

Tissue-specific regulatory elements in mammalian promoters

Transcription factor-binding sites and the cis-regulatory modules they compose are central determinants of gene expression. We previously showed that binding site motifs and modules in proximal promoters can be used to predict a significant portion of mammalian tissue-specific transcription. Here, we report on a systematic analysis of promoters controlling tissue-specific expression in heart, kidney, liver, pancreas, skeletal muscle, testis and CD4 T cells, for both human and mouse. We integrated multiple sources of expression data to compile sets of transcripts with strong evidence for tissue-specific regulation. The analysis of the promoters corresponding to these sets produced a catalog of predicted tissue-specific motifs and modules, and cis-regulatory elements. Predicted regulatory interactions are supported by statistical evidence, and provide a foundation for targeted experiments that will improve our understanding of tissue-specific regulatory networks. In a broader context, methods used to construct the catalog provide a model for the analysis of genomic regions that regulate differentially expressed genes. Introduction Reverse engineering mammalian transcriptional regulatory circuits can be achieved using systematic methodology that includes both computational and experimental techniques, working in tandem to generate, refine and verify hypotheses. Understanding tissue-specific transcription is a necessary step for extending regulatory circuit reverse-engineering efforts from single-cell eukaryotes to metazoans. We recently demonstrated that the information in proximal promoters can predict a significant portion of tissue-specific elevated or inhibited expression (Smith et al, 2006). Here, focusing on tissue-specific regulatory pattern identification and prediction accuracy instead of proof of existence, we use refined analysis and data curation methods to discover and catalog high-confidence regulatory interactions and sites. This catalog will assist experimental efforts to reverse engineer tissue-specific transcriptional regulatory networks from the bottom up. Numerous techniques for analysis of regulatory sequences have been proposed, and the problem of module identification is now receiving due attention (Zhou and Wong, 2004; Gupta and Liu, 2005; Zhu et al, 2005). Previously characterized binding site motifs have been used to infer transcription factor function in certain tissues (Nelander et al, 2005). Xie et al (2005) identified conserved motifs across ortholog promoters of four mammalian genomes. Robertson et al (2006) describe cisRed, a database that integrates genome annotation data, homology data and genome alignments to identify motifs with conserved sites across mammals. We analyzed proximal promoters with evidence for tissue-specific regulation in order to identify tissue-specific motifs, modules and their sites in proximal promoters. We developed a new technique for characterizing tissue-specific modules that ensures that each module component significantly improves tissue-specific module enrichment. We integrated multiple sources of expression data to identify reliable sets of transcripts that are under tissue-specific regulation in human and mouse. Using transcription start site (TSS) annotation in Cold Spring Harbor Mammalian Promoter Database (CSHLmpd) (Xuan et al, 2005), we compiled sets of proximal promoters corresponding to transcripts with evidence for specific regulation in the selected tissues. Our analysis was based on motifs discovered de novo (called novel motifs) using DME (Smith et al, 2005a) and DME-B (Smith et al, 2006), as well as previously characterized vertebrate binding-site motifs (called known motifs) from TRANSFAC (Matys et al, 2003) and JASPAR (Sandelin et al, 2004). We evaluated motifs according to enrichment in tissue-specific promoters relative to other promoters from the same species. We showed that motifs associated with factors with known tissue-specific roles rank high for enrichment, that motif ranks are significantly correlated between human and mouse and that this same set of motifs and their corresponding cis-elements are unlikely to be identified using traditional, order-preserving alignments of ortholog promoter sequences. We constructed modules of interacting motifs (both novel and known motifs), ensuring that each component contributed significantly to the enrichment of the whole module. We annotated tissue-specific promoters with predicted tissue-specific regulatory elements and demonstrated that these sites are in excellent agreement with experimentally annotated liver-specific sites in the human albumin promoter and skeletal-muscle-specific sites in the human α-actin promoter. Both promoters are particularly well annotated with experimentally verified tissue-specific regulatory elements and permit an informative comparison. In other tissues, we gave predicted sites for tissue-specific motifs in representative promoters. The complete data and analysis are available in TCat: The Catalog of Tissue-Specific Regulatory Motifs (http://rulai.cshl.edu/tcat). Results We describe first steps toward cataloging high-confidence tissue-specific motifs, modules and their sites. We first collected and integrated expression and function data from various sources, and identified transcripts that are likely to be under tissue specific regulation. We demonstrated that transcripts with evidence for tissue-specific regulation from multiple expression sources in one species (human or mouse) are significantly more likely to have evidence for tissue-specificity in the other species. We analyzed and annotated proximal-promoter sets in seven representative tissues from both human and mouse, demonstrating that motifs and predicted binding sites are in agreement with experimentally verified data and that analyses in human and mouse are significantly correlated. We also showed that the top-scoring sites in orthologous tissue-specific promoters from human and mouse rarely have significant conservation of site order, suggesting that comparative genomics alone may not be sufficient to decode the regulatory signals in these proximal promoters. Transcripts under tissue-specific regulation Few transcripts have expression restricted to a single tissue, but many transcripts appear to be regulated in a tissue-specific manner (Su et al, 2004), and the corresponding promoters are likely to contain tissue-specific regulatory elements. To circumvent problems associated with individual sources of information, we used a voting system that combined information about expression, function and tissue specificity from different sources. Table I gives the number of transcripts with single and multiple sources of evidence (votes) for tissue-specific regulation in each tissue. Orthologs of transcripts with multiple votes for tissue-specific regulation were more likely to have evidence for specific regulation in that tissue, suggesting that the false-positive rate for calling a transcript tissue specific is lower when based on multiple votes. The number of ortholog transcript pairs with multiple votes for tissue-specific regulation in both species ranged from 1 in CD4 T cells to 69 in liver. Table II lists genes and orthologous transcripts with votes for skeletal-muscle-specific regulation in both human and mouse. Gene and transcript lists for other tissues are given in Supplementary Section 1.5. Enrichment of known tissue-specific motifs Knowledge of factors and corresponding binding sites that regulate tissue-specific transcription can be used to evaluate motif ranking. We measured motif enrichment in tissue-specific promoter sets using balanced error rates, evaluating motifs for their ability to distinguish tissue-specific sets (foreground sets) from background sets that are composed of non-tissue-specific promoter samples from CSHLmpd. Balanced error rates measure proportions of misclassified promoters after normalization of foreground and background sizes. We ranked motifs according to enrichment and determined whether the ranks assigned to binding-site motifs for factors with known tissue-specific roles are significantly elevated in the corresponding tissues. The results presented in Table III demonstrate that binding-site motifs for these factors ranked significantly high (P<0.01) according to a Wilcoxon signed-ranks test in almost all tissues tested. Excluding DBP in human liver and HNF-3 in mouse liver, these factors had evidence for expression in their respective tissues, and their binding-site motifs were highly enriched in our foreground sets. Results are summarized in Table IV, and the motifs with greatest enrichment in each tissue are given in TCat. Table IV also includes information for HNF-6 in liver. HNF-6 is a known liver regulator, but there is no evidence for its expression in liver based on our data, and its binding-site motif was not enriched in our liver foreground sets. In addition, C/G- or A/T-rich motifs are likely to be enriched in foreground sets that are C/G or A/T rich relative to promoter base composition. To eliminate this potential bias, we adjusted the GC content in background sets to match foreground sets. Some known tissue-specific motifs were identified as enriched only after GC content correction. Nuclear receptor binding-site motifs and E-box motifs are among the top enriched motifs in 11 and 10 of the 14 human and mouse tissues, respectively. A Wilcoxon signed-ranks test showed that nuclear receptor and E-box motifs (represented by 54 and 39 TRANSFAC motifs, respectively) are enriched in the union of our foreground sets with P-values below 6.07E−14 and 2.02E−13 (nuclear-receptor motifs) and 2.22E−10 and 4.57E−03 (E-box) in human and mouse, respectively. These results suggest a diversity of tissue-specific roles for nuclear receptors and E-box binders, likely mediated by tissue-specific cofactors. Tissue-specific cis-regulatory elements Highly enriched motifs, and the associated score thresholds identified by our methods, provide a starting point for targeted experimental annotation of tissue-specific promoters. Figure 1 shows known and predicted sites mapped on the −500 to +100 region of the human albumin promoter and the −250 to +50 region of the human skeletal muscle α-actin promoter. Human albumin has known functional binding sites for HNF-1, C/EBP, AFP and NF-Y (Paonessa et al, 1988; Sawadaishi et al, 1988; Frain et al, 1990; Li et al, 1990), all of which were identified among the top predicted motifs or included in top modules for liver. Locations of known sites for C/EBP (at −437) and NF-Y (at −125) do not perfectly align with the corresponding predicted sites (at −462 and −143, respectively), but current knowledge about binding sites for those factors raises the possibility that the predicted locations are more accurate. The only predicted binding site for these factors in the human albumin promoter that is not depicted in the figure is a C/EBP site at −956. Human skeletal muscle α-actin has known sites for SRF, TEF and a known TATA box (Boxer et al, 1989; MacLellan et al, 1994), all among top predicted motifs for human skeletal muscle (TATA box motifs have high similarity with MEF-2 motifs). Figure 2 gives predictions in a representative human promoter in each of the remaining tissues. Comparison to previous results Previous analysis of tissue-specific patterns in regulatory regions includes analysis based on cross-species conservation (Xie et al, 2005) and coexpression (Smith et al, 2006). Xie et al (2005) identified conserved elements in orthologous promoters of four mammalian genomes. They found 59 experimentally validated motifs that are significantly conserved and enriched in at least one human tissue. These include E-box, ETS, MEF2, MEIS1 and NF-1 in skeletal muscle; Chx10 in kidney; NRF-1, ELK-1, GABP and E12 in CD4 T cells; AP-4 and MEF-2 in heart; and NRF-1 in testis. Our results agree with Xie et al (2005) on the enrichment of E-Box and MEF-2 in skeletal muscle, ETS in CD4 T-cells and E-box in pancreas. In previous work (Smith et al, 2006), we tested the hypothesis that proximal promoters contain information that can be used to predict tissue-specific expression. We were not concerned with identifying the most significant tissue-specific motifs, modules and sites. Considering the difference between the goals of the two projects, it is not surprising that the predictive models described by Smith et al (2006) have little similarity to our top motifs and modules. The most significant similarities between our top tissue-specific patterns and the predictive models of Smith et al (2006) include the enrichment of ETS in CD4 T-cell-specific promoters and the enrichment of Smith et al (2006) motifs Novel3 and Novel6 in mouse testis and Novel1 in human testis. The three novel testis motifs are very similar to motifs that rank in the top 100 in our analysis, but the enrichment of these motifs was not sufficiently high for inclusion in TCat. Correlation between human and mouse regulatory regions We compared motif enrichment ranks in each human foreground set to ranks in the corresponding mouse foreground set using Spearman's rank correlation test, and found that enrichment ranks across species are highly correlated (P<0.001) for all but CD4 T cells (Supplementary Table 15). In CD4 T cells, motif enrichment ranks are similar only for few highly enriched motifs. Despite the motif-enrichment ranks correlation, the order of the top predicted binding sites is not usually conserved between orthologous promoters. Fewer than 10% of orthologous pairs showed significant (P<0.01) conservation of site order. Weak site-order conservation suggests that the top tissue-specific sites would be difficult to identify using traditional cross-species alignment alone, and methods that rely on co-linear promoter alignment may have high false-negative detection rates. This evidence is in agreement with Frith et al (2006), who found that homologous transcription start sites can be separated by more than 100 nucleotides. A list of the nine genes (out of 102 candidates) with significant conservation of site order is given in Supplementary Section 2.3. Materials and methods The steps used in creating the catalog include (1) identifying tissue-specific transcripts, (2) identifying factors that are expressed in each tissue, (3) obtaining promoter sequences for tissue-specific transcript, and (4) identifying individual motifs and modules (i.e. sets of interacting motifs) that characterize tissue-specific promoter sets. Identifying tissue-specific transcripts To identify motifs and modules that regulate tissue-specific transcription, we analyzed promoters of transcripts that appear to be regulated in a tissue-specific manner. If an information source indicated that a transcript has restricted expression, unusually high expression, or a specific function in the tissue, that source voted for tissue specificity of the transcript. For each tissue, we sorted the transcripts according to the number of votes received, retaining the top 100 with distinct TSS as tissue specific. Ties in the ranking were broken according to intensity values from the GNF SymAtlas expression data (discussed below), which we have found to be the most complete and the most reliable source of tissue-based expression information. We used the same number of transcripts for each tissue to facilitate comparison across tissues, and 100 sequences provided sufficient information for our analysis while allowing identification of well-known tissue-specific motifs. Microarray data The GNF SymAtlas microarray data were generated using Affymetrix HG-U133A array and the custom GNF1H and GNF1M Affymetrix arrays, and include expression profiles for 79 human and 61 mouse tissues (Su et al, 2004). Among these are the seven tissues we selected to include in the catalog. Tissues were selected with consideration to data availability in GNF and other sources and interest from Zhang lab members and collaborators. A transcript received a vote for tissue specificity from this information source if it was called present and its intensity exceeded its mean across all tissues by 3 standard deviations. The Hughes Toronto microarray data (Zhang et al, 2004), which was generated using custom-built oligonucleotide arrays, provide mouse expression profiles for 55 tissues, including all of our tissues but CD4 T cells. A transcript received a vote for tissue specificity if it was called present in the tissue, and had intensity at least 10 standard deviations above its mean across all 55 tissues. This large number of standard deviations was required to limit the number of transcripts receiving positive votes. The GeneNote expression profiles (Shmueli et al, 2003), which were generated using the Affymetrix GeneChip HG-U95, provide human expression data for 12 tissues, including all of our tissues but CD4 T cells and testis. The GeneNote data were used in the same way as the GNF SymAtlas data, with a transcript being called tissue specific if it is present in that tissue and has intensity at least 3 standard deviations above its mean across all 12 tissues. EST data dbEST is a database of expressed sequence tags (Boguski et al, 1993), and contains source information, such as the tissue of origin, for each EST. This information is used to annotate UniGene clusters with the source data, and a UniGene is said to have restricted expression in a tissue if more than half of the ESTs contributing to that UniGene have the same source tissue. A transcript received one vote for specificity in a particular tissue if the corresponding UniGene cluster is annotated as having expression restricted to that tissue. GO terms We associated a set of GO Terms with each tissue. This was performed by compiling a set of keywords for each tissue (e.g. ‘renal' was associated with kidney; ‘sperm' was associated with testis), and searching GO Term names and definitions for those keywords. This produced, for each tissue, a set of GO Terms that were subsequently reviewed to ensure that the context of the keywords was appropriate. A transcript of a gene annotated with a GO Term that is associated with a tissue received a vote for specificity in that tissue. Selecting promoter sequences Although regulatory elements can exist almost anywhere in the genome, they are concentrated near the TSS (Cooper et al, 2006). We used the CSHLmpd to map transcripts to promoters, using experimentally confirmed promoters from EPD (Perier et al, 1998), DBTSS (Suzuki et al, 2002) and GenBank, as well as computationally predicted promoters. For each promoter, we used the proximal sequence region of −1000 to +100 relative to the TSS. Each part of our analysis is based on comparing the tissue-specific promoter sets to a background of random promoters from the same species. For each tissue, a background set was constructed by selecting 1000 transcripts uniformly at random from the set of RefSeqs for the corresponding species with TSS annotation in CSHLmpd. For each tissue, transcripts with at least one vote for specificity in that tissue were removed from consideration before selecting the background. Because our analysis focused on proximal promoters, −1000 to +100 relative to the TSS, if the TSS annotation is off by several hundred base pairs, important promoter regions might be excluded. AKR1D1, identified as liver specific by our voting system, has two known TSSs within 500 bp of the first exon for the corresponding RefSeq (NM_005989) (Charbonneau and Luu-The, 1999). We used the TSS located upstream of the first exon, but could have chosen to use the other promoter, which was annotated by a generally more reliable source (DBTSS versus GenBank). Currently, in such situations, there is little information that identifies the promoter responsible for observed tissue-specific regulation, but comparative genomics and rapidly improving arrays promise better 5′ end identification, thus improving proximal promoter annotation and association between transcripts and promoters (Kim et al, 2005; Carninci et al, 2006). Negative promoter sets can be used to cancel out patterns that are not related to tissue-specific transcription regulation. We use random negative promoter sets with and without GC-content correction; this correction cancels the inuence of genomic GC-content isochore variability. Identifying and evaluating motifs Given a motif M (represented as a position–frequency matrix) and a sequence S, the max-score of M in S, max-score (M, S) is the score of the top scoring subsequence of S when aligned against the scoring matrix for M. Details on constructing and using scoring matrices can be found in Stormo (2000). For a fixed threshold λ, the max-score classification method classifies S as belonging to the foreground if max-score (M,S)⩾λ, and the background otherwise. Given a set of foreground sequences FG (i.e. the tissue-specific promoters) and a set of background sequences BG, the sensitivity of M and λ under max-score classification is and the specificity is The balanced error rate for M and λ under max-score classification is then The quantity of interest in our analysis corresponds to the optimal value of λ for M in distinguishing FG from BG: Many known motifs are similar to each other, usually owing to similar binding specificities for distinct factors or distinct origins for mofits associated with a single factor. We used MATCOMPARE (Schones et al, 2005) to eliminate redundancies in the sets of known and novel motifs. Identifying and evaluating modules Modules are used to classify sequences based on the max-score values of the motifs they contain. Let ℳ={M1, …, Mk} be a module and Λ={λ1, …, λk} be an associated set of thresholds. The max-score classification for modules assigns sequence S to the foreground if and only if max-score (Mi, S)⩾λi holds for all i (1⩽i⩽k). Given sets of sequences FG and BG, the sensitivity of ℳ and Λ under max-score classification is and the specificity is The balanced-error rate for ℳ and Λ under the max-score classification is As with mofits, we are interested in the optimal value of Λ and define Because modules are intended to describe synergistic function of a set of motifs, we are interested in modules whose performance is better than expected given the performance of the individual motifs composing the module. For a module ℳ composed of k motifs, let ℳ′⊂ℳ minimize B(ℳ′, FG, BG) over all size k−1 modules built from motifs in ℳ, and let ℳ′=ℳ\ℳ′. To assess whether ℳ, with balanced-error rate u, significantly improves over ℳ′ and ℳ′, we use the probability We estimated this probability empirically, by sampling from the distribution of balanced-error rates resulting from intersections of sets with balanced-error rates B(ℳ′, FG, BG) and B(ℳ′, FG, BG). We used MODULATOR, which is available in CREAD (Smith et al, 2005b), to construct modules. Given a set of motifs, a set of foreground sequences and a set of background sequences, MODULATOR identifies those modules composed of the given motifs that have the best balanced-error rates. A branch-and-bound algorithm is used to simultaneously optimize the score thresholds for the motifs in a module. Modules are constructed by adding motifs to existing modules until a user-specified module size is reached or until motif addition does not significantly improve enrichment. Each time a motif is added to a module, the resulting larger module is retained only if the balanced-error rate of the larger module is improved significantly above expectation. The initial modules of size two are obtained by combining pairs of motifs. For modules that are entirely composed of known motifs, the top 100 motifs (before eliminating redundancies) were used. Modules were allowed to contain up to four motifs for reasons of computational feasibility, but many top modules are smaller. Novel modules, which must contain at least one novel motif, were constructed using the top 100 novel motifs and the top 100 known motifs. Redundancies were removed from the lists of top modules using a procedure described in Supplementary Section 2. Measuring the significance of motifs and modules To measure significance of enrichment for top known motifs we used known motifs to classify randomly assembled promoter sets. We constructed 1000 foreground/background pairs for each species by selecting 100 sequences for each foreground and 1000 for each background uniformly at random from CSHLmpd. For each foreground/background pair we calculated the balanced-error rate of each known motif. The best balanced-error rates overall obtained on random samples for human and mouse were 0.364 and 0.368, respectively. We used the distribution of these error rates to identify the q-value (Storey and Tibshirani, 2003) significance of the error rate of each motif. Tissues whose highest ranking motifs fail the q<0.05 test include CD4 T cells and heart in human, and CD4 T cells in mouse. TCat includes q-value annotation for each ranked known motif. The full set of motifs for which q<0.05 is estimated to include five false leads per 100 predictions. We did not obtain statistical significance measures for novel motifs, because this will require running DME and DME-B more times than is computationally feasible. Modules were identified by combining motifs whose cooccurrence was enriched in the foreground sets. We measured enrichment of modules using the balanced-error rate (analogous to that of motifs), and we required that each motif in a module contributes significantly to the enrichment of the module as a whole. To test significance, we randomly selected 100 of the 1000 foreground/background pairs used to evaluate individual known motifs, and performed the module identification procedure on each of the 100 selected pairs. The best balanced-error rates for modules that are entirely composed of known motifs (called known modules) in random human and mouse sets were 0.3145 and 0.304, respectively. We used these balanced-error rates as an estimate of the critical value for P<0.01. We opted for using P-value cutoffs instead of computing q-values because accurate q-value estimation for modules is computationally prohibitive. Top known modules in human kidney (0.2955), liver (0.3105), pancreas (0.3125) and testis (0.3) scored better than the cutoff, as did top known modules in mouse kidney (0.3005), liver (0.3025) and testis (0.2945). Supplementary Material Supplementary information

Dev_Genes_Evol-4-1-2292471

Hedgehog signaling pathway function conserved in Tribolium segmentation

In Drosophila, maintenance of parasegmental boundaries and formation of segmental grooves depend on interactions between segment polarity genes. Wingless and Engrailed appear to have similar roles in both short and long germ segmentation, but relatively little is known about the extent to which Hedgehog signaling is conserved. In a companion study to the Tribolium genome project, we analyzed the expression and function of hedgehog, smoothened, patched, and cubitus interruptus orthologs during segmentation in Tribolium. Their expression was largely conserved between Drosophila and Tribolium. Parental RNAi analysis of positive regulators of the pathway (Tc-hh, Tc-smo, or Tc-ci) resulted in small spherical cuticles with little or no evidence of segmental grooves. Segmental Engrailed expression in these embryos was initiated but not maintained. Wingless-independent Engrailed expression in the CNS was maintained and became highly compacted during germ band retraction, providing evidence that derivatives from every segment were present in these small spherical embryos. On the other hand, RNAi analysis of a negative regulator (Tc-ptc) resulted in embryos with ectopic segmental grooves visible during germband elongation but not discernible in the first instar larval cuticles. These transient grooves formed adjacent to Engrailed expressing cells that encircled wider than normal wg domains in the Tc-ptc RNAi embryos. These results suggest that the en–wg–hh gene circuit is functionally conserved in the maintenance of segmental boundaries during germ band retraction and groove formation in Tribolium and that the segment polarity genes form a robust genetic regulatory module in the segmentation of this short germ insect. Introduction The ontogenic stage at which the body plans of animals belonging to the same phylum reach maximum morphological similarity is called the phylotypic stage. In insects and other arthropods, the phylotypic stage is the elongated germband at which the three germ layers have formed and segments are morphologically evident along the entire anterior–posterior axis (Sander 1997). In Drosophila, segment polarity genes, most of which are components of two major signal transduction pathways (the Wingless and Hedgehog signaling pathways), control the formation of grooves between segments and anterior–posterior patterning within each segment. Initially, pair-rule genes activate engrailed (en) expression at the anterior boundary of each parasegment and wingless (wg) at the posterior boundary DiNardo and O'Farrell (1987; Howard et al. 1988; Ingham et al. 1988). Engrailed protein activates expression of hedgehog (hh), which encodes a secreted protein that signals to surrounding cells (Hidalgo and Ingham 1990; Ingham and Hidalgo 1993). Hh signaling leads to the continued activation of wg, whose secreted protein product is necessary for the continued activation of en. This positive feedback loop ensures the interdependence of these three genes for the maintenance of each other’s expression until embryonic stage 9–10 (Forbes et al. 1993). At the end of stage 10, en expression becomes independent of wg, and it is around this stage that segmental boundaries are morphologically visible at the posterior edge of cells expressing en and hh. Evidence that this segment polarity network might be conserved is primarily based on the expression patterns of en, wg, and more recently, Hh pathway component genes in other insects as well as non-insect arthropods and annelids (Patel et al. 1989; Brown et al. 1994; Kraft and Jackle 1994; Nagy and Carroll 1994; Grbic et al. 1996; Peterson et al. 1998; Damen 2002; Dhawan and Gopinathan 2003; Simonnet et al. 2004). There is also limited data suggesting this network is functionally conserved among insects. Ectopic expression of Drosophila wg in Tribolium induces ectopic en in the anterior half of the parasegment suggesting functional conservation of the wg–en interaction (Oppenheimer et al. 1999). wg and/or en have been implicated by RNAi analyses in the proper formation of segmental boundaries in the milkweed bug Oncopeltus faciatus (Angelini and Kaufman 2005), the honey bee Apis mellifera (Beye et al. 2002), the blowfly Lucilia sericata (Mellenthin et al. 2006) and the beetle Tribolium (Ober and Jockusch 2006). While RNAi analysis of wg and hh in the cricket Gryllus (Miyawaki et al. 2004) failed to reveal significant effects on segmentation, RNAi analysis of armadillo, a wg pathway component, does implicate wg signaling in segmentation in this short germ insect. In the long germ embryo of Drosophila, where all segments initiate virtually simultaneously, loss of any one of these three genes destabilizes the expression of the others and they eventually fade, resulting in shorter embryos that, in addition to the loss of segmental grooves, also show misspecified epidermal cell fates (Ingham et al. 1991; Forbes et al. 1993). We have investigated genes that encode the Hh-signaling pathway components hedgehog (hh), patched (ptc), smoothened (smo), and cubitus interruptus (ci) to determine whether they function with en and wg in the formation of segmental boundaries in this short germ band insect. The Hh-signaling pathway is well-conserved between insects and vertebrates (Huangfu and Anderson 2006) and is thus likely to be conserved in Tribolium. The main components were first elucidated in Drosophila, where Hh is secreted by cells in the posterior compartment of embryonic segments and larval imaginal discs. It diffuses to the anterior compartment (Lee et al. 1992; Tabata et al. 1992; Tashiro et al. 1993) where the signal is controlled by two membrane proteins: Patched (Ptc), a twelve pass transmembrane protein (Hooper and Scott 1989; Nakano et al. 1989) and Smoothened (Smo), a seven pass transmembrane protein (Alcedo et al. 1996; van den Heuvel and Ingham 1996). In the absence of Hh signal, Ptc represses Smo activity (Chen and Struhl 1996). Signaling is initiated by binding of Hh to its receptor Ptc, which relieves this repression and allows Smo to signal to a multimeric complex inside the cell. This complex is composed of the serine threonine kinase Fused (Alves et al. 1998), the kinesin related protein Costal-2 (Sisson et al. 1997), a novel cytoplasmic protein Suppressor of fused (Monnier et al. 1998) and a zinc finger transcription factor Cubitus Interruptus (Ci; Motzny and Holmgren 1995). In unstimulated cells, this complex sequesters ci, inhibits nuclear import of the full-length 155 kDa protein and promotes its cleavage to generate an N-terminal 75-kDa form containing the Zn finger DNA-binding domain, which can enter the nucleus and repress transcription of Hh target genes (Aza-Blanc et al. 1997). When Hh signal is transduced, activation of Smo inhibits ci cleavage and activates the full-length protein, which then translocates to the nucleus, resulting in the transcription of Hh-responsive target genes including wg, ptc, gooseberry, and decapentaplegic (Alexandre et al. 1996; Dominguez et al. 1996; Hepker et al. 1997; Ingham and McMahon 2001). Consistent with reports on other arthropods, we found that the expression patterns of hh, ci, smo, and ptc were largely conserved in Tribolium. Using RNAi to study the function of these genes during segmentation in Tribolium, we followed embryonic development in these embryos using En as a marker of segment development and integrity. When the Hh signal was depleted by RNAi, segments were specified normally in the posterior growth zone and the embryos elongated as fully as wild-type. En and wg expression in these embryos, although properly initiated, was not maintained and defects appeared during germ band retraction, resulting in tiny, sphere-shaped embryos lacking segmental grooves. On the other hand, overactivation of the pathway by ptc RNAi produced embryos with transient ectopic segmental grooves and embryonic cuticles with enlarged heads and thoracic appendages. All together, these results indicate that Hedgehog signaling is an essential component of the segment polarity network in Tribolium, which is necessary to maintain segmental integrity during germband retraction after the segments have been enumerated in the growth zone. The conserved function of an en–wg–hh gene circuit during segmentation suggests that the segment polarity genes constitute a robust gene regulatory module in this short germ insect. Materials and methods Beetle husbandry Tribolium castaneum strain GA-1 was reared in whole wheat flour supplemented with 5% dried yeast at 30 C (Beeman et al. 1989). Identification of hedgehog pathway component genes in the Tribolium genome Partial cDNAs of tc-hh, tc-ci, tc-smo and tc-ptc, cloned into the pCR4TOPO vector (Invitrogen), were obtained from Y. Tomoyasu. Orthologs of each gene were identified in the annotation of the Tribolium genome (the Tribolium genome consortium, in review). The sequences of the partial cDNAs matched those deduced from the gene models with minor differences. hh, glean gene number tc01364 or NCBI mRNA accession number XM961615, is located on LG 2; smo, TC05545 or XM966834, is on LG 8; ci, TC03000 or XM965017, is on LG 3 and ptc, TC04745 or XM962700 is on LG 1 = X. In situ hybridization and immunostaining Whole mount in situ hybridizations were performed according to established protocols (Tautz and Pfeifle 1989). Expression of Engrailed in Tribolium embryos was determined using the α-Invected antibody, 4D9 which cross-reacts with Tc-En (Brown et al. 1994). Double staining for the different mRNAs in addition to En protein was performed simultaneously according to the protocol of Nagaso et al. (2001). RNA interference (RNAi) Templates for dsRNA synthesis were amplified as described (Tomoyasu et al. 2007). Double stranded RNA was synthesized using the T7 MEGAscript kit (Ambion) and purified using the MEGAclear kit (Ambion). Different amounts of dsRNA (Table 1) were mixed with injection buffer (5 mM KCl, 0.1 mM KPO4 pH 6.8) prior to injection. Parental RNAi was performed and affected embryos were analyzed as previously described (Bucher et al. 2002). Microscopy and imaging Stained embryos and larval cuticles were documented with a Nikon Digital DXM 1200F camera on an Olympus BX50 microscope using Nikon ACT-1 version 2.62 software. Brightness and contrast of all images were adjusted and some were placed on a white background using Adobe Photoshop 7.0.1 software. Table 1 Summary of RNAi effects Gene DsRNA μg/μl Class I (%) Class II (%) Class III (%) Undeveloped (%) Wild type (%) Totals (n) Tchh 3.0 0 6 83 11 0 464 1.5 18 73 0 9 0 477 Tcsmo 3.0 0 0 89 11 0 579 1.5 0 43 43 14 0 477 0.75 38 44 0 20 0 230 Tcci 3.0 0 33 61 5 0 694 1.5 0 30 63 7 0 405 0.75 73 19 0 8 0 293 Tcptc 4.0 0 76 – 24 0 271 2.0 13 68 – 19 0 189 Embryos were collected every 48 h for the first 2 weeks after injection. Phenotypic classes are unique for each gene. See text for details. Percentages have been rounded up. Results Expression patterns of Tc-hh, Tc-smo, and Tc-ci Tc-hh transcripts are first detected in the presumptive head lobes on either side of the ventral mesoderm (arrowhead in Fig. 1a) and at the posterior end of the embryo. As the embryonic rudiment condenses, faint stripes of Tc-hh expression appear immediately posterior to the intense stripes in the head lobes and in the presumptive mandibular segment (arrowheads in Fig. 1b). Gnathal and trunk stripes appear in an anterior to posterior progression (Fig. 1c). Double staining for En expression revealed that Tc-hh and Tc-En are coexpressed in cells of the posterior compartment in each segment (Fig. 1d). During germband elongation, expression at the posterior end of the embryo resolves into spots on either side of the mesoderm and eventually into a ring surrounding the proctodeum (Fig. 1b–d and arrowhead in f). In the head, twin spots appear on either side of the presumptive stomodeum. As the head lobes mature, expression in the anterior region of the developing stomodeum increases (Fig. 1c,d and black arrowhead in e), while the anterior-most stripes of Tc-hh expression resolve into spots in the brain that overlap with the Tc-En-expressing cells (Fig. 1c,d and blue arrowhead in e). Expression at the posterior end of the embryo continues throughout germ band extension (Fig. 1b,d and f) eventually surrounding the proctodeum as it invaginates (Fig. 1f). As segments mature, Tc-hh expression at the ventral midline clears and Tc-hh is not expressed in the CNS. In total, there are three gnathal, three thoracic, and ten abdominal Tc-hh stripes with additional expression in the stomodeum, proctodeum, antennae, and brain (Fig. 1d). Fig. 1Expression of Tc-hh, Tc-ci, and Tc-smo in Tribolium during segmentation. In these ventral views, anterior is to the left. a–cTc-hh; d–fTc-hh (purple) and Tc-En (gold); g–hTc-smo; i–kTc-ci; l, mTc-ci (purple) and Tc-En (gold); a Anterior stripes of Tc-hh (arrowhead) in the presumptive head lobes of a blastoderm embryo. Expression at the posterior end of the embryo is not in the plane of focus. b Weak stripes appear in the antennal and mandibular segments (arrowheads) posterior to the dark stripes in the head lobes. Twin spots of expression flank the mesoderm at the posterior end of the embryo and near the stomodeum. c In addition to expression in the head lobes and antennae, three gnathal stripes and one trunk stripes appear in an elongating germband embryo. d Coexpression of Tc-En (gold) and Tc-hh (purple) in the posterior compartment of each segment. Note Tc-En expression at the ventral midline in the absence of Tc-hh. e–fTc-hh expression in the stomodeum and the proctodeum (arrowheads) in the absence of Tc-En. Expression of Tc-hh in the head lobes has resolved into spots that overlap Tc-En expression (blue arrowhead). g Ubiquitous expression of Tc-smo in an early germ band embryo. h In an elongating germ band embryo, Tc-smo expression appears ubiquitous with some segmental modulation (arrowheads). i Expression of Tc-ci at the anterior edge of the head lobes (arrowhead) of a young germband embryo. j Gnathal stripes appear first in a slightly older embryo. Expression throughout the posterior region of the embryo fades anteriorly. k Wide Tc-ci stripes in the head lobes and antennal segments as well as in the gnathal and thoracic segments of an elongating germ band embryo. l Tc-En (gold) expression does not overlap Tc-ci (purple) expression in cells of the anterior compartment of each segment. m Enlarged view of a few thoracic segments from the embryo shown in l reveals a gap of two or three rows of cells anterior to the Tc-En stripes that do not express Tc-ci (arrowhead) Tc-smo is expressed ubiquitously in the germband (Fig. 1g) in Tribolium. As the germ band elongates, expression appears to modulate slightly within each segment (arrowheads in Fig. 1h). smo transcripts are expressed in a similar manner in Drosophila (Alcedo et al. 1996). Tc-ci expression is first detected at the anterior edge of each head lobe in the early embryonic rudiment (arrowhead in Fig. 1i). Dynamic expression of Tc-ci in the head lobes eventually resolves to a wedge of cells in the lateral eye field (Fig. 1j). Expression is detected in the labrum (Fig. 1l) and in a broad posterior region, which fades toward the anterior (Fig. 1j). As the germ band elongates, Tc-ci is expressed in a broad stripe in every segment in an anterior to posterior progression (Fig. 1i–k). Double staining for Tc-En indicates that Tc-ci is expressed in the anterior compartment where the anterior-most Tc-ci expressing cells are immediately posterior to the Tc-En-expressing cells of the preceding segment (Fig. 1m). Segmentally reiterated stripes of ci expression are also observed in the mulberry silkworm Bombyx mori (Dhawan and Gopinathan 2002), the spider Cupiennius salei (Damen 2002), and the millipede Glomeris (Janssen et al. 2004), suggesting that the segmental expression of ci in the anterior region of each segment is conserved among arthropods. Closer examination of Tc-ci expression revealed a gap of two or three rows of cells immediately anterior of the Tc-En-expressing cells that do not express Tc-ci (Fig. lm). In contrast, expression of ci throughout the anterior compartment of each segment in Drosophila is thought to be essential to the function of the Hh-signaling pathway in maintaining wg expression (Orenic et al. 1990; Hepker et al. 1997). Expression of ci immediately anterior to En-expressing cells is conserved in the spider (Damen 2002), but has not been reported for Bombyx or Glomeris. Thus, it is not clear whether this unusual expression pattern is unique to Tribolium. RNAi analysis of Tc-hh, Tc-smo, and Tc-ci The conserved expression patterns of Tc-hh and Tc-smo are consistent with the hypothesis that the Hh function in segmentation is conserved in Tribolium, but the lack of Tc-ci gene expression in cells immediately anterior to Tc-hh is not. To determine whether the Hh-signaling pathway is required for proper segmentation, we performed parental RNAi analysis of these Hh pathway components. Three different amounts of Tc-hh and Tc-smo dsRNA were injected, which uncovered a range of hypomorphic phenotypes (Table 1). Similar RNAi phenotypes were produced for both genes, and the RNAi cuticles were classified into three different categories (Class I, II, and III) based on severity as listed in Table 1. Regardless of the severity, none of the RNAi embryos hatched and the cuticle had to be dissected out of the vitelline membranes. Class I cuticles display the weakest phenotypes, in which the head is severely reduced and contains only rudimentary limb structures. The legs are present but slightly warped, and segmental grooves in the abdomen are occasionally fused but all eight abdominal segments are present (Fig. 2b (Tc-hh), f (Tc-smo)). Class II cuticles are small and spherical, with a large protuberance at the anterior end and three small warped pairs of limbs. The presence of the spiracle on the second thoracic segment (arrowhead in Fig. 2c and g) allowed us to identify these structures as rudimentary legs. The small amount of cuticle posterior to these small warped legs is smooth and lacked any abdominal features (Fig. 2c (Tc-hh), g (Tc-smo)). The most severely affected embryos, Class III, produced small spherical cuticles with a large protuberance at the anterior end and no obvious head structures or thoracic limbs (Fig. 2d (Tc-hh), h (Tc-smo)). These cuticles are very smooth on all sides, with no sign of grooves and are quite small relative to the size of similarly aged wild-type cuticles (Fig. 2a Tc-hh, e Tc-smo). The head and gnathal appendages appear to be more sensitive to the depletion of Tc-hh or Tc-smo than the legs, as even in the weakly affected individuals the head, including gnathal segments, failed to form properly (Fig. 2b,f). Fig. 2Tc-hh, Tc-smo, and Tc-ci RNAi phenotypes in first instar larval cuticles. a–dTc-hh RNAi; e–hTc-smo RNAi; i–lTc-ci RNAi. a Comparison of a severely affected small spherical Tc-hh RNAi cuticle and a wild-type cuticle at the same magnification. b Lateral view of a mildly affected Class I cuticle containing reduced gnathal appendages, normal legs and all abdominal segments. c Ventral view of a Class II cuticle that is similar in size and shape to more severely affected Class III cuticles but retains three pairs of thoracic appendages. Note spiracle on second thoracic segment (arrowhead). d Ventral view of a small spherical Class III cuticle with rudimentary head structures, no appendages and no segmental grooves. e Comparison of a severely affected small spherical Tc-smo RNAi cuticle and a wild-type cuticle at the same magnification. f Class I cuticle with highly reduced head structures and mouthparts but a full complement of body segments. g Class II cuticle with three pairs of legs and rudimentary head structures. Note spiracle on second thoracic segment (arrowhead). h A severely affected Class III Tc-smo RNAi cuticle phenotypically similar to the most severely affected Tc-hh RNAi cuticle. i Comparison of a severely affected small spherical Tc-ci RNAi cuticle and a wild-type cuticle at the same magnification. j Mildly affected Class I cuticle with reduced head structures, three pairs of legs and mildly fused abdominal segments. k Class II cuticles are slightly longer than intermediate Tc-hh or Tc-smo RNAi cuticles with highly fused abdominal segments. l A severely affected Class III Tc-ci RNAi spherical cuticle with three pairs of legs The phenotypes of Tc-ci RNAi embryos are not as severe as those observed for Tc-hh or Tc-smo. The most weakly affected, Class I Tc-ci RNAi cuticles (Fig. 2j), are shorter and fatter than wild-type with severely reduced heads, but contain all thoracic and abdominal segments. The Class II Tc-ci RNAi cuticles are also shorter than wild-type with a protuberance at the anterior end and fairly normal thoracic limbs, but little to no sign of segmental grooves in the abdomen (Fig. 2k). Similar to Class III RNAi embryos of Tc-hh and Tc-smo, Class III Tc-ci RNAi embryos produced smooth unsegmented cuticles lacking heads and gnathal appendages (Fig. 2l) that are considerably smaller than wild-type cuticles (Fig. 2i). Unlike Tc-hh and Tc-smo Class III RNAi embryos, they still produced fairly normal legs (Fig. 2l). Interestingly, loss of function mutants of ci in Drosophila also produce milder limb phenotypes than do hh mutants (Methot and Basler 1999). To understand how the segmentation process is affected by loss of Hh signaling, we followed Tc-En expression during elongation and retraction in Class III RNAi embryos (Fig. 3). In RNAi embryos for all three genes, segmental stripes of Tc-En initiate normally during germ band elongation (blue arrowheads in Fig. 3a,d and g). Tc-En expression fades as the segments matured (black arrowheads Fig. 3a,d and g), suggesting that Hh signaling is required for the maintenance of Tc-En. However, Tc-En expression in cells along the midline, presumably in the developing CNS (wild-type, Fig. 3i), is maintained in the RNAi embryos, which allowed us to follow segmentation in RNAi embryos. Tc-hh, Tc-smo, and Tc-ci RNAi embryos completed elongation more or less normally (Fig. 3a,d and g), but abnormalities became evident during retraction. The head lobes fail to mature, and there is no evidence of antennal Tc-En stripes. There is also a protuberance at the anterior end of the embryo that is likely to correspond to the anterior protuberance seen in the RNAi cuticles (Fig. 2d,h and l, and arrow in 3 h). In embryos that completed retraction, the unsegmented germbands are highly compacted, and Tc-En expression pattern is very irregular (Fig. 3c,e and h). Compared to wild-type germ bands at a similar stage, these germ bands occupy only a portion of the egg (Fig. 3j,k). In addition, loss of early Hh signaling at the ventral midline affected cell fate, as indicated by the loss of Tc-En expression here (Fig. 3b,e). Loss of Hh signal in Drosophila similarly affects En expression in midline cells (Bossing and Brand 2006). We also found that Tc-wg expression was initiated but not maintained in these embryos (data not shown), suggesting Tc-wg is a target of the Hh pathway. Fig. 3En staining in wild-type and severely affected Tc-hh, Tc-smo and Tc-ci RNAi embryos during elongation and retraction. a–cTc-hh RNAi; d–f, kTc-smo RNAi; g, hTc-ci RNAi; i, j wild-type. a Tc-En expression initiated normally in the posterior segments of an elongating Tc-hh RNAi germband embryo (blue arrowhead) but did not persist in older, more anterior segments (black arrowhead). b During germband retraction, patches of Tc-En expression are visible near the ventral midline, and laterally in the posterior segments. c In a highly compacted germ band with fused segments, Tc-En expression is disrupted along the ventral midline. d Expression of Tc-En in an elongating Tc-smo RNAi embryo initiated normally (blue arrowhead) but failed to persist in older, more anterior segments (black arrowhead). e During germband retraction, Tc-En is expressed in patches along the ventral midline, but has faded laterally. f In a highly compacted germ band, persists in a disrupted pattern along the ventral midline. g In an elongating Tc-ci RNAi embryo, similar to Tc-hh and Tc-smo RNAi, Tc-En initiated normally (blue arrowhead) but is not maintained (black arrowhead). h Tc-En expression at the ventral midline of a retracted germband embryo. Note the extended stomodeum (arrowhead) near the anterior end. i Tc-En expression in the CNS along the ventral midline and in the lateral ectoderm of each segment of a wild-type embryo during germband retraction. j Lateral view of a wild-type embryo inside the vitelline membrane at the end of germ band retraction. Tc-En expression persists laterally to the edge of the germband. k Ventral view of a severely affected Tc-smo RNAi embryo located near one end of the egg hedgehog was first isolated in a screen for mutations that disrupt the Drosophila larval cuticle pattern and identified as one of the segment polarity genes with a ‘lawn of denticles’ phenotype (Nusslein-Volhard and Wieschaus 1980). In segment polarity mutants (e.g., wg, hh, and smo), deletion of a portion of the larval epidermis in each segment is accompanied by a mirror image duplication of the remaining structures. As a result, they contain the normal number of segments, but are smaller than wild-type due to partial deletion of each segment. In our study, we found Tc-hh and Tc-smo RNAi embryos to be smaller in size than the wild-type Tribolium larva (Fig. 2a,e), which may be due to cell death, fusion, or failure of cell division in segments during germ band retraction. The few random bristles produced in severely affected RNAi cuticles do not have any definable polarity suggesting that, unlike Drosophila, loss of function of these genes in Tribolium does not appear to produce mirror image duplications or affect polarity within the segments. Analysis of Tc-ptc and overactivation of the Hh pathway Transcripts of Tc-ptc are first detected in a broad domain in the posterior regions of the head lobes in the embryonic rudiment encompassing the antennal segments and the stomodeum, and at the posterior end of the embryo (Fig. 4a). Broad segmental stripes of Tc-ptc appear in an anterior to posterior progression during germband elongation (Fig. 4b). Expression fades in the middle of each initial stripe resulting in two narrow Tc-ptc stripes per segment (Fig. 4c). Double staining with Tc-En and Tc-ptc indicates that in each segment, one of the narrow Tc-ptc stripes marks the anterior boundary of a segment while the other is located immediately anterior to Tc-En-expressing cells (Fig. 4c and arrowhead in inset). This pattern persists even after germ band retraction; the anterior stripe appears stronger than the posterior stripe in each segment. ptc expression during segmentation in Drosophila is similarly dynamic (Nakano et al. 1989; Hidalgo and Ingham 1990). Fig. 4Analysis of Tc-ptc in Tribolium.a–cTc-ptc expression in wild-type embryos; d–fTc-ptc RNAi cuticles; g wild-type; h–mTc-ptc RNAi. a Expression of Tc-ptc in three gnathal stripes, in a broad posterior region of the head lobes and in the posterior region of an early germ band embryo. b Segmental stripes appear sequentially during elongation and resolve in double stripes. c Tc-En (gold) expression in the posterior compartment abuts, but does not overlap the double Tc-ptc stripes (extent denoted by black lines) in the anterior compartment of each segment. Inset: enlarged view of Tc-ptc stripes adjacent to a stripe of Tc-En (arrowhead). d A mildly affected Class I Tc-ptc RNAi cuticle with deformed legs and head appendages. e A more severely affected Class II cuticle with enlarged misshapen legs but a normal complement of abdominal segments. f Comparison of a Class II Tc-ptc RNAi cuticle and a wild-type cuticle at the same magnification. g Expression of Tc-En (gold) and Tc-wg (purple) in a wild-type embryo during elongation. h Segmental expression of Tc-En and Tc-wg initiated fairly normally in a Tc-ptc RNAi germband. Tc-wg expression domains in the head lobes and around the proctodeum are expanded. i Expanded expression of Tc-wg in a slightly older embryo. j Ectopic stripes of Tc-En expression (blue arrowhead) in a germband undergoing retraction. k Ectopic expression of Tc-En at the ventral midline (arrowhead) in a close up of the embryo in h. l Transient grooves (arrowheads) around expanded Tc-wg expression domains in a close up of the embryo in i. m Ectopic Tc-En stripes (blue arrowhead) between each set of normal Tc-En stripes (black arrowhead) in a close up of the embryo in j. T1, first thoracic segment As discussed above, Ptc is a negative regulator of the Hh-signaling pathway in Drosophila. Depletion of a negative regulator of the pathway would ectopically activate the pathway. To understand what happens when the Hh pathway is overactivated in Tribolium, we performed parental RNAi using two different amounts of Tc-ptc dsRNA (Table 1). The resulting cuticles (Fig. 4d,e,f) are very different from the ones described above for the other three genes. In mildly affected embryos (Class I), the head appendages are misshapen, while the legs are relatively normal (Fig. 4d). In the most severely affected embryos (Class II), all segments are present, but the head and thoracic appendages are enlarged and misshapen (Fig. 4e,f). To understand the phenotype of the Tc-ptc RNAi embryos, we examined the expression of Tc-wg and Tc-En. In Tc-ptc-depleted embryos, segmental expression of Tc-En and Tc-wg is initiated normally (Fig. 4h), although the domains of Tc-wg expression in the head and at the posterior end of the embryo are expanded. Closer inspection revealed ectopic Tc-En expression at the ventral midline (Fig. 4k). In slightly older embryos, Tc-wg is expressed more intensely than normal in enlarged domains (Fig. 4i,l). Cells surrounding the expanded Tc-wg domains appear to be invaginating, as if beginning to form grooves. Closer inspection revealed a row of non-Tc-wg-expressing cells between the Tc-wg domain and the grooves (Fig. 4l). Tc-En is expressed in ectopic stripes that, in combination with the normal Tc-En stripes, would surround the expanded Tc-wg domains (Fig. 4j,m). In addition to the normal grooves that form posterior to the normal Tc-En stripe, ectopic grooves initiate anterior to the ectopic Tc-En stripes. Thus, overactivation of the Hh-signaling pathway in Tribolium leads to overexpression of Tc-wg, and ectopic expression of Tc-En, which results in ectopic groove formation. In Drosophila, binding of Hh to Ptc receptor relieves repression of wg and allows expression of target genes. In Drosophila ptc mutants, ectopic induction of wg and En result in the formation of extra grooves (Nakano et al. 1989), suggesting that the functional role of Ptc is highly conserved between these two species. However, unlike Drosophila, the ectopic grooves in Tribolium are transient and it appears that a late regulatory action restores the normal number of segments in this insect. Discussion Expression patterns suggest that the function of the Hh-signaling pathway is conserved in several developmental pathways in Tribolium. In Tribolium, the expression patterns of the four Hh-signaling pathway components we examined are highly similar to those of their Drosophila counterparts. In both Drosophila and Tribolium, the domains of Tc-hh are very similar to those of Tc-En. However, there are some significant differences in their expression dynamics. For example, the antennal Tc-En stripes appear after the three gnathal Tc-En stripes, whereas antennal Tc-hh stripes appear before the gnathal Tc-hh stripes. Each metameric stripe of Tc-hh is laterally continuous across the width of the germband after mesoderm invagination. Later, Tc-hh expression has disappeared at the ventral midline in anterior segments, while in posterior segments the stripes are still continuous. In contrast, Tc-En expression continues in the CNS after Tc-hh expression has faded there. Tc-hh expression in the stomodeum and at the posterior end of the blastoderm embryo in the absence of Tc-En suggests that, similar to hh in Drosophila (Lee et al. 1992; Mohler and Vani 1992; Tashiro et al. 1993), Tc-hh is involved in some En-independent processes in these regions. In both Drosophila and Tribolium, expression of segment polarity genes is initiated by pair-rule genes (recently reviewed by Damen 2007). Soon thereafter, their expression is controlled by interactions between the segment polarity genes themselves. In Drosophila, Ptc is constitutively active unless or until Hh represses Ptc activity. In the absence of Hh, unbound Ptc keeps the pathway switched off. One of the targets of the Hh pathway is ptc itself. In cells where its activity is antagonized upon binding of Hh, ptc continues to be expressed, but ptc expression disappears in cells that do not receive the signal. Thus, an initially broad domain of ptc expression resolves into two narrow stripes flanking the En expression domain. Tc-ptc expression pattern is similar: each broad stripe splits into two, such that each En stripe is bracketed by two Tc-ptc stripes, suggesting that Tc-ptc itself might also be a target of the Hh pathway in Tribolium. The expression patterns of Hh pathway component genes are highly conserved between Drosophila and Tribolium, except for that of Tc-ci (Fig. 1l,m). In Drosophila, ci transcripts are initially expressed uniformly in the early cellular blastoderm and persist until the end of germ band elongation. At that point, ci expression is directly repressed by En in cells of the posterior compartment in each segment. In Tribolium, expression of Tc-ci is somewhat different in that there is a narrow region of two to three cells between the Tc-ci and En-expressing cells that does not express Tc-ci. In Tribolium, the absence of Tc-ci transcripts in cells just anterior to En-expressing cells might suggest the existence of an En-independent mechanism regulating Tc-ci expression. Alternatively, it is possible that Tc-ci transcripts turn over rapidly in these cells, but the expression pattern is conserved at the protein level. In Drosophila, ci is regulated post-transcriptionally. At stage 11, ci transcripts are localized throughout the anterior compartment of each segment whereas protein levels are lower at the center of each transcriptional stripe and higher in cells that bracket the En-expressing cells (Motzny and Holmgren 1995; Slusarski et al. 1995) much like what we describe here for ptc transcription in Tribolium and previously for Ptc protein levels in Drosophila. It will be interesting to see if Tc-ci is also post-transcriptionally regulated in Tribolium. Functional analysis of Hh pathway component genes supports conserved roles in segment boundary formation. In Drosophila, mutations in genes encoding positive regulators of the Hh pathway including hh, smo, and ci produce smaller than wild-type embryos with asegmental phenotypes (Nusslein-Volhard and Wieschaus 1980). In Tribolium, Tc-hh, and Tc-smo RNAi embryonic phenotypes are nearly identical; both produce highly compacted spherical cuticles with no evidence of appendages or segmental grooves. The most severe Tc-ci RNAi phenotypes are not as severe as those of Tc-hh or Tc-smo RNAi. Interestingly, the ci94 allele in Drosophila is a null allele (Slusarski et al. 1995; Methot and Basler 1999) and these mutant embryos differ considerably from hh mutants. hh mutants are much shorter in length and have a continuous ‘lawn of denticles’ phenotype whereas ci94 mutants are almost normal in size and have alternating naked cuticle and denticle belts on the ventral surface. Target genes of hh are partially derepressed in the absence of ci, producing a ci phenotype that is milder than that of hh (Methot and Basler 2001). An analogous situation has been described for the Wingless signaling pathway in Drosophila where derepression of target genes in the absence of pangolin results in a milder segment polarity phenotype compared to that of wg null mutants (Cavallo et al. 1998; Waltzer and Bienz 1998). In hh and smo mutants, only the repressor function of ci remains, producing the catastrophic phenotype. In contrast, loss of both the activating and repressor forms results in the milder phenotype seen in ci null mutants. In Tribolium, the most severe Tc-ci RNAi phenotype is milder than the most severe Tc-hh RNAi phenotype, suggesting similar regulation of the Hh pathway in the beetle. In Drosophila, loss of Wg or Hh-signaling results in larvae that are smaller than wild-type due, at least in part, to epidermal cell death during and after germband retraction (Martinez-Arias and Lawrence 1985) or a combination of transformation and cell death (Klingensmith et al. 1989). In Tribolium, hh, smo, and ci RNAi individuals are greatly reduced in size compared to the wild-type (Fig. 2). These embryos go through the events of early embryogenesis normally, producing the full complement of segments and initiating Tc-En and Tc-wg expression. Tc-En and Tc-wg expression fade and the segmental remnants become highly compacted along the anterior–posterior axis during retraction. Tribolium embryos lacking Tc-wg also elongate normally but fail to maintain En expression and form shorter than wild-type embryos during retraction (Ober and Jockusch 2006). While it is likely that cell proliferation and programmed cell death both contribute to shaping the embryo in Tribolium, during normal development cells divide randomly throughout the elongating germ band (Brown et al. 1994); organized patterns of cell division or cell death have not been reported. Similarly, it is likely that excessive cell death or the lack of cell proliferation contributes to the severely compacted terminal phenotype of Tc-hh, Tc-smo or Tc-ci RNAi embryos, but closer examination will be required to determine if this is so. In Drosophila ptc mutants, deletion of the midregion of each segment is accompanied by a mirror image duplication of the remaining denticles (Nusslein-Volhard and Wieschaus 1980). In contrast, Tribolium Tc-ptc RNAi embryos, which also display the correct number of segments, are characterized by distended gnathal and thoracic appendages. Although there are some random bristles, we could not identify any noteworthy difference in the polarity of these bristles that could be attributed to a characteristic loss of function phenotype for genes belonging to this class. This suggests that unlike Drosophila, the loss of segment polarity gene function in Tribolium does not result in any morphologically identifiable polarity defect in the cuticle. In the absence of ptc in Drosophila (DiNardo et al. 1988) and Tribolium, En expression is established properly; but later, de novo En stripes appear between the normal stripes. Ectopic expression of En in Drosophila is not due to regulation of ptc by pair-rule factors (DiNardo et al. 1988). In the absence of ptc, the wg expression domain broadens anteriorly. Expanded wg expression induces ectopic En stripes, which cause ectopic groove formation, suggesting that the principal function of Ptc is to repress wg. Similar expression of Tc-ptc in Tribolium, considered with the expanded Tc-wg domains that are surrounded by ectopic Tc-En-expressing cells and the ectopic grooves transiently formed in Tc-ptc RNAi embryos suggest that the role of Tc-ptc is likely to be functionally conserved between Drosophila and Tribolium. However, in Tc-ptc RNAi embryos, ectopic Tc-En is also detected along the ventral midline, a phenotype that has not been described for Drosophila ptc mutants. hh RNAi has been attempted in the orthopteran Gryllus bimaculatus (Miyawaki et al. 2004). Unfortunately, this organism seems to be resistant to hh dsRNA. In the RNAi embryos, the level of hh is not reduced. The embryos develop normally and hatched larvae show no cuticular defects. Lack of ptc analysis in other insects makes it difficult to speculate as to whether this novel expression of Tc-En along the ventral midline is specific to Tribolium, or a general feature related to short germ development. Finally, although ectopic grooves appear to form around the expanded Tc-wg domains, they are not detected in the terminal cuticles, implying that events late in embryogenesis restore the normal number of segmental grooves. Conservation of the hh–wg–en gene circuit in short germ segmentation Several lines of evidence suggest that the segment polarity gene circuit, in which the expression of the hh, wg, and en genes are dependent upon one another in the long germ mode of segmentation elucidated in Drosophila, is conserved in the short germ mode of segmentation found in Tribolium. As in Drosophila, in the absence of smo, hh, ci (this paper), or wg (Ober and Jockusch 2006) in Tribolium, En expression is not maintained. In addition, Tc-wg mRNA fails to persist in Tc-hh, Tc-smo and Tc-ci RNAi embryos (data not shown). Furthermore, expression patterns and RNAi phenotypes of the Hh pathway components we examined (Tc-hh, Tc-smo, Tc-ci, and Tc-ptc) suggest that the regulation of the Hh pathway is also conserved. Segment polarity genes, which function last in the segmentation gene hierarchy, are expressed in segmental fields that have been predefined by genes at higher levels (gap and pair rule). Conservation of the segment polarity gene circuit in Tribolium suggests that that segment polarity genes form a robust regulatory module in the short germ mode of segmentation in this beetle and their expression patterns in numerous insects and chilicerates suggest that this module is likely to be conserved among the Insecta, and perhaps the Arthropoda. While the expression patterns of segment polarity orthologs are highly conserved, the expression patterns of pair-rule gene orthologs vary greatly among insects and other arthropods (recently reviewed in Tautz 2004; Peel et al. 2005; Damen 2007). Functional analysis in Tribolium indicates that interactions among pair-rule gene homologs differ from those of their Drosophila counterparts (Choe et al. 2006) and that some secondary pair-rule genes function in opposite parasegmental registers in Drosophila and Tribolium (Choe and Brown 2006). These findings suggest that the inputs from pair-rule genes to the segment polarity gene module are likely to be quite different in each of these insects. Functional analysis of how pair-rule genes regulate the alternating expression of en and wg in Tribolium will provide insight into these differences, which will ultimately help us understand the evolution of genetic regulatory networks. Interestingly, the segment polarity gene module seems to be resilient enough to withstand such evolutionary changes in input from the pair-rule gene module.

Pediatr_Nephrol-3-1-1994209

Genetic approaches to human renal agenesis/hypoplasia and dysplasia

Congenital abnormalities of the kidney and urinary tract are frequently observed in children and represent a significant cause of morbidity and mortality. These conditions are phenotypically variable, often affecting several segments of the urinary tract simultaneously, making clinical classification and diagnosis difficult. Renal agenesis/hypoplasia and dysplasia account for a significant portion of these anomalies, and a genetic contribution to its cause is being increasingly recognized. Nevertheless, overlap between diseases and challenges in clinical diagnosis complicate studies attempting to discover new genes underlying this anomaly. Most of the insights in kidney development derive from studies in mouse models or from rare, syndromic forms of human developmental disorders of the kidney and urinary tract. The genes implicated have been shown to regulate the reciprocal induction between the ureteric bud and the metanephric mesenchyme. Strategies to find genes causing renal agenesis/hypoplasia and dysplasia vary depending on the characteristics of the study population available. The approaches range from candidate gene association or resequencing studies to traditional linkage studies, using outbred pedigrees or genetic isolates, to search for structural variation in the genome. Each of these strategies has advantages and pitfalls and some have led to significant discoveries in human disease. However, renal agenesis/hypoplasia and dysplasia still represents a challenge, both for the clinicians who attempt a precise diagnosis and for the geneticist who tries to unravel the genetic basis, and a better classification requires molecular definition to be retrospectively improved. The goal appears to be feasible with the large multicentric collaborative groups that share the same objectives and resources. Introduction and definition Congenital abnormalities of the kidney and urinary tract are frequently observed in the first year of life, when they collectively represent a significant cause of morbidity [1] and mortality. Data from birth defects registries [Metropolitan Atlanta Congenital Defects Program (MACDP); California Birth Defects Monitoring Program (CBDMP) [2] indicate an overall frequency from three to six per 1,000 births, and the abnormalities seriously impact life expectancy (http://www.marchofdimes.com). Human urinary tract abnormalities are phenotypically variable and may affect several segments simultaneously, often aggregating to form complex phenotypes. Hence, clinical classification and diagnosis may be difficult. As a consequence of the overlap between anatomical defects, many investigators have opted to group renal and urologic malformations under the single label of Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) [3]. This broad classification is supported by the fact that a mutation in a single gene can have pleiotropic effects on the development of the urogenital tract. For example, mutations in the PAX2 gene cause the renal-coloboma syndrome, but the clinical features of the trait vary significantly between affected individuals, ranging from renal agenesis/hypoplasia to vesicoureteral reflux (VUR) and secondary obstruction [4]. Conversely, mutations in different genes can result in similar renal phenotypes, e.g., EYA1 and PAX2 mutations both can cause the development of hypoplastic kidneys [5]. Hence, improved classification of urinary tract malformations may require understanding of primary molecular defects. A broad but clinically useful diagnostic scheme consists of classifying malformations depending on whether the kidney, the collecting system, or both are affected. This scheme stems from the fact that the upper tract (glomeruli and tubules) is derived from the metanephric mesenchyme (MM), and the lower urinary tract (collecting duct, renal pelvis, ureter) is derived from the ureteric bud [1]. Even if this is in contrast with more recent data about the reciprocal interaction between the ureteric bud and the MM (see below), this classification can be clinically useful to partition patients with different types of urinary tract abnormalities. In this review, we focus on the malformations that primarily involve a reduction of renal parenchyma in the form of renal agenesis and/or hypoplasia/dysplasia, occurring both as isolated forms or in association with other malformations of the lower urinary tract (see below). Primary renal agenesis Bilateral renal agenesis is a rare and fatal event, usually associated with severe oligohydramnios, which produces a characteristic clinical pattern with facial compression and pulmonary hypoplasia (Potter syndrome). An estimate of the incidence of bilateral agenesis is 0.1/1000 births. Unilateral renal agenesis is more common, although the frequency is difficult to estimate, as it is usually clinically silent and is commonly detected as a chance observation by autopsy or by prenatal ultrasound [6]. Primary renal hypoplasia and dysplasia Strictly speaking, renal hypoplasia is defined as a small kidney, which contains intact nephrons that are reduced in number, whereas a dysplastic kidney contains disorganized elements and maldifferentiated tissue. Noninvasive imaging studies such as ultrasounds and dimercaptosuccinic acid (DMSA) scan offer limited information to help distinguish a hypoplastic kidney from a dysplastic one. Unequivocal distinction between these two entities therefore depends on histological examination of renal tissue obtained from kidney biopsy or surgical nephrectomy, which are rarely performed. A further confounding factor is the reduction of kidney size due to chronic injury and scarring from VUR. Most of the time, a DMSA scan helps differentiate primary hypoplasia or dysplasia from small kidneys secondary to VUR. However, a DMSA scan has a low negative predictive value in distinguishing primary hypoplasia or dysplasia from a secondary reduction in kidney size from VUR when scars or areas of negative isotope uptake are present. In practice, the diagnosis of primary renal hypoplasia is favored when the following criteria are satisfied: (a) a reduction of renal size by 2 standard deviations (SDS) from the mean size for the age, (b) exclusion of renal scarring by DMSA scan, and (c) a presence of compensatory hypertrophy of the contralateral kidney. In all cases, the exclusion of renal cysts by ultrasonography is mandatory to avoid confusion with primary renal hypoplasia associated with fibrosis and cysts, nephronophthisis being the most pertinent example. The presence of VUR and/or ureteropelvic junction obstruction (UPJO) does not automatically exclude the diagnosis of hypoplasia, as both conditions are frequently associated with primary renal-size defects. It is clear that this problem is difficult to resolve if the ureteral defect presents ipsilateral to renal hypoplasia. For example, severe antenatal hydronephrosis due to UPJO can determine the involution of the renal parenchyma and lead to an erroneous diagnosis of primary renal agenesis after birth. In bilateral cases, syndromic traits as well as inherited disorders such as medullary cystic kidney disease/nephronophthisis have to be excluded. Unequivocal exclusion of renal dysplasia is usually not feasible except in rare cases for which histology is available. It is possible that in the near future, molecular genetic advances could modify our present understanding and allow for a more direct separation of the two pathological entities based on laboratory tests. These challenges in clinical diagnosis of renal hypoplasia complicate studies attempting to discover new genes underlying this anomaly. For research purposes, we utilize a tentative classification scheme for categorizing our subjects for genetic studies: (1) isolated bilateral hypoplasia/dysplasia, (2) isolated unilateral hypoplasia/dysplasia, and (3) hypoplasia/dysplasia associated with lower tract abnormalities such as VUR or UPJO. Once the genetic basis of different subsets of urinary tract malformations is identified, the classification will likely be retrospectively changed and improved. Kidney development and mouse models The development of mammalian kidney derives from reciprocally inductive events between two tissue compartments of the embryonic metanephros: the ureteric bud (UB), an outgrowth of the nephric duct, and the MM. The ureteric bud invades the metanephric blastema at embryonic day 10.5–11 in the mouse and 35–37 in humans. The MM induces the ureteric bud to grow and branch while the ureteric bud induces the MM to transdifferentiate and form the nephrons’ epithelia (see recent reviews in kidney development in human and mice [7, 8]). In recent years, many factors, specific for either the UB or the MM, have been demonstrated to induce and regulate the epithelial conversion in the mesenchymal cells and the UB branching, leading to the development of the final structure and function of the kidney. Most data constituting the basis of our current knowledge on the topic are based on gene targeting studies in mice (Table 1). A partial list of genes includes protooncogenes RET and Wingless-related 11 (WNT11) that are well recognized UB-specific molecules, whereas glial cell-line-derived neurotrophic factor (GDNF), Wilms tumor 1 (WT1), and Eyes absent 1 (EYA1) represent important examples of MM-specific factors. The paired-box gene 2 (PAX2) appears to be expressed in both structures during kidney development [7, 9]. It is noteworthy that almost half of the genes on the list are transcriptional factors or encode for proteins that are involved in the mesenchymal to epithelial conversion. GDNF signaling through the RET receptor is one of the best studied pathways, representing a critical step in the normal growth and branching of the UB during kidney development [10]. Perturbation of Gdnf/Ret signaling has been shown to be the downstream mechanism underlying impaired nephrogenesis in many other mutant models (e.g., in Gdf11 and Six1 null mice). Numerous factors other than the Gdnf/Ret pathway also participate in kidney and urologic development (e.g. Wnt signaling), as evidenced by the long list of mutant mice with malformations in the kidney and urologic tract (Table 1). Table 1Principal genes targeted in mice leading to renal agenesis, hypoplasia, dysplasiaGeneHuman homologKidney phenotypeReferenceFoxd1FOXD1Small, fused, undifferentiated kidneysHatini et al. [59]Eya1EYA1Absent kidneysJohnson et al. [60]Xu et al. [61]Emx2EMX2Absent kidneysMiyamoto et al. [62]Hoxa11/Hoxd11HOXA11/HOXD11Small or absent kidneysDavis et al. [63]Lhx1LHX1Absent kidneysShawlot and Behringer [64]Pax2PAX2Small or absent kidneysTorres et al. [65]Wt1WT1Absent kidneysKreidberg et al. [66]Agtr2AGTR2Multiple urinary tract malformationsNishimura et al. [67]Bmp4BMP4Altered ureteric bud (UB) branchingMiyazaki et al. [68]Bmp7BMP7Disrupted nephrogenesisDudley et al. [69]Wnt4WNT4Undifferentiated kidneysStark et al. [70]RetRETAbsent kidneys, severe dysgenesisSchuchardt et al. [71]GdnfGDNFAbsent kidneys, severe dysgenesisSanchez et al. [72]Moore et al. [73]Pichel et al. [74]Six1SIX1Absent kidneysXu et al. [75]Six2SIX2Small kidneysSelf et al. [76]Sall1SALL1Absent kidneysNishinakamura et al. [77]Fgfr1/Fgfr2FGFR1/FGFR2Absent kidneysPoladia et al. [78]Slit3SLIT3Small or absent kidneysLiu et al. [79]Pbx1PBX1Small or absent kidneysSchnabel et al. [80]Fgf8FGF8Small kidneysPerantoni et al. [81]Rara/Rarb2RARA/RARB2Small kidneysMendelsohn et al. [82]Lim1LIM1Absent kidneysKobayashi et al. [83] The interdependence between developmental pathways explains why defects in different genes result in similar phenotypes and why morphologic classification of abnormalities alone cannot predict the location or nature of primary defects. Available data thus suggest a large list of candidate genes for human renal and urologic malformations, highlighting the potential for genetic heterogeneity of the trait. Genetic contribution to human renal agenesis/hypoplasia and dysplasia A genetic contribution to the development of renal hypoplasia/dysplasia has been recognized for many years. For the isolated, nonsyndromic renal agenesis/hypoplasia and dysplasia, only segregation studies have been performed, and no loci and/or genes have been mapped so far. Much more is known about rare syndromic forms, for which several genes have been already implicated. Syndromic forms Syndromic forms of renal hypoplasia/dysplasia include rare disorders affecting extrarenal organs such as the eye, the central nervous system, the skin, the limbs, and others. The list of syndromes that include the renal agenesis/hypoplasia/dysplasia phenotype consists of at least 73 clinical conditions (for more details, see Limwongse and Cassidy [11]). Several genes underlying these defects having been identified (Table 2). Renal-coloboma syndrome, orofaciodigital syndrome, branchiootorenal syndrome, renal cysts and diabetes syndrome, and Fraser syndrome are the most frequent syndromes associated with renal parenchymal defects. It seems clinically relevant that the renal abnormalities may represent the first manifestation of the disease, thus requiring a detailed evaluation of other organs. A list of extrarenal signs and symptoms that clinicians should look for to define these syndromes include retinal coloboma [4], deafness, external ear abnormalities including cysts and fistulas [12, 13], anus imperforates and limb and ear anomalies [14], diabetes and renal cystic dysplasia [15], and others. Finally, renal agenesis/hypoplasia is frequently part of chromosomal disorders (Table 3) that must be recognized for genetic counseling. Most common syndromes that should be considered in the initial differential diagnosis are listed in Tables 2 and 3, and we suggest referring to popular Web sites for further details (links provided at the end). Table 2List of human malformation syndromes with kidney hypoplasia/dysplasiaGeneHuman syndromeKidney phenotypeOMIMJAG1, NOTCH2Alagille syndromeMCDK, kidney dysplasia, kidney mesangiolipidosis#118450#610205BBS1-BBS11Bardet-Biedl syndromeRenal dysplasia and calyceal malformations#209900EYA1, SIX1, SIX2Branchiootorenal syndromeRenal agenesis/dysplasia#113650SOX9Campomelic dysplasiaDiverse renal malformations#114290CHD7CHARGE syndromeDiverse urinary tract malformations#214800Del. 22q11Di George syndromeRenal agenesis, dysplasia, VUR#188400GATA3Hypothyroidism, sensorial deafness, renal anomalies (HDR)Renal agenesis, dysplasia, VUR#146255DNA repairFanconi anemiaRenal agenesis#227650FRAS1, FREM2Fraser syndromeRenal agenesis, dysplasia#219000KALL1, FGFR1Kallman’s syndromeRenal agenesis, dysplasia#308700, #147950PAX2Renal coloboma syndromeRenal hypoplasia, MCDK, VUR#120330TCF2Renal cysts and diabetes syndromeRenal dysplasia, cysts#137920GPC3Simpson-Golabi-Behmel syndromeRenal dysplasia, cysts#300209DHCR7Smith-Lemli-Opitz syndromeRenal dysplasia, cysts#270400SALL1Townes-Brocks syndromeRenal dysplasia, lower urinary tract malformations#107480LMX1BNail-patella syndromeGlomerulus malformation, renal agenesis#161200NIPBLCornelia de Lange syndromeRenal dysplasia#122470CREBBPRubinstein-Taybi syndromeRenal agenesis#180849WNT4Rokitansky syndromeRenal agenesis#277000PEX-familyZellweger syndromeRenal dysplasia, cysts#214100GLI3Pallister-Hall syndromeRenal agenesis, dysplasia#146510p57(KIP2)Beckwith-Wiedemann syndromeRenal dysplasia#130650SALL4Okihiro syndromeRenal ectopia with or without fusion, lower urinary tract malformations#607323TBX3Ulnar-Mammary syndromeRenal agenesis#181450MCDK multicystic dysplastic kidney, VUR vesicoureteral refluxTable 3Common chromosomal disorders associated with urinary tract anomaliesChromosomal disordersRenal agenesisHypoplasiaOther associated anomaliesPatau syndrome (trisomy 13)+Holoprosencephaly, midline anomalies, cleft lip/palateMiller-Dieker syndrome (17p13 deletion)+MR, lissencephaly, microgyria, agyria, typical facie, seizuresEdward syndrome (trisomy 18) 18q deletion+IUGR, CHD, clenched hands, rocker bottom feet SS, MR, microcephaly, narrow external ear canals, long handsDown syndrome (trisomy 21)+MR, hypotonia, CHD, typical face, clinodactylyCateye syndrome (tetrasomy 22p)+MR, CHD, colobomas, anal/digital anomaliesVelocardiofacial syndrome (22q11 deletion)++Conotruncal CHD, thymic aplasia, typical face, cleft palateTurner syndrome (45,X or 46,X,i(Xq))++SS, amenorrhea, webbed neck, cubitus valgus, hypogonadismMR mental retardation, IUGR intrauterine growth retardation, CHD congenital heart disease, SS short stature Nonsyndromic forms It is well known that nonsyndromic renal malformations may occur as hereditary traits and can present with familial aggregation. Evidence in favor of a genetic determination of the disease is raised by an increased recurrence risk among first-degree relatives and by several reports of familial occurrence of multiple malformations, including renal agenesis/hypoplasia and dysplasia. The relative recurrence risk of bilateral and unilateral agenesis has been estimated at 4–9% [6, 16, 17]. For familial cases, in most of the pedigrees, the suggested mode of inheritance was autosomal dominant with reduced penetrance, estimated to range between 50% and 90% [16]. For example, a large pedigree with an autosomal dominant mode form of nonsyndromic renal hypoplasia and dysplasia has recently been described [18]. However, a Somalian kindred in which the trait was segregating in an autosomal recessive fashion has been reported [19]. Nevertheless, until recently, no linkage studies in familial renal agenesis/hypoplasia and dysplasia have been reported. Incomplete penetrance, variable expression and the fact that anatomical defects in many family members can be clinically silent, complicate recruitment of large pedigrees that would be suitable for linkage analysis. Strategies for gene discovery Strategies to find genes causing renal agenesis/hypoplasia and dysplasia vary significantly depending on the characteristics of the study population available. Different data sets of patients have potential advantages and possible pitfalls. Candidate gene studies So far, candidate gene studies have been the only alternative to linkage analysis to find genes underlying both Mendelian and complex traits. Such studies have identified many genes causing rare genetic diseases [20] (The Human Gene Mutation Database, http://www.hgmd.cf.ac.uk/ac/index.php) and most of the genes that are known contribute to susceptibility to common diseases [21, 22]. Large cohorts of sporadic cases or small pedigrees can be utilized in case-control association studies to find common disease associated alleles. Such cohorts can also be screened by resequencing of candidate genes to detect rare variants with large effects that account for disease in a small proportion of the patients. Selection of one approach over the other depends on the expected degree of genetic and allelic heterogeneity of the trait under investigation. Genetic heterogeneity refers to the situation where mutations in different genes account for disease in different affected individuals. Allelic heterogeneity refers to the presence of many independent mutations in a given gene. For a trait with high locus and allelic heterogeneity, the search for common disease-contributing alleles is problematic, and resources would be better directed toward comprehensive resequencing of candidate genes to discovery the rare disease-causing variants. In practice, the heterogeneity parameters are difficult to predict a priori. The resequencing approach has been successfully applied to find several genes causing kidney developmental disorders. As an example, mutations in the uroplakin III gene, which produce VUR in mice [23], explain a small fraction of human renal hypodysplasia [24–28]. Similarly, results from the ESCAPE study recently provided the first comprehensive analysis of renal developmental genes in children affected by nonsyndromic renal hypodysplasia, showing a fairly high prevalence of PAX2 and TCF2 mutations [5, 29]. Another success of the candidate gene approach is the latest discovery of mutations in genes of the renin-angiotensin system (RAS) in severe forms of renal tubular dysgenesis [30]. The search for common variants predisposing to nonsyndromic renal hypodysplasia has not been frequently applied. However, these common predisposing alleles may not be recognized until a comprehensive search is undertaken. As an example, a common noncoding variant in a RET enhancer has recently been shown to be a strong risk allele for Hirschsprung disease, explaining the paucity of coding mutations found in families showing linkage to the RET locus [31]. Traditional linkage studies and genetic isolates The genome-wide linkage analysis/positional cloning approach is a time-tested method used to identify disease-causing mutations, and it has been extremely successful in the past few decades for mapping genes that underlie monogenic Mendelian diseases [32, 33]. This approach hinges on availability of single, uniquely large pedigrees that segregate genes with large effect or a large number of small-sized pedigrees. Mutations in genes underlying Mendelian forms of disease usually account for a fraction of sporadic forms (e.g. PAX2 and TCF2). For renal agenesis/hypoplasia and dysplasia, large pedigrees amenable for linkage analysis are very difficult to ascertain because these traits have incomplete penetrance (due to genetic and environmental modifiers). Moreover, many malformations, such as unilateral agenesis can be clinically silent and will not be detected without systematic screening of family members. As for candidate gene studies, locus heterogeneity is another potentially complicating factor that may dilute the power of linkage studies. Our previous data demonstrated that in the setting of reduced penetrance, variable expressivity, and very high genetic heterogeneity, approaches based on a limited number of uniquely large pedigrees or a very large number of medium-sized kindreds, are more likely to be successful to map a disease gene [34]. As a result of these difficulties, no linkage studies of renal agenesis/hypoplasia have been published so far. These kinds of patient cohorts are very arduous to collect and require multicenter collaborative efforts. We have been able to collect seven multigenerational extended pedigrees segregating congenital anomalies of the kidney and urinary tract, including renal agenesis/hypoplasia, as an autosomal dominant trait with reduced penetrance trait. These families allowed us to localize a gene for this trait to a ~7 Mb interval to chromosome 1p32–33 in a setting of genetic heterogeneity [35]. This work represents the first step toward the discovery of a new gene and, possibly, a new pathway, in kidney development. Genetic isolates represent a population structure that can greatly facilitate gene identification efforts. The genetic isolates are populations that are originated from a limited group of founders with little subsequent immigration into the population. Without an inflow of genes, a long period of time would be required for spontaneous mutations to rebuild genetic diversity. Therefore, genetic isolates are likely to harbor few disease-contributing alleles that have been inherited identical by descent from common ancestors [36–38]. These ancestral mutations can be detected by searching for a shared haplotype signature in affected individuals, representing a powerful shortcut to narrow down a linkage interval to a handful of genes. This strategy, called linkage disequilibrium (LD) mapping, has allowed the identification of several genes for Mendelian disorders [39–41]. Hence, the advantages of studying a genetic isolate rely on: (a) a higher prevalence of certain diseases, allowing traits with reduced penetrance to express and show their hereditary component, (b) a more uniform genetic background, thus reducing the genetic heterogeneity, (c) usually good genealogical records, (d) a more uniform environment, and (e) the possibility of speeding up gene discovery through linkage disequilibrium mapping. We have recently characterized a genetic isolate in an Italian valley, in which different glomerular diseases occurred at a much higher prevalence compared with the general population, in apparently unrelated individuals. The genealogical reconstruction allowed us to reconnect most of the patients to a few founders up to the sixteenth century [42]. This study is an example of how an isolate can allow traits that display reduced penetrance and variable expressivity to express their genetic component and represent a first step to find genes causing or predisposing to such diseases. Further investigation of recognized population isolates for developmental disorders, especially renal agenesis/hypoplasia and dysplasia, might help to accelerate gene mapping. Genome-wide association studies The genome-wide association study is an approach aimed at exhaustively covering the genome to look for causative variants. Similar to genome-wide linkage studies, no assumptions are made about either the location of the causative variant or the biological role of the disease gene. Therefore, this approach represents an unbiased method to find disease-causing genes, with also a very high probability of discovering new genes, thus unraveling new pathophysiological pathways. Genome-wide association studies were not feasible until now because of the lack of information about the variability in the human genome and lack of low-cost, high-throughput genotyping technology. This situation has changed in the past 2 years: dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/) now contains about 5 million SNPs, including most of the SNPs with a minor allele frequency higher than 1% estimated to exist in the human genome [43]. Moreover, the HapMap project [44] represents a fundamental advance to performing efficient and successful genome-wide studies through the determination of LD patterns and haplotype blocks across the genome. Another important step has been the tremendous improvement in genotyping technology, with the development of platforms for fast, high-throughput, low-cost SNPs genotyping. Such platforms allow the simultaneous genotyping of 100–500,000 SNPs in a single assay, allowing a dense coverage of the human genome [45, 46]. Some examples of success of this approach have been recently published. For example, genome-wide association studies on patients affected by age-related macular degeneration allowed the individuation of a common variant in the complement factor H as a major risk-associated allele [47, 48]. Similarly, polymorphisms in the transcription factor TCF7L2 have been found to confer risk to type 2 diabetes in different populations [49, 50]. Whether genome-wide association studies will lead to significant discoveries in renal agenesis/hypoplasia and dysplasia is still unclear, but certainly, this approach represents a very promising strategy to identify common variants conferring susceptibility to more frequent, complex traits. Search for structural variations in the genome A number of urogenital malformations are associated with chromosomal abnormalities. For example, a deletion on chromosome 10q26 has been implicated in urogenital development [51]. Similarly, two distinct loci for renal malformations, including VUR, have been mapped to chromosome 13q by deletion mapping using microsatellites in a limited number of affected individuals [52, 53]. Advances in technology, mainly, genome-scanning array technologies and comparative DNA-sequence analyses, have identified a high prevalence of DNA variations that involve segments that are smaller than those recognized by standard cytogenetics techniques [54]. These structural variations are a common feature of our genomic landscape, encompassing deletions, duplications, inversions, and translocations, which range from a few bases up to hundreds of kilobases. These rearrangements comprise benign polymorphisms, as well as deleterious mutations that can disrupt gene structure or affect gene regulation. Newer techniques now allow for the identification of structural variation at the genome-wide level, enabling examination of single patients to rapidly define a chromosomal region (locus) of interest. Several studies have already reported structural variations associated to human disease, leading in some cases to a molecular definition of a disorder before a recognized clinical syndrome [55–57]. These technologies have also been already successfully applied to developmental disorders. A genome-wide search for structural variations using comparative genomic hybridization (CGH) array allowed the discovery of the gene CHD7 as a cause of CHARGE syndrome, a rare, complex disorder in which congenital anomalies affect in a nonrandom fashion several tissues, including the urinary tract [58]. Careful clinical selection of patients and application of genome-wide methods for searching structural variation in renal agenesis/hypoplasia and dysplasia can help find new loci linked to the disease, confirm and narrow loci obtained by linkage analysis, and speed up the discovery of causative genes. Conclusions Renal agenesis/hypoplasia and dysplasia still represents a challenge for both the clinicians who attempt a precise diagnosis and for the geneticists who try to unravel the genetic basis. Genetic and clinical approaches are now converging toward a common goal, which is the discovery of genetic markers, to make the diagnosis of this trait easier. The final objective is to improve classification, to make a reliable prognosis, and to attempt prevention. Based on advances from the last few years, the goal appears to be more feasible with large multicentric collaborative groups that share the same objectives and resources.

Mol_Biochem_Parasitol-1-5-1906845

An approach to classifying sequence tags sampled from Plasmodium falciparum var genes

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) appears to play an key role as both a virulence factor and as a target of naturally acquired immunity [1,2]. This large family of molecules is encoded by the highly polymorphic superfamily of var genes of which there are 60 variants in every genome [3]. A rapidly growing collection of var sequences is now available from clinical isolates around the world [4–11]. Despite immense diversity both in terms of overall organization and primary sequence, the majority of var genes contain a DBL1α region [3]. The existence of short islands of homology within this region has enabled the design of primers that can be used to sample sequence from most var genes to create DBL1α sequence tags [5]. A standard approach to classification of these sequence tags would enable direct comparisons to be made between different studies. However, the extreme diversity of var genes and the fact that they undergo intra-genic recombination [4,12,13], makes this difficult. Despite the high diversity there does appear to be underlying simplicity to the var genes that supports the use of information present in DBL1α sequence tags in making comparisons between the expression levels in different isolates. Analysis of the fully sequenced genome of a single P. falciparum isolate 3D7 suggests that the genomic location of the 60 var genes promotes genetic structuring and the maintenance of genetically distinct sequence types [14–16]. In addition, structural features of the genes within the single genome of 3D7 closely mirrors the range of structural features among collections of DBL1α sequence tags from clinical parasite isolates [9]. We previously used a small number of key sequence features in an algorithm to classify the DBL1α sequence tags from a single geographical location in Kenya into six groups [9] (see Fig. 1A and below). This var tag grouping system, though it is based on portion of the DBL1α domain (see Supplementary information), corresponded well with whole var gene classification based on the whole genome sequence of the parasite line 3d7 [9]. This grouping system appears to be biologically meaningful. Expression of group 2 sequences was strongly associated with the parasite rosetting phenotype in Kilifi whereas expression of group 1 sequences was negatively associated with the repertoire of antibodies to infected erythrocyte surface antigens carried by the patient at the time of disease [9]. Thus DBL1α sequence tags appear to contain useful information about the genes to which they belong that is currently not directly accessible in field studies of clinical parasite isolates. We have developed a rapid approach to performing the classification using text string analysis functions in Microsoft Excel and Perl (see Supplementary files). This classifies sequence tags directly without the need for prior alignment and can be performed on many sequences simultaneously. The approach is summarized in Fig. 1A. The classification is based around a count of the number of cysteine residues within the tag region and a set of sequence motifs at four positions of limited variability (PoLV 1–4) whose positions within the sequence are fixed in relation to four anchor points (a–d, marked with arrows in Fig. 1A). Thus PoLV1 and PoLV4 are fixed in relation to the 5′ and 3′ ends of the sequence, respectively (anchor points a and d). PoLV2 and PoLV3 are fixed in relation to a “WW” motif (anchor point b). The definition of the groups defined by these features is summarized in the box in Fig. 1. Henceforth we will refer to these groupings as cyteine/PoLV groups. This text string analysis approach was tested on the original set of sequences from Kilifi, Kenya [9] and sequences from 9 other studies (see Fig. 1B–E). The sequences were pre-screened to ensure that they contained a 5′DIGDI and 3′PQFLR consensus sequences. Overall 99.6% of sequences could be classified using this approach. This included 100% of sequences from Malawi (J. Montgomery unpublished), Papua New Guinea [7,17], Mali [10], Solomon Islands [7], and The Philippines [7] together with 100% of sequences from one dataset from Brazil [6]. A dataset from Venezuela (52 non-identical sequences [8]) carried two sequences that could not be classified. A dataset from Brazil (137 non-identical sequences, [18]) carried one sequence that could not be classified. The original dataset from Kilifi (878 non-identical sequences [9]) carried two sequences that could not be classified. All five of these sequences lacked WW or VW motifs required as anchor points within the sequence. Part of the rationale for this grouping system came from a search for PoLV motifs that were associated with sequences with distinct length distributions [9]. Two motifs were identified which were independently associated with short sequences. These are MFK* at PoLV1 and *REY at PoLV2 (an asterisk here denotes any amino acid). We hypothesised that if sequences of different length recombine with each other they will generate a wide range of sequences of different lengths whereas genetically isolated sequences, i.e., those that are not recombining with one another are able to maintain distinct distributions in their length. If these groupings are genuine the sequences classified into different groups should have similar lengths in different settings. As shown in Fig. 1B–E, broadly similar distributions of sequence length are observed within the six different groups between three different continents, suggesting that sequences generated in these different studies shared the same set of structural features. Specifically, MFK* (carried at PoLV1 in group 1) and *REY (carried at PoLV2 in groups 2 and 5) are associated with short sequences in each geographical region. No examples of sequences with both MFK* and *REY motifs were found, suggesting that these motifs are mutually exclusive. In addition, though *REY motifs were found in sequences with 2 or 4 cysteine residues (cys2 or cys4), with the exception of a single cys4 (group 4) sequence from the Philippines, MFK* motifs were found exclusively in cys2 (group 1) sequences. Further support for the cysteine/PoLV groupings comes from recent publications. Trimnell et al. found a good correspondence between cysteine/PoLV groupings of cys2 sequences and groups defined phylogenetically within a globally sampled subset of var genes with a specific upstream control region, upsA [11]. Also evident from sequences reported in that study is the fact that DBL1 from two other globally sampled subsets of var genes can be easily distinguished from DBL1 domains from other vars using unique PoLV motifs. var2csa vars have a unique PoLV2 motif “EVIT”, whereas Type3 vars have a unique PoLV4 motif “PPVV” (data not shown). Kraemer et al. have recently performed an analysis and re-classification of whole var genes from 3D7, HB3 and IT4 [19]. Fig. 2A and B summarizes the relationship between the cysteine/PoLV groupings and whole var gene classification. With the exception of group 6 sequences which were not found in HB3 var genes all sequence groups were represented. In all three genomes cysteine/PoLV group 1 sequences are exclusively found in group A var gene and long genes with >5 domains whereas cysteine/PoLV group 5 are found only in non-group A genes and those with 4–5 domains. Cys2 sequence tags (groups 1–3) were never found in group C var genes. Kyriacou et al. used a phylogenetic approach to compare DBL1α sequence tags from Mali [10]. Visual inspection of the layout of these sequences reveals three main groups and a minor group. There was good correspondence between these groups and the cysteine/PoLV groupings (Fig. 2C [10]). This study showed that cys2 sequence tags were more frequent among parasite isolated from children with cerebral malaria than those from children with hyperparasitaemia. However, division of the sequences into cysteine/PoLV groups suggests that the frequency of group 2 sequences is similar in parasites from these two groups of children (Fig. 2D [10]). At a higher level of resolution, the distinct sequence identifier (DSID) (see Fig. 1A) is a potentially useful method of further classifying sequence tags. This consists of a string of sequence features in the form “PoLV1-PoLV2-PoLV3-number of cysteines-PoLV4-sequence tag length”. The DSID captures more of the overall sequence diversity than the previously described “sequence signature” [9] whilst remaining robust to minor changes introduced by sequencing or PCR errors. Among the 1595 non-identical sequences identified in all the studies described here, there were 1111 DSIDs. Fig. 1F–G illustrates the potential usefulness of this approach to classification. In Fig. 1F, 44 “common” sequences that were shared between more than one study were selected. Fishers exact test was used to determine whether these common sequences were shared between two studies more or less than would be expected by chance (+ or − symbols, respectively). Fig. 1G is the same except that the analysis was done at the level of 157 “common” DSIDs that were shared between more than one study. In contrast to Fig. 1F, there was a highly significant similarity between var genes from South American isolates in support a recent study of Amazonian isolates [18]. In contrast to the low overlap between DSIDs from Kilifi and from South America (Fig. 1G) there is considerable overlap in the constituent PoLV motifs themselves (see Supplementary information). This illustrates the potential for recombination to generate diversity from a limited number of sequence blocks [4,12,13]. Since the cysteine/PoLV system of classification is based on commonly occurring sequence features it is hoped that it will useful for initial analysis and annotation, comparison of different geographical regions over time and identification of unusual sequences.

Mol_Genet_Genomics-4-1-2329726

Transcriptomics and adaptive genomics of the asymptomatic bacteriuria Escherichia coli strain 83972

Escherichia coli strains are the major cause of urinary tract infections in humans. Such strains can be divided into virulent, UPEC strains causing symptomatic infections, and asymptomatic, commensal-like strains causing asymptomatic bacteriuria, ABU. The best-characterized ABU strain is strain 83972. Global gene expression profiling of strain 83972 has been carried out under seven different sets of environmental conditions ranging from laboratory minimal medium to human bladders. The data reveal highly specific gene expression responses to different conditions. A number of potential fitness factors for the human urinary tract could be identified. Also, presence/absence data of the gene expression was used as an adaptive genomics tool to model the gene pool of 83972 using primarily UPEC strain CFT073 as a scaffold. In our analysis, 96% of the transcripts filtered present in strain 83972 can be found in CFT073, and genes on six of the seven pathogenicity islands were expressed in 83972. Despite the very different patient symptom profiles, the two strains seem to be very similar. Genes expressed in CFT073 but not in 83972 were identified and can be considered as virulence factor candidates. Strain 83972 is a deconstructed pathogen rather than a commensal strain that has acquired fitness properties. Introduction Urinary tract infection (UTI) is one of the most common infectious diseases in humans and a major cause of morbidity. It is estimated that 40–50% of adult healthy women have experienced at least one UTI episode (Foxman 2002). UTI can be caused either by pathogenic strains leading to symptomatic UTI or by asymptomatic bacteriuria (ABU) strains resulting in a symptom-free carriage resembling commensalism. Escherichia coli is responsible for more than 80% of all UTIs. Acute pyelonephritis is a severe acute systemic infection caused by uropathogenic E. coli (UPEC) clones with virulence genes clustered on “pathogenicity islands” (PAIs) (Eden et al. 1976; Funfstuck et al. 1986; Stenqvist et al. 1987; Orskov et al. 1988; Johnson 1991; Welch et al. 2002). Paradoxically, a large proportion of UTIs are caused by ABU E. coli. Individuals infected with ABU-class E. coli may carry high urine titres of a single E. coli strain for months or years without provoking a host response. Escherichia coli 83972 is a prototype ABU strain and undoubtedly the best-characterised ABU-class E. coli to date. Strain 83972 was originally isolated in the 1970s from a young girl who had carried it for at least 3 years without symptoms (Lindberg et al. 1975; Andersson et al. 1991). The strain is well adapted for growth in the human urinary tract (UT) where it establishes long-term bacteriuria (Hull et al. 2000). It has been used for prophylactic purposes in numerous studies; as such it has been used as an alternative treatment in patients with recurrent UTI who are refractory to conventional therapy (Hull et al. 2000). An ongoing study on patients infected with strain 83972 has so far reported over 50 patient years with no serious side effects (Sundén et al. 2006). ABU patients may carry a single strain for months or years, creating a condition that resembles commensalism, but with a strain that may have evolved from a pathogenic ancestor. Several lines of evidence support the notion that the ancestor of strain 83972 was a pyelonephritic UPEC strain; it belongs to the B2 clonal group, a group associated with pyelonephritis and other extra-intestinal invasive clinical syndromes such as bacteremia, prostatitis and meningitis; the strain also contains gene clusters in various stages of erosion encoding the three UPEC-class fimbriae, i.e. the fim, pap and sfa/foc clusters (Klemm et al. 2006; Roos et al. 2006a). Several studies have investigated the virulence characteristics of uropathogenic E. coli (UPEC) isolates and ABU isolates, in order to get better understanding of how some UTI strains can cause severe disease, while others can be used prophylactically to prevent the same (Blanco et al. 1996; Dobrindt et al. 2003; Vranes et al. 2003; Johnson et al. 2005; Marrs et al. 2005). ABU strains have been shown to lack many of the virulence-associated phenotypes; many of them are nonhaemolytic, nonadherent and lack haemagglutination ability (Vranes et al. 2003). Strain 83972 lacks many of the virulence-associated phenotypes but has been shown to carry many of the virulence-associated genes, such as kps, iutA, fyuA and malX (Dobrindt et al. 2003). However, apart from the fimbrial clusters, the strain has not been sequenced and it is not known which genes it shares with other E. coli isolates, which of the genes in the E. coli “core genome” it carries and which genes it shares with other UTI isolates. Thus far, although a number of UPEC isolates (i.e. CFT073, 536, UTI89 and F11) have been completely sequenced, no genomic sequencing of any ABU strain has been reported. Comparative genomics profiling using microarray chips designed to cover entire genomes is one strategy to obtain information about variability between different strains of the same species and indication of horizontal gene transfer (Willenbrock et al. 2006). DNA microarray-assisted functional genomics provides the global expression profile of a strain, revealing which genes are expressed under certain conditions. Global gene expression profiling of ABU strain 83972 employing the GeneChip E. coli Genome 2.0 Array (Affymetrix), containing four E. coli genomes including that of the UPEC isolate CFT073, will not only provide information regarding the up- and down-regulation of genes comparing different conditions, but will also reveal which genes are actually present (expressed) in the genome of 83972. Bacterial pathogens differ from commensals by expression of specific virulence factors such as those that mediate histological damage. Commensals, in contrast, have generally been regarded as bacteria lacking such virulence factors or other specific mechanisms for interaction with host tissues. Here we compare the global expression profiles of E. coli ABU strain 83972 grown under a number of different in vitro conditions and in three patients in order to get a representative picture of which genes are present/expressed in the genome of this asymptomatic UTI strain. Materials and methods Bacterial strain Escherichia coli 83972 is a prototype ABU strain and lacks defined O and K surface antigens (Lindberg et al. 1975). It belongs to the ECOR group B2 together with many other UTI strains such as the well-characterized and virulent E. coli isolates CFT073, 536 and J96. Growth conditions and stabilisation of RNA for microarray experiments of E. coli 83972 grown on urine agar plates Human urine was collected from four healthy men and women volunteers who had no history of UTI or antibiotic use in the prior 2 months. The urine was pooled, filter sterilised, stored at 4°C, and used within the following day. E. coli 83972 was grown aerated in triplicates in 10 ml of human urine for 6 h. Thereafter, 100 μl of each culture was spread on urine plates (1:1 ratio of human urine and 0.9% NaCl) containing 1.5% agar. The plates were incubated at 37°C for 16 h. Subsequently, 600 μl of a 1:2 mixture of PBS and RNAprotect™ Bacteria Reagent (QIAGEN AG) was poured on the plates, mixed with the lawn of cells and incubated for 5 min at room temperature to stabilise RNA. The stabilised mixture was then centrifuged and pellets were stored at −80°C. The samples from 83972, grown exponentially in MOPS and urine, in urine biofilms and in patients (>108 CFU/ml) were all treated identically with RNAprotect Bacteria Reagent and have been described previously (Roos and Klemm 2006; Hancock and Klemm 2007). RNA isolation and microarray hybridisation Total RNA was isolated using the RNeasy® Mini Kit (QIAGEN AG) and on-column DNase digestion was performed using RNase-Free DNase Set (QIAGEN AG). The quality of the total RNA was examined by agarose gel electrophoresis and by measuring the absorbance at 260 and 280 nm to ensure intact high-quality RNA. Purified RNA was precipitated with ethanol and stored at −80°C until further use. Conversion of RNA (10 μg per sample) to cDNA, labelling and microarray hybridisation were performed according to the GeneChip Expression Analysis Technical Manual 701023 Rev. 4 (Affymetrix, Inc., Santa Clara, CA). GeneChip E. coli Genome 2.0 Arrays (Affymetrix) were used for hybridisation of the labelled cDNA. The microarrays were scanned using the GeneChip Scanner 3000. Data analysis The raw intensities from the microarray experiments were background corrected and quantile-normalised. All microarray data in the study were obtained from mRNA being converted to cDNA, i.e. no genomic DNA was used for hybridisation. Probe intensities were summarised to yield expression values for each probe set or gene. These calculations were performed using the implementation of GCRMA (Wu et al. 2004) in Bioconductor (Gentleman et al. 2004) (http://www.bioconductor.org, http://www.r-project.org). In order to derive a cut-off expression value for making presence/absence calls, we made use of intensities due to control probe sets with IDs beginning with AFFY. There were 96 such probes. The cut-off value was set so that only the top 1/16th of these control probes would be flagged as present. As a result 4,109 genes in the array were marked as present; the remaining genes are referred to as “absent” throughout this report, i.e. these genes could be truly absent, non-homologous or not expressed during any of the seven different growth conditions. Orthologs of all the genes in the array across E. coli K12 MG1655, E. coli O157:H7 Sakai, E. coli O157:H7 EDL933 and E. coli CFT073 were identified using bidirectional best hit BLAST. Microarray data accession number The supporting microarray data have been deposited in ArrayExpress (http://www.ebi.ac.uk/arrayexpress) with accession numbers E-MEXP-584 (MOPS, urine and patient arrays), E-MEXP-926 (biofilm arrays) and E-MEXP-1453 (urine-agar plate arrays). Results Genes expressed in ABU E. coli 83972 The bacterial transcriptome is a dynamic entity that reflects the organism’s immediate, ongoing response to its environment. DNA microarray-assisted functional genomics provides the global expression profile of the genome. The genomic expression profiles of the urinary tract infectious E. coli isolate 83972 were analysed under several different growth conditions and in different media using the GeneChip E. coli Genome 2.0 Array (Affymetrix). This array contains approximately 10,000 probe sets for all 20,366 genes present in E. coli strains MG1655 (K-12), CFT073 (UPEC), EDL933 (EHEC) and O157:H7-Sakai (EHEC). Due to the high degree of similarity between the E. coli strains, whenever possible, a single probe set is tiled to represent the equivalent ortholog in all the four strains. In total, 21 microarrays were included in the study; arrays in triplicates were hybridised with RNA of the ABU strain 83972 cultured (1) aerobically to exponential phase in MOPS minimal medium, (2) aerobically to exponential phase in pooled human urine, (3) on urine agar plates, (4) statically in urine biofilm on Petri dishes and finally, (5–7) in three patients (Pat1, Pat2 and Pat3) in vivo. Figure 1 shows the expression levels of all CFT073 genes in strain 83972 during growth in the different environments; many genes were similarly expressed during all seven conditions. However, some genes were expressed only during one or a few of the conditions. For example, the genes encoding yersiniabactin in the high pathogenicity island (HPI), i.e. PAI-asnT, were highly expressed in Pat2 (and in biofilm), but much lower during the other conditions. The c2557–c2563 genes (around 2.4 M in Fig. 1), involved in nucleotide sugar and mannose metabolism and encoding hypothetical proteins, were highly expressed in Pat3 but not under any other condition. Another example is the c1968–c1971 genes (around 1.8 M), i.e. ydfI encoding a d-mannonate oxidoreductase, ydfJ encoding a metabolite transport protein and rspAB involved in the starvation response, which also were highly expressed only in Pat3. Fig. 1The expression levels of CFT073 genes in strain 83972 during seven different growth conditions. The outer blue circle shows the calculated absence (0.0) and presence (1.0) of CFT073 genes in ABU strain 83972. The seven PAIs of CFT073 are indicated in red In total, there were 108 genes that were significantly changed in all six urine environments compared with MOPS. Twenty of these genes were up-regulated in all six urine conditions whereof half were related to different iron systems, i.e. iroN, fepA, fecI, iucBC, fhuA and exbD, as well as b3337 and b1995 involved in iron storage and encoding a putative haemin receptor, respectively. The other urine up-regulated genes were marA, a multiple antibiotic resistance gene, sodA, encoding superoxide dismutase, ahpC, encoding hydroperoxide reductase, b1452, c1220, c4210, lysA, rrsG, rrsH and yrbL. Most iron acquisition systems were expressed in all the six urine environments; the enterobactin, salmochelin, aerobactin, haem and sitABCD systems were all expressed in all the six urine conditions (although weaker in the urine plates). Interestingly, the fec system, which is a citrate-dependent iron uptake system found in K-12 but missing in CFT073 and other UPEC strains, was highly expressed in Pat3. Up-regulation of all these iron-uptake systems revealed that the strain has an impressive array of iron acquisition systems and all of these are active in the human bladder. Nineteen of the top 31 highest expressed genes overall were genes involved in ribosomal synthesis. The high expression of ribosomal genes in E. coli 83972 suggests a rapid growth rate; the highest expression values were obtained in Pat1 followed by MOPS, urine and Pat2, indicating a growth rate just as fast in the patients in vivo as in exponential growth phase in a shake flask. This supports our hypothesis that the strain’s optimized growth properties in human urine explain its ability to successfully colonize the human urinary tract in the absence of functional fimbriae (Roos et al. 2006b). Figure 1 reveals that strain 83972 almost exclusively expresses the iron uptake and transport systems in the seven CFT073 PAIs, almost none of the other genes in these islands are expressed. There are only two exceptions; c0300, located in PAI-aspV encoding a hypothetical protein, and c3686–3690, located in PAI-pheV encoding YrbH and KpsEDC. The yrbH gene belongs to the 131 genes that were recently identified as UPEC specific and it was the second highest expressed UPEC-specific gene in mice (Lloyd et al. 2007); in our samples the highest expression was found in the three patients and in MOPS. Outside the PAIs there are a few genes/gene clusters that are highly expressed in all urine samples or only in the patients. The enterobactin system was up-regulated during all urine conditions and the chu cluster (involved in haem uptake and transport) was highest up-regulated in the patients followed by in vitro urine growth. The ycdO and ycdB genes were highly expressed in the three patients; these have recently been identified to encode haemoproteins, probably involved in iron transport, induced at acidic conditions (Sturm et al. 2006). Looking at the significantly changed genes for all six urine conditions compared with MOPS (in total 1,897 genes) revealed that Pat2 and Pat3 shared the largest number of similarly changed genes; 75% of all changed genes in Pat2 are regulated in the same way (i.e. up or down-regulated) in Pat3 (Fig. 2). Interestingly, Pat1 shared the largest number similarly regulated genes with the biofilm growth mode; also for Pat2 and Pat3, the biofilm growth mode showed a larger number of similarly regulated genes than Pat1 or any other condition. This could indicate that the expression profile of strain 83972 during in vivo growth is closer related to biofilm growth than to growth in shake flasks or plates. Fig. 2Number of significantly up- and down-regulated genes in strain 83972 during the different growth conditions (i.e. exponential growth in urine, on urine-agar plates, in urine biofilm, in vivo in three patients) compared with exponential growth in MOPS minimal lab medium. The diagonal boxes (dark blue colour) show the number of significantly changed genes during cultivation in that specific condition compared with MOPS (e.g. 664 genes were up- or down-regulated in urine compared with MOPS and 938 genes were changed in plates compared with MOPS) and the other boxes show the number of significantly changed genes shared between two conditions (e.g. 311 of the 664 and 938 significantly changed genes in urine and plates compared with MOPS were shared between these two conditions, i.e. up- or down-regulated in both urine and plates compared with MOPS). Stronger blue colour indicates larger number of significantly changed genes shared between two conditions Closeness to CFT073 Given the different growth conditions analysed, it is not unrealistic to assume that most genes present in strain 83972 would be expressed, to some extent, under at least one of these seven different conditions/environments, i.e. growth in liquid and on solid media; during exponential phase, in biofilm and during colony-forming conditions; in different growth media (human urine and minimal lab medium); as well as in vivo in three different individuals. Data analysis of the 21 microarrays revealed that of the 8,716 E. coli transcripts on the microarray (not including probes representing intergenic regions and controls), 4,109 transcripts (47%) showed expression levels above detection limit during at least one of the growth conditions investigated (referred to as “present”, see blue, outer circle in Fig. 1). Figure 3 shows the distribution among the four E. coli genomes represented on the microarray of these 4,109 transcripts expressed in E. coli 83972. Not surprisingly, the UTI strain 83972 shows highest similarity with the UPEC isolate CFT073 of the four genomes on the array; the large majority of the 4,109 transcripts found present (96.3%) can be found in CFT073, corresponding to 71% of the CFT073 genome. E. coli 83972 expressed 150 genes that do not exist in CFT073; 85 of these can be found in MG1655 and the remaining 65 genes can be found exclusively in one or both of the two EHEC strains present on the array (Fig. 3). Thirty of the 65 genes homologous to EHEC genes are encoding proteins of cryptic prophages, whereas the large majority of the remaining 35 genes encode unknown or hypothetical proteins. The 85 genes that can be found in MG1655 but not in CFT073 includes the fec cluster encoding an iron citrate transport system (fecABCDEIR). In total, 3,959 CFT073 genes were expressed in strain 83972; this could be compared with 4,162 CFT073 genes present in the UPEC (cystitis) isolate F11 (Lloyd et al. 2007). Fig. 3Venn diagrams showing the distribution of the 4,109 genes filtered present in strain 83972. The percentages indicated below each strain show how large part of the genome of the corresponding strain was filtered present in strain 83972 E. coli core genome There is a large diversity in size of the chromosome of E. coli; in all 32 E. coli (and Shigella) genomes that have been fully sequenced, or at least with an expected coverage of greater than 99%, the size of the chromosome ranges from 4.5 to 5.6 Mbp. The genomes show a considerable amount of diversity, and the estimated size of the current pan-genome was estimated to contain 9,433 different genes (Willenbrock et al. 2008). Several studies have identified sets of “core genes” found in most E. coli genomes. However, the number of these core genes tends to decrease as the full genomic sequences of new E. coli strains become available. The size of the E. coli core genome has recently been predicted to contain 1,563 genes for an infinite number of E. coli strains, and the number of new genes predicted from each new E. coli genome that is sequenced is ∼79 (Willenbrock et al. 2008). In our analysis, 2,472 (60%) of the genes found present in strain 83972 were common in all the four E. coli genomes on the array (Fig. 3), which is well above the estimated E. coli core genome and also above the 2,241 common genes conserved among the 32 sequenced E. coli strains (Willenbrock et al. 2008). Furthermore, considering the fact that the microarray contains only four E. coli genomes, the total number of genes detected present (4,109 genes) in 83972 seems reasonable comparing the size of other sequenced UTI E. coli genomes. The genome size of strain 83972 has been reported to be 4.9±0.2 Mbp (Zdziarski et al. 2007), indicating that the strain contains roughly an additional 800 genes, not identified in the present analysis. Of the 2,734 transcripts on the chip that are present in all the four strains represented on the microarray, 393 transcripts were below detection limit on all 21 microarrays and filtered as “absent” in strain 83972. These included 81 genes encoding hypothetical proteins. Several of the absent genes were found in clusters, many of which are involved in surface structure elements and chemotaxis. These included genes involved in flagellar biosynthesis (flgABCDEFGHIJKL, flhABE, fliACDEFGHIJKLNOPQRSTZ and motAB), curli production (csgABCEFG), colanic acid synthesis (wcaABCDEFGHI and wza) and chemotaxis (cheBRWYZ and tap). Other whole cluster of genes that were not expressed in the ABU strain but found in all the four E. coli present on the chip were hyaBCDEF (hydrogenase I), hycACD (hydrogenase 3), tauABCD (responsible for taurine uptake in E. coli) and b1500–1505 (containing the fimbrial-like genes ydeQRST), as well as the fimEAIC genes which previously have been shown to be absent in strain 83972 (Klemm et al. 2006). UPEC-associated genes present in strain 83972 The four UPEC isolates that have been sequenced, CFT073, UTI89, 536 and F11, contain 5,379, 5,154, 4,766 and 4,467 genes, respectively, on the chromosome. CFT073 and 536 are both O6 strains and yet show a large diversity; the genome of 536 is almost 300 kb smaller than that of CFT073 (Brzuszkiewicz et al. 2006). The genomic differences are mainly restricted to large pathogenicity islands, the additional DNA in CFT073 are genes of five cryptic prophages, which are absent in 536 (Brzuszkiewicz et al. 2006). The 427 genes that are present only in the strain 536, and the 432 genes present only in the two UPEC (compared with other sequenced E. coli) are scattered all over the genome (Brzuszkiewicz et al. 2006). Over 70% of the CFT073 transcripts were present in strain 83972 compared with 89% of the CFT073 transcripts found in strain 536. Figure 4 shows the homology of 16 sequenced E. coli and Shigella isolates including the three sequenced UPEC strains (UTI89, 536 and F11) pasted on the CFT073 genome; the outer, red circle in the figure shows the results from the presence/absence analysis on strain 83972. Many virulence-associated genes are located on the large pathogenicity islands (PAIs) found in different UPEC strains. The large pathogenicity island at pheV in CFT073 (also called PAI ICFT073) encodes haemolysin (hlyCABD), aerobactin biosynthesis proteins (iutA and iucABCD), antigen 43 (c3655) and the secreted autotransporter toxin (sat); these were all filtered present in our analysis, suggesting that strain 83972 harbours a similar island on its chromosome. Interestingly, the aerobactin system is missing in the other three UPEC isolates. Furthermore, this PAI contains genes encoding the uropathogenic-associated P fimbriae (papIBAHCDJKEFG). The pap gene cluster of 83972 has been sequenced (Klemm et al. 2006); the pap genes are all present and show 72–100% sequence homology with the corresponding genes in CFT073. The results of the microarray analysis corresponded very well to the observed sequence homology of the different genes in the cluster (i.e. if a specific gene on the microarray is represented with probes that contain a non-homologous region compared with the corresponding gene in the hybridised sample, that gene will not hybridise and will be filtered absent); the six genes with highest sequence homology were filtered present (i.e. papHCDJKF with 98, 100, 100, 98, 99 and 95% homology, respectively) and the four with least sequence homology were filtered absent (i.e. papIAEG with 94, 83, 77 and 72% homology). Fig. 4BLAST atlas comparing the absent (0.0) and present (1.0) CFT073 genes in strain 83972 with other sequenced E. coli and Shigella strains, including the three sequenced UPEC isolates 536, UTI89 and F11. The UPEC CFT073 genome is used as reference. The outer blue circle represents the calculated absence/presence in 83972 followed by the three UPEC isolates; the six inner circles represent Shigella strains. The seven PAIs of CFT073 are indicated in red. The blow-up shows the presence/absence of the fim cluster (c5391–5400) in strain 83972 The employed microarray contains probes for all ten known and putative fimbriae-encoding gene clusters in CFT073. Together with the pap cluster, two other fimbrial clusters that have been associated with UPEC virulence are known to be present in strain 83972 and have been sequenced, i.e. the fim and sfa/foc clusters. As for the pap cluster, the filtering of absent genes corresponded very well to the actual presence and sequence homology of the genes; strain 83972 contains a large deletion in the fim cluster but shows high sequence homology with the present genes, and all the genes in the deleted part of the cluster, i.e. fimEAIC, were filtered absent (see blow-up in Fig. 4). Also, the sfa/foc cluster in 83972 shows high homology with that in CFT073 (98–100%), and eight of nine genes were filtered present; the putative regulatory gene, sfaC, was filtered absent. Regarding the other fimbrial clusters present on the microarray, none of the genes encoding F9 fimbriae, which appear to be common in UPEC and plays a role in biofilm formation (Ulett et al. 2007), and another putative fimbriae (yehABCD) were expressed and might be absent in strain 83972 (Table 1). Table 1Analysis of fimbriae-encoding genes in strain 83972Descriptionc numberGenesNo of genesNo (%) of absent genesAbsentPutative chaperone-usher fimbrial operonc0166–0172yadN-ecpD-htrE-yadMLKC73 (43)ecpD, yadMKF1Cac1237–1245sfaCB-focAICDFGH91 (11)sfaCF9c1931–1936c1936-34-ydeSRQ66 (100)AllPutative chaperone-usher fimbrial operonc2635–2638yehABCD44 (100)AllPutative chaperone-usher fimbrial operonc2878–2884yfcOPQRSUV75 (71)yfcQRSUVP fimbriaeac3583–3593papIBAHCDJKEFG114 (36)papIAEGbPutative chaperone-usher fimbrial operonc3791–3794ygiLGH-c379441 (25)ygiLAuf fimbriaec4207–4214aufABCDEFG87 (88)aufBCDEFGP fimbriae (2)ac5179–5189papIBAHCDJKEFG2 (papAD)1 (50)papA_2Type 1 fimbriaec5391–5399fimBEAICDFGH94 (44)fimEAICaFimbrial operons present on PAIs. The two pap clusters share the same probes on the array with exception for papA and papD, which are represented by two separate probe sets eachbNone of these pap genes are absent in strain 83972, but papIAEG were filtered absent in the microarray analysis due to non-homologous sequence regions compared with the CFT073 pap probes present on the array Presence of other pathogenicity islands in 83972 Strain 83972 seems to carry most of the pathogenicity islands of CFT073 (or PAIs similar to the ones in CFT073) according to our present/absent analysis (Table 2). The only PAI of CFT073 in which most genes (i.e. 93%) were filtered absent in strain 83972 is PAI-pheU (PAI IICFT073), the island that contains a second pap cluster. The three genes filtered present in this PAI are present in several of the other sequenced E. coli and Shigella strains indicating that these three genes not are unique/characteristic for this island wherefore this PAI is most probably absent in strain 83972. Table 2Analysis of presence of pathogenicity islands in strain 83972Island nameCommon namec numberNo of genesaAbsent (%)Virulence-associated genesbPAI-CFT073-aspVPAI III CFT073c0253–c03689640 (42)cdiA (c0345), picU (c0350)PAI-CFT073-serXc1165–c12939233 (36)mchBCDEF (c1227, c1229–1232), sfa/foc (c1237–c1247), iroNEDCB (c1250–c1254), ag43 (c1273)PAI-CFT073-icdAc1518–c1601425 (12)sitDCBA (c1597–1600)PAI-CFT073-asnTHPI CFT073c2418–c2437193 (16)fyuA (1246)PAI-CFT073-metVc3385–c34102617 (65)hcp (c3391), clpB (c3392)PAI-CFT073-pheVPAI I CFT073c3556–c369811951 (43)hlyA (c3570), pap (c3582–c3593), iha (c3610), sat (c3619), iutA, iucDCBA (c3623–3628), ag43 (c3655), kpsTM (c3697–c3698)PAI-CFT073-pheUPAI II CFT073c5143–c52164643 (93)pap2 (c5179–c5189)aNo of genes in the PAI that were present on the array with unique probes (i.e. genes that are not orthologues to any other E. coli transcripts present on the array)bBoldface indicates genes filtered present in strain 83972 Insertion of the high pathogenicity island (HPI) of Yersinia pestis has been suggested to be one of the earliest events in the evolution of extraintestinal E. coli strains (Welch et al. 2002). The genes of HPI encoding yersiniabactin (Ybt) were all expressed in strain 83972. The HPI genes have been found up-regulated during urine biofilm growth of 83972 indicating that Ybt-mediated iron-uptake might play an important role in biofilm growth (Hancock and Klemm 2007) and a deletion mutant in the Ybt uptake receptor (FyuA) exhibits reduced biofilm formation (Hancock et al. 2008). The HPI genes have also been found up-regulated in vivo in two of the three patients (particularly in Pat2, see Fig. 1) infected with this strain (Roos and Klemm 2006). The pks island, a recently characterised and widely spread genomic island found in, for example, meningitis strains and the uropathogenic strain CFT073, encodes a machinery for the synthesis of peptide–polyketides hybrid compounds (Nougayrede et al. 2006). The presence of the island is associated with the accumulation of double-strand DNA breaks in host cells and has genotoxic activity (Nougayrede et al. 2006). This island was expressed in strain 83972 and up-regulated in urine and in vivo (Table 4; Fig. 1). The pks island is widely distributed within E. coli phylogenetic group B2, and has been found in both pathogenic and commensal isolates; in commensal strains the cell-cycle-blocking activity might slow the turnover of the intestinal epithelium, and therefore prolong colonisation. Presence of positively selected UPEC genes A recent paper comparing the UPEC isolates CFT073 and UTI89 with six other finished E. coli genome sequences presented 29 genes that are under positive selection only in UPEC strains (Chen et al. 2006). These 29 genes are involved in various aspects of cell surface structure, DNA metabolism, nutrient acquisition and UTI. Of these 29 genes, 25 were filtered present in our ABU strain 83972; many of these genes are represented by more than one transcript on the array due to sequence differences among the four strains present on the array, in all cases the gene filtered present in 83972 corresponded to the CFT073 transcript. Four genes were filtered absent, agaI, yjiL, recC and yegO; they encode a putative galactosamine-6-phosphate isomerase, a hypothetical protein, exodeoxyribonuclease V gamma subunit and a hypothetical transport protein, respectively. The genes in the two COG categories that were significantly enriched in the two UPEC strains, i.e. “cell wall/membrane biogenesis” (amiA, cutE, fepE, ompC, ompF and yfaL) and “secondary metabolites biosynthesis, transport and metabolism” (entD, entF and yojI) (Chen et al. 2006), were all present in strain 83972. Functional analysis of MG1655 transcripts of ABU E. coli 83972 To gain more information concerning what type of genes were absent, the MG1655 genes were grouped into functional categories defined by the clusters of orthologous groups (COGs) of proteins (Tatusov et al. 1997). Previous studies have, in attempts to identify essential genes and the E. coli core genome, found that groups with genes involved in metabolism and various cellular processes (excluding cell motility) contain a substantially higher percentage of conserved and essential genes, while COGs with genes of unknown function and external origin as well as genes involved in signalling and motility contain fewer essential genes (Anjum et al. 2003; Gerdes et al. 2003). Classification of the absent genes of strain 83972 revealed that the groups “cell motility”, “defence mechanisms” and “not in COGs” had a significant overrepresentation of absent genes (Table 3). A significantly lower proportion of absent genes were found in the groups: “cell cycle control”, “posttranslational modification” and “translation”. This is in agreement with a previously published study of pathogenic E. coli; Anjum et al. (2003) studied 26 strains of E. coli and found that the two groups with largest proportion of absent genes were “not in COGs” and “cell motility”, while the six groups with the lowest proportion of absent genes were “translation”, “cell division”, “posttranslational modification”, “coenzyme metabolism”, “nucleotide transport and metabolism” and “energy production and conversion”, which all, with exception for the last group, contained significantly fewer absent genes in strain 83972 (Table 3). This suggests that strain 83972 utilises a similar set of core genes as other E. coli strains. Table 3Distribution of absent genes in functional categoriesFunctional categoryAbsentTotalZ testNo.%P valueAmino acid transport and metabolism9833.12960.636Carbohydrate transport and metabolism12642.32980.025Cell cycle control, cell division and chromosome partitioning516.1310.000Cell motility7784.6910.000Cell wall/membrane/envelope biogenesis8140.52000.087Coenzyme transport and metabolism2523.11080.001Defense mechanisms1748.6350.000Energy production and conversion8836.72400.563Function unknown6124.72470.003General function prediction only8330.92690.255Inorganic ion transport and metabolism5534.41600.921Intracellular trafficking, secretion and vesicular transport822.2360.000Lipid transport and metabolism2129.6710.129Nucleotide transport and metabolism1924.4780.002Posttranslational modification, protein turnover, chaperones2420.21190.000Replication, recombination and repair7042.41650.023Secondary metabolites biosynthesis, transport and catabolism2240.7540.075Signal transduction mechanisms3933.61160.748Transcription7833.22350.654Translation, ribosomal structure and biogenesis2113.51560.000Not in COGs57754.210650.0001,59539.24,070 CFT073 genes absent in strain 83972 There were 1,636 CFT073 genes that could not be detected according to our expression profiling in ABU strain 83972; 961 of these genes are exclusively found in CFT073, i.e. not present in the other three strains represented on the array. The majority, 645 genes, corresponded to hypothetical, putative or unknown proteins. Considering the very different patient symptom profiles of strains CFT073 and 83972 (one being a true pathogen, while the latter is a commensal-like strain), genes that are present in UPEC isolate CFT073 but not expressed in ABU strain 83972 can be considered as virulence factor candidates. However, most genes associated with UPEC pathogenesis were expressed in strain 83972 and up-regulated during growth in urine, e.g. all iron-related genes encoding uptake and transport of aerobactin, salmochelin, yersiniabactin and haem/haemoglobin (Table 4). Two exceptions were the ireA gene encoding an iron-regulated outer-membrane protein that was filtered absent as well as the tsx gene encoding a nucleoside-binding outer-membrane protein. Although the tsx gene has not previously been associated with UPEC virulence, it has just recently been identified together with more well-known UPEC genes as involved in movement from the intestinal tract to the bladder and vagina (i.e. occurred significantly more often in multiple-site isolates than in rectal site-only isolates) (Xie et al. 2006); furthermore, Tsx was also recently identified together with 22 other outer-membrane proteins from CFT073 cells grown under conditions mimicking the urinary tract (Hagan and Mobley 2007). Table 4Characteristics of ABU isolate 83972 compared with UPEC isolates CFT073, UTI89 and 536CharacteristicaCFT073UTI8953683972Expression in 83972bSerotypeO6O18O6O?cCapsuleK2K1K15K?cChu++++U, BF, PatEnt++++U, BF, PatFep++++Pl, U, BF, PatFeo++++BF, PatFhu++++Pl, U, BF, PatIro++++U, PatIuc+−−+Pl, U, BF, PatIutA+−−+U, BF, PatSit++++U, BF, PatFyuA++++U, BF, PatIha+−−+U, PatIreA+−−−Pks island++++U, PatRfaH++++BFd-serine++++PatPap+++−Fim+++−Foc/sfa+++−Vat++++BF, PatSat+−−+UTsx+++−Biofilm formation1.01.314.4aBoldface indicates genes that were filtered absent in strain 83972bUp-regulation in urine (U), biofilm (BF), plates (Pl) and patients (Pat) compared with MOPS minimal mediumcUndefined. Extensive electron microscopy analysis of the strain has never reported any capsule Type IV fimbriae are assembled by the type II general secretory pathway. They occur in a wide range of species and frequently are associated with diseases. The ppdD and hofBC genes (b0106–0108), which encode type IV prepilin and are present in CFT073, EDL933 and MG1655, were filtered absent in strain 83972. CFT073 genes present in strain 83972 but not found in other UPEC strains The majority of the genes that are absent in the other three UPEC isolates (i.e. 536, UTI89 and F11) were filtered absent in strain 83972 as well (gaps in Fig. 4). However, there are a few exceptions where a gene that is not found in any of the other UPEC strains is filtered present in strain 83972. The aerobactin system belongs to one of the exceptions, indicating that strain 83972 is particularly well equipped with iron uptake systems. The other exceptions are all but one located on PAIs and they all encode hypothetical proteins: c1194–c1204 (on PAI-serX), c1522–c1528 (on PAI-icdA), c3394–c3396 (on PAI-metV), c3681–c3682 (on PAI-pheV where the aerobactin genes also are found) and c5372–c5382. c3394–c3396 and c5372–c5382 are not present in any of the 16 sequenced E. coli and Shigella strains represented in Fig. 4, indicating that some genes unique to CFT073 can be found in strain 83972 as well. Discussion Bacterial genomes are under constant change. New genes are acquired by horizontal transfer and old ones are lost by mutations. It is generally believed that commensal E. coli can become pathogenic through the acquisition of novel genes encoding virulence factors and niche-adaptation factors (Kaper et al. 2004). In contrast to organisms that have acquired genes for pathogenesis, E. coli 83972 is an example of an organism that has adapted to a commensal-like existence through gene deletions and point mutations. Using primarily the CFT073 as a scaffold, we used presence/absence data from seven sets of different gene expression profiles (in total 21 microarrays) to model the gene pool of strain 83972. Given the limitations of the approach, i.e. genes not present on the employed chip have been ignored, a substantial body of information was gathered concerning the genomic content of the strain. As it turned out the strain was highly similar to CFT073; 96% (3,959) of the genes found to be expressed on the employed microarray by 83972 are also found in CFT073, and genes on six of the seven pathogenicity islands of CFT073 were expressed by 83972; furthermore, CFT073 genes not found in any other UPEC isolate were expressed by 83972. An estimated ∼900 CFT073 genes are not expressed by 83972. Arguably, in the light of the difference in patient symptoms invoked by encounters with the two strains, this list represents virulence gene candidates. Although strain 83972 seems to be a deconstructed uropathogen and does not provoke symptoms in the human host it grows fast in urine and is an excellent colonizer of the human bladder (Roos and Klemm 2006; Roos et al. 2006b; Klemm et al. 2007). It can do so because it has kept a large assortment of fitness factors required for this particular ecological niche. Among the genes expressed under realistic environmental conditions such as in the human bladder are candidates for fitness factor genes, e.g. the many iron acquisitions systems expressed by the strain and many genes involved in sugar acid and amino acid metabolism. Interestingly, many of the known and putative virulence factors of the urinary tract are expressed by strain 83972 and might therefore be considered as fitness factors rather than virulence factors; these include 25 of 29 positively selected UPEC genes as well as the newly characterised pks island inducing breaks in double-stranded DNA in host cells. Also, virulence-associated genes such as cdiA, mchBCDEF, flu, hcp, rfaH, sat, picU and vat were all expressed by strain 83972. Very few of the known or putative virulence factors were absent in (or not expressed by) strain 83972. The pap, fim and foc/sfa clusters encoding UPEC-class fimbriae are dysfunctional in strain 83972 and the clpB, ireA and tsx genes were not expressed in the ABU strain. These stand out as potential virulence candidates together with a number of uncharacterised genes encoding hypothetical proteins. Thus from the analyses performed here we can make predictions about several gene categories such as potential virulence genes, fitness genes and “household-class” genes. It is also noteworthy that the information reported herein complements a potential genome sequence of strain 83972. Whole genome sequencing can identify the presence of genes but is unable to reveal if they are transcribed. Genes can be silenced not only due to lesions in the actual gene and its promoter but also due to mutations of genes encoding regulatory factors. The methodology employed in the present work reveals the active genome of strain 83972. ABU strain 83972 is closely related to fully virulent uropathogenic strains. All evidences suggest that the strain is a deconstructed pathogen. This study dispels the commonly held idea that ABU strains are commensals that have picked up niche-adaptation genes by horizontal gene transfer. Rather, strain 83972 was originally a true pathogenic strain that has lost whole or part of operons that contribute to virulence.

Plant_Mol_Biol-4-1-2268730

Deoxymugineic acid increases Zn translocation in Zn-deficient rice plants

Deoxymugineic acid (DMA) is a member of the mugineic acid family phytosiderophores (MAs), which are natural metal chelators produced by graminaceous plants. Rice secretes DMA in response to Fe deficiency to take up Fe in the form of Fe(III)–MAs complex. In contrast with barley, the roots of which secrete MAs in response to Zn deficiency, the amount of DMA secreted by rice roots was slightly decreased under conditions of low Zn supply. There was a concomitant increase in endogenous DMA in rice shoots, suggesting that DMA plays a role in the translocation of Zn within Zn-deficient rice plants. The expression of OsNAS1 and OsNAS2 was not increased in Zn-deficient roots but that of OsNAS3 was increased in Zn-deficient roots and shoots. The expression of OsNAAT1 was also increased in Zn-deficient roots and dramatically increased in shoots; correspondingly, HPLC analysis was unable to detect nicotianamine in Zn-deficient shoots. The expression of OsDMAS1 was increased in Zn-deficient shoots. Analyses using the positron-emitting tracer imaging system (PETIS) showed that Zn-deficient rice roots absorbed less 62Zn-DMA than 62Zn2+. Importantly, supply of 62Zn-DMA rather than 62Zn2+ increased the translocation of 62Zn into the leaves of Zn-deficient plants. This was especially evident in the discrimination center (DC). These results suggest that DMA in Zn-deficient rice plants has an important role in the distribution of Zn within the plant rather than in the absorption of Zn from the soil. Introduction Zn, an essential element for plant growth, is absorbed from the rhizosphere by higher plants through specific transporters. The Zinc-regulated and Iron-regulated transporter Proteins (ZIP) have been isolated from both graminaceous (Ramesh et al. 2003; Ishimaru et al. 2005) and non-graminaceous plants (Grotz et al. 1998; Lasat et al. 2000; Moreau et al. 2002). Several ZIP transporters have been shown to be transcriptionally upregulated in Zn-deficient roots, but their contribution to the absorption of Zn from the rhizosphere into roots has not yet been demonstrated. Zn is also absorbed from the rhizosphere as complexes of Zn(II)–mugineic acid family phytosiderophores (MAs). The maize protein YS1 transports Fe(III)–MAs complexes (Curie et al. 2001), as well as other micronutrient-MAs complexes, including Zn(II)–MAs (Schaaf et al. 2004). von Wirén et al. (1996) reported that the ys1 mutant absorbed less 65Zn(II)-MAs than did the wild type, and Suzuki et al. (2006) showed that Zn-deficient barley absorbed more 62Zn(II)-DMA than 62Zn2+. These two studies also showed that free Zn2+ ions are absorbed into roots to some extent, suggesting that graminaceous plant roots absorb both Zn-DMA and Zn2+. Graminaceous plants secrete MAs into the rhizosphere from their roots, although the amount differs among species in the order of: barley > wheat = rye > oat > maize > sorghum > rice (Takagi 1993). While the amount of MAs secreted is increased dramatically by Fe deficiency, it is not clear if Zn deficiency increases the secretion of MAs. Zn deficiency has been reported to increase (Cakmak et al. 1994; Walter et al. 1994; Zhang et al. 1989) or have no effect on the secretion of MAs from wheat and barley roots into the rhizosphere (Gries et al. 1995; Pedler et al. 2000). The biosynthesis of MAs and their corresponding genes have been characterized (Fig. 3b) as being synthesized from methionine (Mori and Nishizawa 1987). Three molecules of S-adenosyl-l-methionine (SAM) are combined to form one molecule of nicotianamine (NA) by nicotianamine synthase (NAS). NA is then converted to the 3′′-keto acid by NA aminotransferase (NAAT), and 2′-deoxymugineic acid (DMA) is synthesized by DMA synthase (DMAS). In some graminaceous species, including barley, DMA is further hydroxylated by two dioxygenases, IDS2 and IDS3 (Kobayashi et al. 2001; Nakanishi et al. 2000). The genes encoding the enzymes involved in DMA synthesis have been well-characterized in Fe-deficient rice and barley. The expression of HvNAS1, HvNAAT-A, HvNAAT-B, HvDMAS1, HvIDS2, and HvIDS3 is increased in Fe-deficient barley roots (Nakanishi et al. 1993; Okumura et al. 1994; Higuchi et al. 1999; Takahashi et al. 1999; Bashir et al. 2006). In rice, the expression of OsNAS1, OsNAS2, OsNAAT1, and OsDMAS1 is increased in both roots and shoots by Fe deficiency (Inoue et al. 2003; Bashir et al. 2006). The expression of OsNAS3 is increased in Fe-deficient roots, but decreased in Fe-deficient shoots (Inoue et al. 2003). Promoter-GUS analysis suggests that OsNAS1 and OsNAS2 are involved in DMA secretion, because those genes are expressed in all root cells. On the other hand, OsNAS3 may not be involved in DMA secretion because its expression is restricted to the pericycle and companion cells of the roots (Inoue et al. 2003). Recently, we showed that the expression of NASHOR2, a NAS gene in barley (Herbik et al. 1999), and HvNAAT-B was increased in Zn-deficient barley shoots, but that the expression of IDS2 and IDS3 was not detected in Zn-deficient shoots (Suzuki et al. 2006). On the other hand, the expression of HvNAS1, HvNAAT-A, HvNAAT-B, IDS2, and IDS3 was increased in both Zn- and Fe-deficient barley roots, while the expression of these genes was not evident in Fe-deficient shoots (Suzuki et al. 2006). This suggests that in barley Zn deficiency induces DMA synthesis in shoots, while both Zn and Fe deficiency induce MAs synthesis and secretion in roots. The aims of the present study were to examine if the stimulation of MAs secretion by Zn deficiency is common in graminaceous plants and roles of MAs in Zn homeostasis of rice plants. In this report, we shows that DMA secretion is decreased in rice roots, while the concentration of endogenous DMA is increased in Zn-deficient rice shoots. Materials and methods Plant material and growth conditions Rice seeds (Oryza sativa L. cv. Nipponbare) were germinated for 7 days at room temperature on paper towels soaked with distilled water. After germination, the seedlings were transferred to a Saran net floating on nutrient solution (Higuchi et al. 2001) and grown in a glasshouse. After 4 days, 270 seedlings were transferred to six 20-l plastic containers containing nutrient solution. After a further 5 days, 90 seedlings were transferred to two 20-l plastic containers containing nutrient solution without Zn followed 7 days later when a further 90 seedlings were transferred to two 20-l plastic containers containing nutrient solution without Fe. All samples were harvested 5 days later for analysis. Consequently, the Zn-deficient treatment was performed for 12 days, and Fe-deficient treatment for 5 days since Fe deficiency has a much more rapid deleterious effect. The nutrient solutions were renewed weekly with adjustment to pH 5.5 every 2 days with KOH. Measurement of DMA secretion and endogenous levels of DMA and NA DMA secretion was measured as described by Suzuki et al. (2006) and the concentrations of endogenous DMA and NA were measured as described by Higuchi et al. (2001). Briefly, the roots of 12 plants were rinsed with deionized water and then placed in 600 ml of deionized water at sunrise. Root exudates were collected for two periods of 2 and 3 h. After collection, Micropur (Katadyn, Switzerland), an antimicrobial agent, was added to prevent microbial degradation of the MAs Both solutions containing root exudates were combined and filtered (Advantec 5C, Toyo Roshi Kaisha, Ltd., Japan). The shoots of the plants from which root exudates were collected were ground in liquid nitrogen using a mortar and pestle. Approximately 1 g of material was suspended in 40 ml of distilled water at 80°C for 30 min and filtered. The cationic fraction of the samples was prepared as a 2-M NH4OH eluate from Amberlite IR(H+)120 (Rohm and Haas Co., USA). The condensed and microfiltered samples were subjected to HPLC analysis, as described previously (Mori et al. 1987; Shojima et al. 1989). To ensure better separation of DMA, the pH of 0.15-N Li-citrate buffer was decreased to 2.97 and the pH of 0.2-N Li-citrate buffer increased to 3.30 for better separation of NA. Northern blot analysis The samples for Northern blot analysis were harvested 3 h after sunrise. The roots and shoots were separated immediately and stored at −80°C. Total RNA was extracted from the roots and shoots using SDS-phenol. Ten micrograms of total RNA were electrophoresed in 1.2% (w/v) agarose gels containing 0.66 M formaldehyde, then transferred to a Hybond-N+ membrane (Amersham, USA) and hybridized with probes at 65°C. The blots were analyzed using a Bio-imaging Analyzer System (BAS) 3000 (Fuji Film, Japan). The following specific primers were used to prepare specific probes: OsNAS1, 5′-GTCTAACAGCCGGACGATCGAAAGG-3′ and 5′-TTTCTCACTGTCATACACAGATGGC-3′; OsNAS2, 5′-TGAGTGCGTGCATAGTAATCCTGGC-3′ and 5′-CAGACGGTCACAAACACCTCTTGC-3′; OsNAS3, 5′-GACTGCTTCCATCGCTTGCTACCTC-3′ and 5′-CGCAACAGAGACAATGGTTGATTGT-3′; OsNAAT1, 5′-TAAGAGGATAATTGATTTGCTTAC-3′ and 5′-CTGATCATTCCAATCCTAGTACAAT-3′; and OsDMAS1, 5′-GCCGGCATCCCGCAGCGGAAGATCA-3′ and 5′-CTCTCTCTCTCGCACCTGCTAGCGT-3′. The accession numbers of the genes are AB021746 (OsNAS1), AB023818 (OsNAS2), AB023819 (OsNAS3), AB206814 (OsNAAT1), and AB269906 (OsDMAS1). PETIS analysis of 62Zn translocation PETIS experiment was established by Kume et al. (1997). 62Zn (half life: 9.13 h) was produced by the method of Watanabe et al. (2001). After adjustment of the 62Zn2+ solution to about pH 4 with 1 M KOH, 62Zn2+ was chelated with DMA in darkness for more than 3 h. Prior to the analysis, Zn-deficient rice plants were grown as described above but in a growth chamber (day: 30°C, 14 h of light at 320 μmol photons m−2 sec−1; night: 10 h at 25°C). The Zn-deficient rice plants were each supplied with 15 ml of culture solution lacking Zn in a polyethylene bag. The plants and bags were fixed between two acrylic boards and placed between a pair of PETIS detectors in a chamber set at 30°C, 65% humidity, and a light density of 320 μmol m−2 s−1. In the root absorption experiment, 62Zn (0.6 nmol) with DMA (79 nmol) was dissolved in 1 ml of distilled water and added to the culture solution. In the leaf absorption experiment, 62Zn (0.5 nmol) with DMA (33 nmol) was dissolved in 4.5 ml of nutrient solution containing cold Zn (2.5 μmol) and additional DMA (308 μmol), and supplied at the cut end of the second newest leaf. The individual leaves were cut to ensure a constant distance from discrimination center (DC) which is the junction of shoot and root (Kiyomiya et al. 2001). The γ-rays emitted from decaying positrons from 62Zn were counted over time using the coincident method with the paired detectors. The data were automatically corrected using 9.13 h as the half-life of 62Zn. The radioactivity of selected region was measured as region of interest (ROI) analysis. After PETIS analysis for 8 h, the plants were removed from the polyethylene bags, and the roots were gently washed for 1 min in 50 ml of 0.01 mM EDTA. Next, each plant was placed under a Bio-Imaging plate inside a cassette. The plate was scanned using an image analysis system (BAS-1500, Fuji Film, Japan). The plants were then cut into pieces, and the absolute amount of radioactivity was analyzed using γ-ray spectrometry with an ORTEC HPGe detector (Seiko EG & G Co., Ltd., Japan). Each experiment was conducted in triplicate. Due to the very short half-life of 62Zn, the radioactivity was different in each experiment. Therefore, images of individual plants and PETIS results are representative of those obtained. Results Plant growth, DMA synthesis and gene expression In contrast with the good growth of the control rice plants, Zn or Fe deficiency had a markedly detrimental effect when plants were grown for 12 d or 5 d in solutions containing no Zn or Fe, respectively. Shoot growth was reduced by 50% in the absence of Zn and by 10% without Fe (Fig. 1). The corresponding figure for the Zn-deficient roots was a reduction of 20%, while the absence of Fe supply for 5 d had no significant effect on root growth. As previously established in our studies (Suzuki et al. 2006), there was no measurable effect of no Zn supply during the first week of growth (data not shown), but thereafter plants developed symptoms of Zn deficiency and growth declined. Chlorotic symptoms of Fe deficiency developed after 5 days of treatment while the fresh weights of the shoots and roots were almost the same as in the control plants. Fig. 1Fresh weights of rice shoots and roots. 31-days old rice seedlings grown in hydroponically were harvested. Zn-deficient treatment (-Zn) was performed for 12 days, and Fe-deficient treatment (-Fe) was performed for 5 days. Values are mean ± SD (n = 4) The amount of DMA secreted from the roots of Fe-deficient rice was much higher than that of the control plants (Fig. 2). On the other hand, Zn deficiency slightly decreased the level of DMA secretion. In fact, the amount of DMA secreted per plant in Zn-deficient rice was significantly decreased compared to that in the control plants (P < 0.05). Fig. 2Amount of DMA secreted from rice roots. The growth condition and harvest time are the same in Fig. 1. Root exudates were collected for 5 h after sunrise. -Zn, zinc deficiency; -Fe, iron deficiency. Values are mean ± SD (n = 4) We next analyzed the expression pattern of the genes participated in DMA synthesis in roots and shoots under Zn or Fe deficiency (Fig. 3a, b). As reported previously, the transcription of OsNAS1, OsNAS2, OsNAS3, OsNAAT1, and OsDMAS1 in roots was increased by Fe deficiency (Inoue et al. 2003, 2008; Bashir et al. 2006). On the other hand, Zn deficiency enhanced the transcription of OsNAS3 and OsNAAT1, but not that of OsNAS1, OsNAS2, and OsDMAS1. The expression of OsNAS2 in roots was slightly decreased by Zn deficiency. The expression patterns of OsNAS1 and OsNAS2 were well correlated with the amount of DMA secreted from the roots of Fe-deficient or Zn-deficient rice plants (Figs. 2, 3a). Fig. 3Expression patterns of genes involved in DMA synthesis (a) Northern blot analysis of each gene involved in DMA synthesis. Each lane contained 10 μg of total RNA. C, control; -Zn, zinc deficiency; -Fe, iron deficiency. (b) DMA synthesis from S-adenosyl-l-methionine (SAM) In the shoots, Fe deficiency increased the expression of OsNAS1, OsNAS2, OsNAAT1, and OsDMAS1, but decreased that of OsNAS3. In comparison, Zn deficiency increased the expression of OsNAS3 and especially that of OsNAAT1. The expression of OsDMAS1 was slightly increased in Zn-deficient shoots. Transcripts of OsNAS1 and OsNAS2 were not detected in control or Zn-deficient shoots (Fig. 3a). Higuchi et al. (2001) reported that the concentration of endogenous DMA was increased in Fe-deficient rice shoots. We confirmed that the concentration of endogenous DMA in Fe-deficient shoots was about three times higher than that in control shoots (Fig. 4a). The concentration of endogenous DMA in Zn-deficient shoots was about two times higher than that in control shoots (Fig. 4b). Although the concentration of endogenous DMA in Zn-deficient shoots was lower than that in Fe-deficient shoots, the expression of OsNAAT1, which encodes the enzyme that converts nicotianamine to its 3′′-keto acid, in Zn-deficient shoots was much higher than that in Fe-deficient shoots (Fig. 3a). Therefore, we measured the endogenous NA, which is an intermediate of DMA. Corresponding to the expression pattern of OsNAAT1, endogenous NA in Zn-deficient shoots was under the detection limit by HPLC, while in Fe-deficient shoots NA was lower than in the control shoots (Fig. 4b). Fig. 4Concentration of endogenous DMA and NA in rice shoots (a) Concentration of endogenous DMA. (b) Concentration of endogenous NA. The plants were harvested after collection of root exudates (Fig. 2), then DMA or NA was measured using HPLC. -Zn, zinc deficiency; -Fe, iron deficiency; N.D., not detected. Values are mean ± SD (n = 4) Contribution of DMA to Zn translocation in Zn-deficient rice To investigate whether DMA is involved in Zn absorption and translocation, a PETIS experiment was performed using Zn-deficient rice (Fig. 5; supplemental movie 1, supplemental Fig. 1). 62Zn chelated with DMA (62Zn-DMA) or 62Zn without DMA (62Zn2+) was supplied to Zn-deficient rice roots. Images of the plants and the localization of 62Zn after 6 h of absorption are shown in Fig. 5a. In both plants, most of the 62Zn absorbed into the plant was observed at the DC and in the bottom of the leaf sheath (Fig. 5a). The radioactivity of the plant supplied with 62Zn2+ was higher than that of the plant supplied with 62Zn-DMA both in the roots and shoots (Fig. 5b, c). This indicates that compared to Zn-DMA, Zn2+ is the preferred form for root absorption in Zn-deficient rice. Real-time imaging of the 62Zn absorption was observed by PETIS analysis (Fig. 5d). More 62Zn was observed in the bottom of the leaf sheath supplied with 62Zn2+ than in that supplied with 62Zn-DMA. This observation was quantified by ROI analysis (Fig. 5e) On the other hand, beginning at 90 min after the start of the experiment, more 62Zn was observed in the newest leaf of the plant supplied with 62Zn-DMA than in that supplied with 62Zn2+ (Fig. 5d, f). This indicates that Zn-DMA is preferred over Zn2+ for translocation within Zn-deficient rice plants. Continuous PETIS imaging is shown in supplemental movie 1. We performed three experiments of 62Zn absorption. Due to the very short half-life of 62Zn, however, the radioactivity was different in each experiment. Therefore, we showed the relative radioactivity of each experiment in supplemental Fig. 1. In each experiment, 62Zn2+ was more absorbed than 62Zn-DMA. Fig. 562Zn movement from roots in Zn-deficient rice using PETIS (a) Plant image (left) and BAS image (right). (b) Radioactivity in roots after 6 h of absorption. (c) Radioactivity in shoots after 6 h of absorption. (d) Time course of the accumulation of radioactivity as determined by PETIS analysis. The data were scored every 3 min, and the images shown were taken at 90-min intervals for 6 h after supplying 62Zn through the roots. (e) Region of interest (ROI) analysis of 62Zn movement in the bottom of the sheath white boxed in (d). (f) ROI analysis in the newest leaf yellow boxed in (d) We conducted an additional PETIS experiment in which Zn-DMA or Zn2+ was supplied to Zn-deficient rice at the cut second newest leaf (Fig. 6; supplemental movie 2, supplemental Fig. 2). Plant images and the localization of 62Zn after 6 h of absorption are shown in Fig. 6a. Most of the 62Zn absorbed from the second newest leaf remained in that leaf, but some 62Zn was translocated to the other leaves and roots as well as to the DC and leaf sheath. The level of radioactivity in the entire plant, except for the second newest leaf, was higher in the plant supplied with 62Zn-DMA than in the plant supplied with 62Zn2+ (Fig. 6b), while that of the second newest leaf was not higher in the plant supplied with 62Zn-DMA than in the plant supplied with 62Zn2+ (supplemental Fig. 3). PETIS analysis showed that more 62Zn appeared continuously in the plant supplied with 62Zn-DMA than in the plant supplied with 62Zn2+. This suggests that 62Zn-DMA is translocated more rapidly than 62Zn2+ within the plant. Fig. 662Zn movement from cut leaf in Zn-deficient rice using PETIS (a) Plant image (left) and BAS image (right). Arrows indicate the point where 62Zn was absorbed. (b) Radioactivity in the entire plant except for the second newest leaf after 8 h of absorption. (c) Time course of the accumulation of radioactivity as determined by PETIS analysis. The data were scored every 3 min, and the images shown were taken at 120-min intervals for 8 h after supplying 62Zn through the shoots Discussion The concentration of endogenous DMA is increased in Zn-deficient shoots, but DMA secretion from the roots is not increased Previously we reported increased MAs secretion in Zn-deficient barley (Suzuki et al. 2006); however, in rice, Zn deficiency did not increase the secretion of DMA, but rather slightly decreased it (Fig. 2). Fe deficiency increased MAs secretion in both rice and barley (Suzuki et al. 2006; Fig. 2). In barley, expression of the genes encoding the MAs biosynthetic enzymes was increased in the roots of both Zn- and Fe-deficient plants (Suzuki et al. 2006). On the other hand, in rice, the expression pattern of the genes involved in DMA synthesis in roots was increased in Fe-deficient roots, while the expression of OsNAS1, OsNAS2, and OsDMAS1 was not increased in Zn-deficient roots (Fig. 3a). Inoue et al. (2003) suggested that OsNAS1 and OsNAS2 are involved in DMA secretion. Supporting this idea, the expression pattern of OsNAS1 and OsNAS2 in roots was in good correlation with the amount of DMA secreted (Figs. 2, 3a). On the other hand, OsNAS3 is thought not to be involved in DMA secretion, because the expression of OsNAS3 is restricted to the pericycle cells adjacent to the protoxylem and companion cells but not in other cortical cells (Inoue et al. 2003). The expression of OsNAS3 is observed in whole leaf tissues in the control plants (Inoue et al. 2003). The expression of OsNAS3 was increased in Zn-deficient roots and shoots (Fig. 3a); therefore, OsNAS3 may function to synthesize NA or subsequent components, such as DMA, for the distribution of Zn within Zn-deficient plants. It is noteworthy that Zn deficiency enhanced the expression of OsNAS3 in shoots, while Fe deficiency enhanced the expression of OsNAS1 and OsNAS2. This suggests that NA and DMA are involved both in Fe and Zn homeostasis, but that their synthesis under conditions of Fe and Zn deficiency is catalyzed by different isozymes. The concentration of endogenous DMA in the shoots was increased by Zn and Fe deficiencies (Fig. 4a). Although the induction of OsNAAT1 in Zn-deficient shoots was much higher than that in Fe-deficient shoots, the concentration of DMA in Zn-deficient shoots was lower than that in Fe-deficient shoots. In accordance with the high level of OsNAAT1 expression in Zn-deficient shoots, the concentration of NA in Zn-deficient shoots was low (Fig. 4b). This result led to us hypothesize that OsDMAS1 is a rate-limiting enzyme for DMA synthesis in Zn-deficient rice. Zn-DMA is the preferred form for long-distance transport of Zn in Zn-deficient rice Recently, we showed that Zn-deficient barley absorbs more Zn-DMA than Zn2+ from its roots (Suzuki et al. 2006). In the present paper, however, less Zn-DMA than Zn2+ was absorbed from the roots of Zn-deficient rice (Fig. 5). These findings are correlated with the change in the amount of MAs secreted under Zn deficiency. In barley, MAs secretion is increased by Zn-deficiency, and this contributes to the absorption of Zn from the soil. On the other hand, in rice, the level of DMA secretion was slightly decreased (Fig. 2). This suggested that rice may prefer to absorb Zn2+ rather than Zn-DMA. Our PETIS experiment clearly showed that more 62Zn was translocated to the newest leaf when 62Zn-DMA was supplied than when 62Zn2+ was supplied, while the opposite tendency was observed at the bottom of the leaf sheath (Fig. 5, supplemental movie 1). This finding suggests that Zn-DMA is the preferred form for long-distance transport of Zn in Zn-deficient rice. The preferential translocation of 62Zn-DMA was also observed in the PETIS experiment in which 62Zn-DMA and 62Zn2+ were supplied from the Zn-deficient leaf (Fig. 6, supplemental movie 2). These data also support the idea that Zn-DMA is the preferred form for long-distance transport of Zn in Zn-deficient rice. Since DMA synthesis might be promoted by Zn deficiency in barley shoots (Suzuki et al. 2006), graminaceous plants have a common system for Zn translocation within the plant, while the contribution of MAs to Zn uptake from the soil differs among species. Further studies will enable us to understand the mechanism of Zn transport, to manipulate the distribution of Zn in order to produce crops that tolerate Zn-deficient stress, and to enhance the Zn content of crop seeds. Electronic supplementary material (EPS 348 kb) (EPS 362 kb) (EPS 353 kb) (MOV 2400 kb) (MOV 2257 kb)

Pediatr_Nephrol-4-1-2214826

Rituximab therapy for juvenile-onset systemic lupus erythematosus

Rituximab (RTX), an anti-CD20 monoclonal antibody, has been proposed for use in the therapy of systemic lupus erythematosus (SLE). We present the initial long-term experience of the safety and efficacy of rituximab for treatment of SLE in children. Eighteen patients (mean age 14 ± 3 years) with severe SLE were treated with rituximab after demonstrating resistance or toxicity to conventional regimens. There was a predominance of female (16/18) and ethnic African (13/18) patients. All had lupus nephritis [World Health Organization (WHO) classes 3–5] and systemic manifestations of vasculitis. Clinical disease activity of the SLE was scored with the SLE-disease activity index 2K (SLEDAI-2K). Patients were followed-up for an average of 3.0 ± 1.3 years (range 0.5 to 4.8 years). B-cell depletion occurred within 2 weeks in all patients and persisted for up to 1 year in some. Clinical activity scores, double-stranded DNA (dsDNA) antibodies, renal function and proteinuria [urine protein to creatinine ratio (Upr/cr)] improved in 93% of the patients. Five patients required multiple courses of RTX for relapse, with B-cell repopulation. One died of infectious endocarditis related to severe immunosuppression. In conclusion, our data support the efficacy of rituximab as adjunctive treatment for SLE in children. Although rituximab was well tolerated by the majority of patients, randomized controlled trials are required to establish its long-term safety and efficacy. Introduction Systemic lupus erythematosus (SLE) in children is known to carry a worse prognosis than in adults, especially in those of African and Hispanic ethnicity [1, 2]. Although treatment protocols for proliferative lupus nephritis have evolved during the past two decades, pediatric regimens have been center based and selectively derived from adult protocols [3]. These protocols primarily include cyclophosphamide (CYC), which carries long-term toxicities related to malignancy and gonadal dysfunction [4]. Most recently, B-lymphocyte function has been recognized as a major component in the pathogenesis of autoimmune diseases [5]. Rituximab (RTX), a chimeric monoclonal antibody directed against CD20 lymphocytes (commonly called B-cells), was developed as a primary treatment against B-cell lymphoma [6] and is now approved for use in the treatment of rheumatoid arthritis [7]. Although clinical trials of the use of RTX in adults with SLE are substantial and ongoing [8], studies in children have been limited [9, 10]. A collaborative retrospective and uncontrolled multicenter experience from Europe reported mixed results [11]. Unfortunately, that study failed to use a single protocol, and many patients were treated concurrently with more toxic medications, which made safety analysis unreliable [11]. The purpose of our study was to determine the safety and efficacy of RTX therapy in the treatment of children with severe SLE. Over the course of 7 years, we retrospectively assessed the indications and responses to RTX therapy in a cohort of children with active SLE that was refractory to conventional therapies or who had clinical indications prohibiting the use of more toxic immunosuppressant drugs. Patients and methods A retrospective analysis was performed on a cohort of 51 patients diagnosed with SLE and lupus nephritis who received their care at Holtz Children’s Hospital, University of Miami Miller School of Medicine, between January 1996 and June 2007. The study was approved by the institutional review board, with waiver of consent authorization, and all subjects were assured anonymity in compliance with the Health Insurance Portability and Accountability Act (HIPAA). The medical records were reviewed for patients’ demographic characteristics, age at diagnosis, prior and current medical treatments, type of lupus nephritis and serologic and clinical response to therapy. Children were considered eligible for inclusion in the analysis if they fulfilled the following criteria: (1) American College of Rheumatology (ACR) criteria for the diagnosis of SLE [12]; (2) age < 16 years at the time of diagnosis of SLE; (3) treatment with the anti-CD20 monoclonal antibody, rituximab, during the observation period from January 2000 through October 2007. Clinical protocol Rituximab (RTX) was administered weekly for two to four doses. The initial dose was 188 mg/m2, infused over 4 h. Subsequent doses were 375 mg/m2 per dose, infused over 6 h to 8 h. Premedication initially consisted of diphenhydramine and acetaminophen, administered 30 min before the rituximab infusion. However, mild infusion reactions, including pruritis, nausea and emesis, resulted in our adding hydrocortisone 50–100 mg intravenously 30 min prior to each infusion. Lymphocyte subsets were assayed before and after each course of RTX therapy at 1- to 3-month intervals. This included B cells assayed as CD19+ lymphocytes and T-cells assayed as CD4 and CD8 lymphocytes as total and percent of the total lymphocyte cell count. The serum immunoglobulin levels were measured every 1 to 6 months prior to and after the course of RTX. Adverse events were recorded from the time of the first infusion until 1 year following the treatment course. Lupus disease activity index and serology Clinical and biological parameters were assessed prior to the RTX infusion course, at 1- to 3-month intervals thereafter. The clinical disease activity was scored with the SLE-disease activity index 2K (SLEDAI-2K), which has a maximum activity score of 105 [13]. Laboratory evaluations included serum complement components (C3 and C4) and anti-double stranded DNA (Anti-dsDNA) antibody and anti-nuclear antibody (ANA). Lupus nephritis and renal disease activity Renal biopsies were classified according to the new proposed modifications of the World Health Organization (WHO) classifications [14]. Three patients were treated at the time they were on hemodialysis for extra-renal manifestations of SLE. For those patients not on dialysis, degree of proteinuria and renal function were assessed in response to RTX therapy. Degree of proteinuria was determined by the random urine protein to creatinine ratio (Upr/cr) in milligrams per milligram (mg/mg), with normal <0.2 and nephrotic range >1.0 [15, 16]. Serum albumin (Salb) was assayed as an indirect measure of the nephrotic syndrome. Serum creatinine (Scr) was assayed, and an estimation of glomerular filtration rate (eGFR) was derived by the height index formula of Schwartz, expressed as milliliters per minute per 1.73 meters squared (ml/min per 1.73 m2) [17]. Complete renal remission was defined as normalization of Upr/cr and eGFR. Partial remission was defined as > 50% decline in Upr/cr. All serum and urine chemical analyses and immunoassays were performed in the central hospital clinical laboratory or the Quest referral laboratories. Statistical methods All data sets were analyzed for Gaussian distribution using the D’Agostino–Pearson omnibus test for normality. The data were calculated into average values at baseline and at 2 months, 4 months, and 6 months after initiation of treatment. Variables at baseline, during and after the treatment were compared by the Wilcoxon matched-pairs signed-ranks test. Differences in parameters between the 2 independent groups were analyzed using Mann-Whitney and the Friedman test with Dunn’s multiple comparisons post tests for repeated measures of nonparametric data. P values of less than 0.05 were considered significant. All results are expressed as mean ± standard deviation (SD). Results Patients’ demographics During the period of observation 18 patients were treated with RTX for at least one course of two to four doses. There were 16 female patients and two male patients. Mean age at the time of diagnosis of SLE was 10.7 ± 2.5 years (range 7 to 14 years). Mean duration of disease prior to treatment with RTX was 3.1 ± 2.5 years (range 2 months to 8 years). Age at the time of first RTX treatment was 14.2 ± 3.3 years, and current age at the time of this report was 17 ± 5.0 years. All patients had clinical evidence of lupus nephritis. Individual patient demographics, clinical manifestations of SLE and indications for rituximab therapy for patients not on hemodialysis are shown in Table 1. All patients received concurrent therapy with low-dose corticosteroids (CSs) and hydroxychloroquine (HCQ). Sixteen patients had failed or had suffered toxic effects from multiple courses of intravenous therapy with cyclophosphamide (CYC) and corticosteroids. Treatment with RTX included concurrent or subsequent treatment with maintenance doses of mycophenolate mofetil (MMF) or azathioprine (AZT) similar to recently published adult protocols [18]. Table 1Patients’ baseline characteristics, indications for rituximab treatment, and clinical course (F female, M male, H Hispanic, B Black, HT hypertension, CYC cyclophosphamide, CyA cyclosporine A, AZT azathioprine, MMF mycophenolate mofetil, IVIG intravenous therapy with gamma globulin, CS corticosteroids, HXQ hydroxychloroquine, FR full remission, PR partial remission)PatientGenderAge at diagnosis (years)RaceIndication for rituximabNephritis classPrevious and/or concurrent therapiesaUpr/cr (mg/mg)OutcomeBefore RTXAfter RTX1F12.0BNephritis, serositis, Coomb’s (+) anemia, CYC-toxicityIVCYC, MMF13.00.1FR2M10.0WNephritis, serositis, CS toxicityIVCYC, MMF2.10.6PR3F11.0BNephritis, serositis, Coomb’s(+) anemia, CYC- toxicity sepsisIVCYC, MMF4.01.7PR4F8.0HPulmonary HT, nephritis, Coomb’s(+) anemiaIV-VCYC, MMF, IVIG2.60.2FR5F12.0BOphthalmic vasculitis, nephritisIV-VCYC, AZT0.40.1FR6F14.0BNephritis, Coomb’s(+) anemia, CS toxicityNACYC, AZT, MMF, IVIG0.60.3PR7F12.0BNephritis, serositis, CYC-toxicity sepsisIV-VCYC, AZT, MMF, IVIG2.6NADied8F12.0BNephritis, serositis, Coomb’s(+) anemiaIVCYC, AZT, IVIG3.61.9PR9F14.0BNephritis, serositis, ataxia, miliary tuberculosisIIIIVIG1.70.2FR10F7.0HNephritis, serositis, arthritis, CYC-toxicity sepsisIVCYC, MMF, IVIG3.00.7PR11F12.8HNephritis, serositis, arthritis, obesityVMMF9.70.2FR12F7.0BNephritis, thrombocytopenia, CYC toxicityIV-VCYC, AZT, MMF, IVIG, CyA5.40.2FR13F7.0BNephritis, Coomb’s(+) anemia, skin vasculitisIVMMF1.50.2FR14M12.5HNephritis, serositis, arthritis, CYC-toxicity sepsisIIICYC, MMF, IVIG2.30.8PR15F13.0BNephritis, arthritis, skin vasculitis, Coomb’s(+) anemiaIVCYC, MMF, IVIG6.80.9PRaAll patients were treated with CS and HXQ All three patients on hemodialysis responded positively to the RTX therapy. Patient HD-1 had Raynaud’s syndrome and finger necrosis; her symptoms resolved, and she recently received a transplant, 4 years after RTX treatment. Patient HD-2, who had been on ventilator support with pneumonitis, pleuritis and carditis for 1 month prior to receiving RTX, recovered and has been stable on hemodialysis as an outpatient for >4 years since RTX therapy. Patient HD-3, after 3 years on hemodialysis, developed debilitating chorea with increased lupus serology. She did not respond to anti-convulsant therapy. The chorea resolved within weeks of the RTX therapy. She remains on dialysis without recurrence of chorea. B-cell depletion and re-population Figure 1 shows the average B-cell depletion relative to the beginning of the first dose of RTX. Patients received an average of 4.3 ± 1.6 doses overall. B-cell depletion was achieved within 2 weeks of the first dose of RTX and lasted an average of 3 ± 4 months (range 3 to 12 months). Five patients (28%) received repeated courses of RTX therapy ranging from two to four courses of one to four additional doses following re-population of the B-cell line in the setting of clinical relapse of SLE. Overall follow-up period from the initial dose of RTX to this report was 3.0 ± 1.3 years. It should be noted that those patients who did not have clinical relapse were not routinely assayed for lymphocyte subsets. Therefore, it is not known if or when those patients with persistent clinical remission had their B-lymphocyte lineage re-populated. Fig. 1Graph of B-cell depletion over time from before rituximab therapy to 12–18 months after rituximab therapy. Asterisks indicate significant difference from the post-rituximab values; P < 0.01 Table 2 describes the changes in the lymphocyte subsets from baseline to the nadir drop in the B-lymphocyte lineage. Total lymphocyte counts, as well as CD4 and CD8 T-cell counts, tended to be lower before RTX treatment and increased after RTX therapy. Similarly, the levels of serum immunoglobulins remained normal in comparison with the pre-RTX values. Table 2Clinical parameters before and after rituximab therapy (NS not significant)ParameterBefore rituximab therapyAfter rituximab therapyPTotal lymphocyte count1044 ± 5961734 ± 1535NSCD4+ lymphocytes344 ± 217556 ± 264NSCD19+ lymphocytes243 ± 22374 ± 710.005Immunoglobulin IgG1603 ± 8481596 ± 1055NSImmunoglobulin IgM273 ± 514283 ± 531NSUpr/cr (mg/mg)4.0 ± 3.50.6 ± 0.60.001Serum albumin (g/dl)2.6 ± 0.73.5 ± 0.60.001Scr (mg/dl)1.2 ± 0.40.6 ± 0.20.001eGFR (ml/min/1.73m2)86 ± 32144 ± 370.0003Corticosteroid dose (mg/m2 per day)79 ± 2613 ± 20<0.0001SLEDAI-2K score47 ± 1925 ± 140.0004 Lupus activity and auto-antibody response Table 2 and Fig. 2 summarize the clinical response to RTX therapy. The average lupus activity score (SLEDAI) improved in all patients. Average dsDNA antibody decreased and serum C3 complement increased significantly after RTX therapy. Other serological parameters showed a trend towards improvement as well. Fig. 2Composite graphs depicting response to rituximab therapy on clinical parameters. Panela SLEDAI [13] scores before and after rituximab therapy. Panelb proteinuria (Upr/cr) before and after rituximab. Panelc auto-antibody titers before and after rituximab. Paneld serum complements as C3 and C4 complements. Asterisks indicate significant differences from the post-rituximab values; P < 0.01 All 15 patients not on dialysis had active lupus nephritis with significant proteinuria. Table 1 provides detailed information regarding the classification of the lupus nephritis for each patient, together with extra-renal manifestations and concurrent or previous therapies, as well as their final outcome. All parameters improved or stabilized in 14/15 (93%) patients. Seven had total remission of proteinuria, while the other seven had partial remission, with significant improvement in proteinuria and renal function. Coincident with the improvement in renal disease, the requirement for maintenance corticosteroid therapy was reduced in most patients, with an average fall in corticosteroid dosing from 79 ± 29 mg/m2 per day before RTX therapy to 13 ± 20 mg/m2 per day after RTX therapy (P < 0.0001). Adverse events One patient (#7) received three doses of RTX shortly after a cycle of intravenous cyclophosphamide (CYC) administration in an effort to control lupus serositis and nephritis. She developed Staphylococcus aureus endocarditis, with vegetations on the mitral and aortic valves. She died after open heart surgery. This was considered a death from overwhelming infection most likely related to excessive immune suppression. Her CD4 count prior to RTX was 220/mm3, possibly related to prior CYC therapy. At the time of her death her serum immunoglobulin levels were normal. Patient #13 had systemic vasculitis and class IV lupus nephritis. Since she was only 7 years of age, it was decided to treat her with intravenous administration of Solu-Medrol followed by weekly RTX for four doses and concurrent mycophenolate mofetil (MMF) as induction therapy. She responded well to RTX, with resolution of her proteinuria and clinical remission of the lupus nephritis. However, she developed symptoms of cerebral vasculitis, with seizures and a cerebral infarct, demonstrated by magnetic resonance imaging (MRI) after her fourth dose of RTX. She was treated with intravenously administered gamma globulin (IVIG) and two doses of cyclophosphamide. Her neurologic deficits resolved and her vasculitis reversed, as demonstrated by MRI. She remains in remission of the lupus nephritis and cerebritis 6 months after RTX therapy. Mild adverse reactions occurred in approximately half the patients and included pruritis, nausea, or malaise associated with the infusions. These became less when hydrocortisone was added to the pre-medication regimen and the infusion rate was slowed to 6 to 8 hours. Discussion To date, this report has provided the largest long-term experience in children treated with targeted B-cell depletion using the monoclonal antibody, rituximab, for severe SLE. We showed a beneficial effect of the drug in over 90% of the children in this series, which is consistent with that of previous reports [8, 19–21]. Moreover, we were able to demonstrate efficacy in controlling proteinuria in conjunction with a fall in auto-antibodies in those patients with active lupus nephritis, which mirrored that in a series of adult patients [19]. B-lymphocyte dysregulation is central in the pathogenesis of systemic lupus, with disruption of an individual’s immune homeostasis. These mechanisms are multi-dimensional and are not limited to the single role of B cells in antibody production [22]. Hence, the elimination of the B-cell lineage, with potential re-population with a “healthier” line, is theoretically curative to the patient. The safety of rituximab in young lupus patients remains unanswered. Most of the patients in our cohort tolerated the rituximab well. However, as in the European trial [11], our patients were on variable multi-drug regimens. The one patient who died did so from infectious complications in conjunction with severe immune suppression from multiple cytotoxic agents, including both rituximab and cyclophosphamide. Another young patient developed symptoms of cerebral vasculitis, with seizures and encephalopathy. The differential diagnosis included the dreaded progressive multifocal leukoencephalopathy (PML), recently reported to occur in patients treated with rituximab [23]. Although she showed clinical recovery after cyclophosphamide therapy, the case emphasizes the uncharted risks imposed by the use of drugs without well-studied indications. The efficacy of rituximab may be short-lived in some patients. Resistance to RTX B-cell depletion may be related to antibodies against the human-mouse chimeric protein [8]. It may also be genetically influenced by polymorphisms of the FcRIIIa gene, responsible for the Fc-γ receptor required for RTX binding to phagocytes [24]. In our series, all patients had some degree of B-cell depletion after the initial course of RTX. However, five of the 18 patients (28%) developed clinical relapse in association with re-population of the B-cell lineage. All but one of these responded to additional courses of RTX. This is similar to recent long-term reports in a series of adults [25]. Treatment regimens in children with SLE do not conform with those of adults developed by carefully designed randomized controlled trials. This may be partially attributable to the fact that children ≤ 16 years of age are excluded from these trials. This has resulted in an ambiguous and often anecdotal approach to pediatric patients, especially those with early onset and severe disease. Although intravenous cyclophosphamide treatment was established as induction therapy for proliferative lupus nephritis over two decades ago [26], its short- and long-term toxicity delayed its incorporation into pediatric regimens [27]. Paradoxically, those with severe disease have received excessive courses of toxic medications in futile efforts to preserve renal function [27]. More recently, less toxic alternative treatment regimens, including mycophenolate mofetil for both induction and maintenance therapy, have resulted in equal or improved renal survival over conventional therapies [18, 28]. Since treatment protocols of children do not conform to conventional protocols for adults, attempts at reporting multicenter experiences are hampered by a lack of consistency in treatment regimens [11, 22]. Therefore, anecdotal center experiences such as our own are necessary in developing treatment protocols tailored towards efficacy while decreasing long-term risks on growth, fertility and malignancy. In conclusion, RTX therapy offers a potential innovation in the current treatment regimens for children with aggressive SLE that includes both renal and extra-renal manifestations. The development of a randomized controlled treatment protocol for induction and maintenance therapy in pediatric patients is very much warranted.

Matern_Child_Health_J-2-2-1592247

A Perspective of Preconception Health Activities in the United States

Objectives: Information regarding the type and scope of preconception care programs in the United States is scant. We evaluated State Title V measurement and indicator data and abstracts presented at the National Summit on Preconception Care (June 2005) in order to identify existing programs and innovative strategies for preconception health promotion. Introduction The June 2005 National Summit on Preconception Care, sponsored by the Centers for Disease Control and Prevention (CDC) and the March of Dimes, assembled an array of health care providers, public health practitioners, and researchers in an effort to promote the exchange of information and ideas related to preconception care at the local, state, and national levels. A key issue emerging from conference discussions was the perceived lack of information pertaining to the nature and scope of preconception health services currently being provided in the United States, whether explicitly defined or falling under another umbrella of services. Another issue was the recognition that a compilation of local, state, and national resources would provide important information related to the ability of current public health systems to address preconception care needs, as well as allow for the description of innovative models for the delivery of such services. Attended by more than 400 registered participants representing a myriad of agencies and organizations, the summit generated significant response within the public health and clinical communities and reflects a perspective that cannot be readily gleaned from literature reviews or the inspection of existing programmatic guidelines: that is, the topic of preconception health has garnered substantial interest and support and has already been incorporated into numerous existing programs, policies, and strategies in the United States. Unique to the summit was an opportunity to learn about the development and administration of preconception care programs within clinical and population-based settings as Medline, PubMed, and other Internet searches have revealed very little information regarding the development and implementation of preconception care programs within a local context. However, as evidenced by the numerous programmatic components featured in the conference abstracts, such programs are being introduced in many different formats, among various private and public organizations, and across the entire nation. Although little is now known at this writing about the scope and extent of U.S. programs and projects to promote preconception health, this article uses readily available sources (in the published professional literature and the gray literature of government and organization reports) to provide a snapshot of related activities in the United States. Thus, we are able to report the findings of a scan of State Title V Performance Measures and Priority Needs pertaining to preconception care and a detailed summary of the abstracts of the papers presented at the Summit.Fig. 1State Title V priority needs focused on preconception health and health care, U.S. 2005 Methods The Maternal and Child Health Bureau website (http://mchb.hrsa.gov/) was used to ascertain the proportion of states including preconception health indicators in State Performance Measures and Priority Needs, as reported in the State Title V Maternal and Child Health Service (MCH) Block Grant FY 2004 Annual Report and FY 2006 Application, Form 14. The web-based Title V Information System (https://perfdata.hrsa.gov/mchb/mchreports/Search/core/ measureindicatemenu.asp) was used to search State Title V Performance Measures and Priority Needs for indicators related to preconception health. Initial search parameters included all keywords and all populations and were further refined by selecting for birth outcomes, reproductive health, and women. Those indicators for which infants, newborns, children, men, or service providers represented the sole target population were excluded. The remaining measures were evaluated based on their relevancy to preconception health. Indicators using the term “preconception” or “preconceptual” and those that did not use such terminology but adequately captured the concept were considered to fall within the domain of preconception health and health care. States identifying preconception health Priority Needs were depicted on a national map. States reporting relevant Performance Measures were identified and selected measures were described in tabular format. Finally, a comprehensive list of State Title V Performance Measures and Priority Needs related to preconception health, that reflected the tone and language of multiple states was developed. All abstracts submitted and accepted for presentation at the National Summit on Preconception Care (June 2005) were pooled and subjectively categorized into five broad topic areas: research pertaining to preconception risk assessment and outcome evaluation; preconception care programs and activities; tools intended for provider or patient preconception health education; clinical practice strategies; and public policy related to preconception care. Results State Title V Performance Measures and Priority Needs The federal MCH Block Grant guidance requires states to report annually on National “Core” Performance Measures and to assess needs and set priorities every 5 years. In addition, each state must develop 7 to 10 additional Performance Measures relevant to its own priority needs and programs. Priority Needs reflect a state’s focus for programmatic efforts throughout the next five years and can be adjusted during interim years in response to variations in needs. Performance Measures describe a specific maternal and child need that, when effectively addressed, can lead to improvements in health. In 2005, states completed the 5-year needs assessments and set out priorities and targets for 2010. A previous analysis of the 2000 priorities and measures found that some states, but not all, included measures for preconception care or visits, as did Healthy People 2000 National Health Promotion and Disease Prevention Objectives [1]. Twenty-three states reported a Priority Need that focused on preconception health and health care (Fig. 1). “Preconception” or “preconceptual” health was mentioned by 10 states as a Priority Need in maternal and child health. In other states, Priority Needs indirectly addressed preconception care via broad objectives such as improving the health of women of childbearing age, promoting reproductive health and sexual responsibility, and advancing a holistic care continuum for women’s health. A total of 42 states and jurisdictions reported at least one Performance Measure related to preconception care, neural tube defects, birth spacing, family planning, unintended pregnancy, and healthy weight/obesity (Table 1). Unintended pregnancy and healthy weight or obesity were the measures identified most frequently (43% and 19%, respectively). The majority of the measures pertaining to birth spacing or parity, or both, addressed repeat pregnancies among adolescent populations. A total of thirty states or territories reported measures on tobacco use during pregnancy; however, only one focused on the proportion of women who smoked during 3 months prior to pregnancy. Measures pertaining to smoking rates among women of childbearing age were reported by two states. Four states addressed mental health among women of childbearing age and postpartum depression was identified in three others. Two states included measures pertaining to preventive oral health services among women of childbearing age (data not shown). Table 2 provides examples of selected State Performance Measures and Priority Needs derived from 18 states. Variations in state-selected preconception health indicators are evident, with some states detailing various components of preconception care and others employing a broader focus on reproductive health. Table 1States or U.S. jurisdictions reporting Performance Measures related to selected preconception health topicsPerformance measureStatePreconception careNeural tube defects/folic acidBirth spacing/parityFamily planningUnintended pregnancyHealthy weight/obesityArizona×Arkansas×California×Delaware×District of Columbia×a×Florida×aGeorgia××aGuam×Hawaii×Illinois×Indiana×Kentucky×Louisiana×Maine×Marshall Islands×bMaryland×Massachusetts××Michigan×Minnesota×Mississippi×aMontana×Nebraska×Nevada×New Jersey×aNew Mexico××New York×North Carolina×××North Dakota×Northern Mariana Islands×Ohio×Oklahoma×Puerto Rico×South Carolina×TennesseeTexas×South Dakota×Wyoming×cUtah×Vermont×Washington×bWisconsin×aMeasure references repeat births/pregnancies among adolescents onlybMeasure references family planning counseling or postpartum birth control planning as addressed during routine prenatal carecMeasure references multivitamin use in month prior to conceptionTable 2Sample of selected state Performance Measures and Priority Needs related to preconception healthSelected performance measures and priority needsPreconception health and health care Performance measure  •Percentage of women of childbearing age who receive preconception health care in the local health department Priority need  •Enhance preconception care and work toward eliminating disparities in infant and maternal morbidity and mortality  •Improve women’s preconception and interconception health  •Improve indicators of health in the preconception and perinatal periods, including but not limited to smoking, alcohol, folic acid use,   family violence, intention of pregnancy, access to and use of health care  •Promote sexual responsibility and reproductive healthNeural tube defects/folic acid Performance Measure  •Percent of women of reproductive age who consume at least 400 mcg of folic acid daily  •Percent of women of childbearing age taking folic acid regularlyBirth spacing and parity Performance Measure  •Percentage of live births to women who have another birth in less than 18 months  •Percentage of repeat pregnancies among adolescents 15–19 years of age Priority need  •Reduce repeat teenage pregnancies, unintended pregnancies and inadequate spacing of births through widely available, adequately    funded comprehensive, efficiently run family planning and support services  •Increase the proportion of births that are intended, including promotion of healthy interpregnancy spacingFamily planning Performance measure  •Proportion of low-income women who receive reproductive health/family planning services Priority need  •To improve access to and utilization of contraceptive servicesUnintended pregnancy Performance measure  •Percentage of pregnancies (live births, fetal deaths, abortions) that are unintended  •Percentage of women responding to the Pregnancy Risk Assessment Monitoring System (PRAMS) survey that they wanted to be   pregnant later or not then or at any time in the future Priority need  •Improve the health of children and families by increasing the percent of births that are intended  •Promote planned pregnancies and child spacingHealthy weight/obesity Performance measure  •Percent of women (18–44) with healthy weight (BMI)  •To increase the percentage of Family Planning clients with BMI greater than the 85th percentile who receive educational materials in    the Family Planning Clinics, and are referred to community sources Priority need  •To reduce proportion of women of childbearing ages, pregnant women and children and youth with BMIs in the underweight,    overweight and obese categories Abstracts from the National Summit on Preconception Care A total of 59 abstracts accepted for presentation at the National Summit on Preconception Care were reviewed and categorized according to primary area of focus. Approximately 32% addressed preconception health research; 27% described preconception care programs and activities; 22% outlined tools for provider or patient education; 15% detailed clinical practice strategies; and 3% highlighted policy-based strategies for increasing access to preconception care services. Among the research abstracts, the topics noted most frequently pertained to methods for preconception risk assessment, potential predictors of adverse pregnancy outcomes, and variations in preconception health indicators across high-risk populations. Three abstracts described the use of the Perinatal Periods of Risk (PPOR) model [2] for assessing risks among various populations. Other issues included folic acid awareness among women and health care providers, maternal nutrition, pregnancy planning and risk behaviors, preconception hepatitis B prevention, and workplace hazards. The PPOR model facilitates the identification of four contributors to infant mortality: maternal health and prematurity, maternal care, newborn care, and infant health. Excess rates of fetal and infant mortality in any of the four components indicate a need for targeted interventions within that construct [2–4]. CityMatCH’s 2000–2002 PPOR Practice Collaborative provided public health workers in 14 U.S. cities with the skills, knowledge, and support to implement the PPOR method in various urban communities [2]. The PPOR approach is also is being used statewide by Florida and Ohio and in selected areas across the country [2–4]. The Fountain Project in Kansas City, Missouri linked PPOR techniques with the Fetal Infant Mortality Review (FIMR) process in an effort to identify the causes of consistently higher rates of infant mortality among African-American residents compared with their non-Hispanic White counterparts. The findings of the analysis indicated an excess rate of maternal health or prematurity-related deaths among African-American infants. In light of the contributing social factors identified by the FIMR, the development of a Women’s and Children’s Wellness Center was proposed to provide social and clinical services for high-risk families (Cook BE, Guillory VJ, Cai J, Hoff GL, Manning J, unpublished data, 2005). In an effort to facilitate the analysis of various perinatal health outcomes, a group of nine counties surrounding the San Francisco Bay (California) formed the Maternal, Child and Adolescent Health Bay Area Data Collaborative (BADC). The group used the PPOR model to evaluate recent county and regional fetal-infant mortality rates and reported substantial excess in the proportion of deaths among African-American infants, particularly in the prematurity and maternal health component of the model. Using a national comparison group, these investigators were able to determine that approximately 56% of the excess deaths among African-American infants could be prevented by targeting preconception and interconception risk factors (Stein EJ, Abramowitz A, Brown J, Chabra A, unpublished data, 2005). Federally funded Healthy Start projects were highly represented among the 16 abstracts detailing preconception care programs and activities presented at the Summit. Of these, seven abstracts described the modification or expansion of existing programs to include direct services or community-based interventions for high-risk postpartum mothers or all women of reproductive age. The Magnolia Project in Jacksonville, Florida is an example of a Healthy Start program in which a comprehensive array of health services was offered in an effort to improve birth outcomes. Services included, but were not limited to, case management, education and risk reduction, and well-woman care. The target population was high-risk African-American women aged 15–44 years who lived in five zip code areas of Jacksonville. The project operates as a collaborative between the Northeast Florida Healthy Start Coalition, the Duval County Health Department, and local community-based organizations. As reported, a 2004 assessment of project services conducted by the Health Resources and Services Administration (HRSA) Office of Performance Review found high rates of success (>70%) in the resolution of key risks (for example, lack of family planning, and rates of repeat sexually transmitted diseases) among the project participants (Brady, CM, unpublished data, 2005). The Missouri Bootheel Healthy Start project, a provider of referral and education services in a five county area of southeast Missouri, uses community-based education and interventions to improve adverse perinatal outcomes. The project has been one of few in the area that has promoted a family-focused approach to health education through the inclusion of various services directed at men. The curriculum for fathers has addressed a variety of issues, such as communication with the mother, addressing stress in the relationship, and the impact of nutrition on birth outcomes (Dean CG, Campbell T, Frazier V, Washington J, unpublished data, 2005). Title X family planning clinics were identified as providers of preconception primary care and folic acid awareness interventions in two programs and activities abstracts. The Women Enjoying Life Longer (WELL) Project was initiated by the Maryland Department of Health and Mental Hygiene following a community needs assessment. Comprehensive preventive women’s health services were added to three Title X family planning clinics in Baltimore County, using Title X and Maternal and Child Health funding. Augmented services included nutrition and physical activity counseling, adult immunizations, smoking cessation interventions, and preconception counseling, as well as referrals for problems such as substance abuse, depression, domestic violence, and chronic disease. Early evaluation data indicated that patients and staff have responded positively to new services, patient knowledge of women’s health has improved, and patient volume has increased 37% (Cheng D, unpublished data, 2005). The Oklahoma Birth Defects Registry developed, implemented, and evaluated a preconception “women’s health appraisal” project. The intervention was comprised of a 3-page health appraisal for women, with follow-up risk counseling, in selected family planning clinics (two rural and one urban). Pre-evaluation results indicated that 84% of nurses found the questionnaire helpful in assessing risk factors, 90% of nurses found it helpful as a guide to counseling and referrals, and 86% of patients increased their understanding of risk factors. Post-evaluation results found that 62% of patients modified one to three risk factors in a three-month period (Feuerborn VR, Pearson K, unpublished data, 2005). The remaining programs were funded either through grants, private organizations, or state/local health departments and comprised comprehensive health care programs for women with a history of preterm delivery or a low birth weight infant and broad women’s wellness interventions aimed at improving women’s health or folic acid utilization. For example, the Interpregnancy Care Program at Grady Memorial Hospital in Atlanta, Georgia employs a “resource mother” to coordinate the provision of primary health care and dental services, enhanced nurse case management, and other outreach services to African-American women who delivered a very low birth weight infant at the hospital and who qualified for indigent or charity care. For 24 months postpartum, the women were offered health care visits every 1–3 months in order to address risks known to be associated with the delivery of a low birth weight infant such as poorly controlled chronic disease, short intervals between pregnancies, reproductive tract infections, periodontal disease, nutritional deficits, substance abuse, and stress. Preliminary evaluation of the 22 women retained in the pilot program indicated that approximately one-quarter of them were affected by unrecognized or poorly managed chronic health problems and none of the participants wanted to become pregnant during the next 2 years (Dunlop A, unpublished data, 2005). Thirteen abstracts (22%) described the creation, dissemination, and evaluation of tools for patient or provider education. Of those, eight described marketing campaigns and toolkits for patient education, three specifically referenced provider education, and three assessed the use of women’s health appraisals by health care providers. For example, the California Preconception Care Initiative, Every Woman, Every Time, was created in 1989 through a partnership between Sutter Medical Center in Sacramento and the March of Dimes. The project conducted a metanalysis of the preconception care literature [5] and used this information in a consensus development process to produce a marketing packet for providers. Key components of the packet included the rationale for providing preconception care, a description of the essential elements of care, patient education materials, and information on billing methods. More than 9,000 packets were distributed statewide. An evaluation found that, among 187 providers responding, 75% indicated the information was very useful, 80% said they would distribute materials to patients, and 72% said they would use the billing codes provided. A further impact evaluation is being considered (Cullum AS, unpublished data, 2005). A research project conducted at a New York inner-city hospital served by Albert Einstein College of Medicine/Montefiore Medical Center sought to evaluate the knowledge and awareness of providers regarding preconception care. A pre-intervention chart review and a provider survey were conducted to evaluate delivery or preconception care. The two-part intervention included: [1] a 1-h lecture for all providers, and [2] a standardized preconception care form inserted into all charts. A post-intervention chart review of a convenience sample and repeated provider survey were then conducted. The result was a significant improvement in documentation of the delivery of preconception care (p<0.05); however, provider knowledge and attitudes, as measured by the survey, did not change significantly (Bernstein P, unpublished data, 2005). Clinical practice strategies were discussed in nine abstracts and included topics such as genomics, group care, screening for environmental exposures and maternal depression, smoking cessation, and provider knowledge of current practice guidelines. An example of a promising practice strategy presented at the Summit is the use of a group model of prenatal care to address topics relevant to preconception care. In 2002, the Comprehensive Family Care Center at Montefiore Medical Center implemented the group model of prenatal care developed by the Centering Pregnancy and Parenting Association. Traditional prenatal visits were replaced with group appointments lasting approximately 2 hours and attended by 10 to12 women. Typical clinical care services were provided during the visits and were supplemented with group discussions on pregnancy-related topics. Issues related to preconception health were covered during many of the meetings and included nutrition, substance abuse, contraception, and family planning. Since its inception, the group care model has been employed for 14 patient groups with high levels of patient and provider satisfaction reported, particularly related to enhanced opportunities for patient education (Bernstein P, Rising SS, Dolan S, Pardanani S, Merkatz IR, unpublished data, 2005). Finally, policy-based strategies for the funding of preconception care were described in a small number of abstracts. One pair of abstracts highlighted Illinois’ efforts to promote preconception health using public policy and funding through Medicaid waivers, Title V, and other resources (Murphy AM, unpublished data, 2005; Saunders SE, unpublished data, 2005). Identified as a priority area, preconception care has been integrated into a number of programs, most notably the Illinois Healthy Women program. This Medicaid waiver program initiative extends coverage for family planning services to women who would otherwise lose their benefits after 60 days postpartum and to all women 19 through 44 years of age who were previously enrolled in Medicaid but who had lost their benefits (Murphy AM, unpublished data, 2005). Additional efforts have been made through the Illinois Family Planning Program. Preconception education is currently provided at all state-funded family planning clinics, with 16 state family planning agencies offering additional counseling and referral for high-risk clients [6]. Other strategies employed by the Illinois Department of Human Services include statewide genetic counseling programs and folic acid campaigns, screening mothers of children enrolled in the state’s Medicaid program for perinatal depression, and programs aimed at increasing birth spacing and promoting the health of teen parents (Saunders SE, unpublished data, 2005). Conclusions In public health, effective programs are those developed in response to a well-defined problem analysis, are mindful of the local context, and apply proven interventions. Although the scientific evidence in support of some preconception interventions is relatively well described within the literature, topics discussed during the National Summit on Preconception Care reframed the issues in light of new findings regarding unmet needs, health disparities, service delivery models, and tools such as the PPOR to assess risks among populations. Variations in the type and scope of policies, programs, and strategies were evident both in the diversity of the summit abstracts and in the state Title V indicators. Clearly, a preconception focus is being woven into existing programs and guiding the development of new initiatives. Innovative strategies are being implemented that target a variety of populations including community groups, health care providers, medical students, adolescents, and fathers. Finally, many state indicators are adopting a broader language to encompass the health needs of women of childbearing age and support the notion of a lifespan approach to preconception health. It should be noted that the findings reported in this brief are limited by a number of factors. First, state measures and priorities reported by the federal Maternal and Child Health Bureau might not reflect the programs and projects actually being implemented by states and communities; they are only an expression of interest and concern. Next, the content of the abstracts accepted for the National Summit on Preconception Care presented a selection of current research, policies, and programs and, therefore, does not fully represent the scope of national preconception care projects. Finally, the identification of preconception health state Title V indicators required a certain amount of subjective interpretation regarding the actual focus of the Priority Need or Performance Measure. Although the authors have considerable knowledge of the definition and content of preconception care, it is possible that certain indicators might have been incorrectly excluded or included due to misinterpretation. Notwithstanding these limitations, the results of the present analysis indicate a widespread recognition of the need for a continuum of care that starts well before birth, involves both men and women, and employs a lifespan approach to emotional and physical well-being. Because preconception interventions can take place at any time during a woman’s reproductive life and across a variety of dimensions, countless opportunities exist for positively influencing the health of women, children, and families. The variations among domestic preconception health projects and related public health priorities are reflective of the true breadth of the topic. If a national agenda for preconception health is to be moved forward, it is imperative that these efforts continue to be fostered and expanded in all settings and across a multitude of disciplines.

Anal_Bioanal_Chem-3-1-1764596

Instrumental methods and challenges in quantifying polybrominated diphenyl ethers in environmental extracts: a review

Increased interest in the fate, transport and toxicity of polybrominated diphenyl ethers (PBDEs) over the past few years has led to a variety of studies reporting different methods of analysis for these persistent organic pollutants. Because PBDEs encompass a range of vapor pressures, molecular weights and degrees of bromine substitution, various analytical methods can lead to discrimination of some PBDE congeners. Recent improvements in injection techniques and mass spectrometer ionization methods have led to a variety of options to determine PBDEs in environmental samples. The purpose of this paper is therefore to review the available literature describing the advantages and disadvantages in choosing an injection technique, gas chromatography column and detector. Additional discussion is given to the challenges in measuring PBDEs, including potential chromatographic interferences and the lack of commercial standards for higher brominated congeners, which provides difficulties in examining degradation and debromination of BDE congeners, particularly for BDE 209. Introduction Over the past 5 years there has been a surge in the number of studies investigating polybrominated diphenyl ethers (PBDEs) in the environment. In 1981 the first report on PBDEs was made in biota samples from a Swedish river [1], which was followed by a handful of studies throughout the 1980s and 1990s. Then, in 2000 Noren and Meironyte [2] published a paper on organohalogen temporal trends in human breast milk from Sweden that generated rapid interest in the fate of PBDEs. This study demonstrated that PBDE levels appeared to be increasing at an exponential rate, and since then the number of studies investigating PBDEs has increased as well. PBDEs are used as brominated flame retardant additives which are incorporated into a number of polymers and resins found in a majority of consumer products. PBDEs are the most widely used additive flame retardant and, until recently, almost 70,000 t was produced every year, half of which was used in products sold in the USA and Canada. In 2004 the European Union phased out the use of two of the three PBDE commercial mixtures, PentaBDE and OctaBDE. Following this action, and owing to growing concerns in some US state governments, the sole US chemical manufacturer voluntarily agreed to stop manufacturing these two PBDE commercial mixtures beginning in January 2005 [3]. Presently, the only remaining unregulated PBDE mixture in production is DecaBDE, with an annual global market demand of 56,000 t (http://www.bsef.com). DecaBDE is composed almost exclusively (more than 97%) of decabromodiphenyl ether (BDE 209), a fully brominated BDE congener. Much of the concern regarding PBDEs has been focused on the elevated levels measured in human tissues within the US population [4]. The BDE congeners typically measured in human tissues are associated primarily with the PentaBDE mixture, and to some extent with the OctaBDE mixtures. While production of PentaBDE and OctaBDE has been halted, there are still a number of products left on the market and in use today that contain these mixtures. On the basis of their persistence and current reservoirs, the fate of PBDEs will most likely need to be monitored for years to come. One of the greatest challenges to measuring PBDEs in environmental samples has been developing methods to accurately quantify BDE 209. While analytical methods are readily available for quantifying tribrominated through heptabrominated congeners found in the PentaBDE and OctaBDE mixtures, the analysis of higher brominated compounds (i.e., congeners with eight or more bromines) has proven to be difficult. In 1999–2000, the first worldwide interlaboratory comparison on PBDE measurements was conducted. Laboratory agreements were acceptable for most of the lower brominated congeners; however, reported values for BDE 209 varied over almost 2 orders of magnitude [5]. Over the past few years, improved methods have been developed to measure BDE 209 with increased accuracy and precision, and with the use of these methods, more data on BDE 209 have been generated. A number of papers have described analytical methods for extracting and quantifying environmental samples for the determination of PBDEs [6–11]. In 2003 Covaci et al. [9] published an excellent review on extraction methods, cleanup techniques and analytical detection methods for brominated flame retardants. Since the publication of that review, several interesting papers have been published which highlight improvements in injection techniques, analyte resolution and selectivity, particularly for BDE 209. When choosing a method one must find the best compromise in considering cost, reproducibility, chromatographic and mass resolution, sensitivity, and analysis time. The aim of this paper is to review and disseminate the latest information available and discuss the advantages and disadvantages found when using different analytical techniques to determine PBDEs in environmental and human samples. The physical-chemical properties of PBDEs are very similar to those of PCBs [12], and as such, PBDEs are typically extracted using nonpolar solvents (e.g., hexane, dichloromethane) in methods typically employed for PCB analysis. In this paper all PBDE congeners are labeled and numbered according to the same scheme developed by Ballschmitter and Zell [13], for PCB congeners. A variety of extraction and cleanup methods have been reported [6, 9, 14–16], but for the purpose of this paper, we will focus on instrumental methods of analysis for PBDE congeners. Injection techniques Owing to their vapor pressures and polarity, gas chromatography (GC) has become a standard analytical separation method employed for the analysis of PBDEs. The injection of samples into the GC system is an important and crucial step for the accurate and optimal determination of compounds with relatively high boiling points like PBDEs. Various injection methods have been reported which introduce sample analytes into the GC analytical column. The three most common injection techniques for PBDE analysis are split/splitless injection, on-column injection and programmable temperature vaporization (PTV) injection. All three methods possess advantages and disadvantages that are dictated primarily by availability, price, acceptable detection limits and discrimination of congeners on the basis of molecular weight. Split/splitless injection is the most routinely used, and is capable of analyzing dirty samples; however, this method can be limited by small injection volumes (typically 1–3 μl) and high injection temperatures (250–300 °C). During split/splitless injection the analytes are immediately vaporized in the inlet liner and are transferred to the column in the gas phase. In some cases, the high inlet temperature can lead to thermal degradation and discrimination of higher molecular weight PBDEs, particularly the fully brominated BDE 209. Evaporation of the sample can begin inside the injection needle or on the tip, and differences in evaporation rates can lead to discrimination of the higher molecular weight congeners. Inefficient transfer of the sample to the column can be difficult for analytes with high boiling points. Furthermore, active sites on dirty liners can result in thermal degradation; therefore, the temperature of the injection port and the time spent in the injection port prior to transfer are important mediating factors in the response of the PBDE congeners. A detailed study by Björklund et al. [17] found that the optimal injector temperature and splitless time should be kept as high as possible, in their case, 325 °C and 4 min, respectively (Fig. 1). Using these optimal setpoints, Björklund et al. demonstrated that the BDE 209 response increased by almost 25% when compared with average settings used in an interlaboratory comparison [5]. However, they also noted a significant decrease in the precision of higher molecular weight congeners, particularly BDE 209, compared with the case for on-column techniques, which are less prone to discrimination effects. Fig. 1Detector response for BDE 209 as a function of splitless time and injector temperature, with pressure held constant at 2.3 bar. BDE brominated diphenyl ether. (Reprinted from Björklund et al. [17] with permission) On-column injection has become a popular method for introducing samples into the gas chromatograph for PBDE analysis [18–20] and is comparable in cost to split/splitless injectors. The design is much simpler relative to a split/splitless injection port and involves direct injection of the sample, dissolved in a carrier solvent, onto the head of the column. This reduces the potential for thermal degradation and discrimination experienced in split/splitless injectors. In the same study mentioned previously, Björklund et al. [17] observed the highest precision in PBDE measurements, particularly for BDE 209, when using on-column injection compared with split/splitless and PTV injectors. However, when using on-column injection, care must be employed to ensure a clean sample, to prevent coextracted compounds from building up in the retention gap and column leading to increased noise, peak tailing, retention shifts and reduced lifetimes of the columns. On-column injections are also limited by injection volumes (typically 1–2 μl) to prevent excess solvent from overwhelming the detectors. PTV inlets have become a more popular choice for injection over the past 5 years, particularly for the analysis of PBDEs [21–23]. The primary advantage in using PTV injectors is an increase in injection volumes (up to 125 μl), which can drastically improve detection limits, and can be useful in measuring low-level samples such as human serum [14, 24–26]. With this type of injection, multiple injections are made into a liner while the solvent is vented off, trapping the analytes and increasing the mass of analytes present in the liner. Transfer of the analytes to the head of the column occurs after the solvent vent is closed, and rapid heating of the injection port (200–700 °C/min) aids the carrier gas in transferring the analytes to the column. Several studies have demonstrated that the PTV injectors must be optimized prior to use as they are more complicated than the conventional split/splitless injector. Analyte discrimination can result if the following are not optimized: injection rate or flow, injection temperature, vent flow, solvent elimination time, injection volume, transfer time and transfer temperature. Tollbäck et al. [25] published a review on optimization parameters for PTV injection and their optimal values are reported in Table 1. In general, it is important that the rate of injection is equivalent to the rate of solvent evaporation to avoid flooding the injector. Care must also be taken to avoid discriminating low molecular weight compounds through solvent vent losses. The rate of injection will influence the solvent introduction mode (i.e., drops versus spray), which can discriminate congeners of different masses on the basis of the point (vertical position) of solvent evaporation within the injection liner. BDE 209 responses can be particularly sensitive to injection flow rate. To balance the solvent removal from the injector, the temperature and vent flow must also be optimized. At elevated injector temperatures, low molecular weight compoundss can be lost through solvent venting and at high flow rates the risk of analyte loss increases. The type of liner used in the injector can also influence PBDE responses. Some liners contain activated sites, which can lead to irreversible adsorption or catalysis of thermal degradation of BDEs, particularly the high molecular weight congeners. Tollbäck et al. [25] recommended adding 0.1–0.35% (v/v) of dodecane with multibaffled liners to trap low molecular weight congeners and changing the liner every 200 injections. Transfer of analytes to the GC column is accomplished by rapid heating of the injector port after the solvent vent is closed. PBDE responses are not significantly affected by the analyte transfer temperature ramp; therefore, steep ramps (700 °C/min) should be used to increase the rate of transfer to GC columns. A final injector temperature of 325 °C was found to be a good compromise to balance transfer efficiency of high molecular weight analytes relative to the risk of their thermal degradation in the injector. Table 1Optimized injector parameters for polybrominated diphenyl ether (PBDE) analysis using programmable temperature vaporization as reported by Tollbäck et al. [25]ParameterDomain Injection rate300 μl/minInjection temperature80 °CVent flow150 ml/minTemperature rate700 °C/minTransfer temperature325 °CTransfer time0.3–0.6 minSolvent elimination time0.1 minCarrier gas pressure650 kPa While split/splitless, on-column and PTV injections are the most common methods used in PBDE analysis, other injection techniques have been reported. Large volume injections up to 500 μl have been reported using both loop-type [26] and automated rotary valves [27]. Loop-type injectors can easily be constructed and used but require day-to-day checks for stability and reproducibility owing to problems with the injection pressure and temperature which can result in peak distortion. Björklund et al. [27] used a stainless steel high-performance liquid chromatography (LC) automated rotary valve to inject PBDEs into a GC system. This technique reduces discrimination of higher molecular weight congeners and thermal degradation similar to on-column injection, but offers the advantage of injecting larger volumes (up to 50 μl). However, care must be taken to incorporate a solvent vent system for certain detector systems, particularly mass spectrometry (MS). Lastly, Sjodin [28] used a septum-equipped programmable injector (SPI), which resulted in reproducible results with minimal congener discrimination; however, these injectors are no longer produced by the manufacturer. GC column selection The next crucial step in the method development is the selection of an appropriate column system. The optimal chromatographic column is one that optimizes resolution and discrimination of congeners with the greatest sensitivity in the shortest amount of time. Of the three primary injection methods described earlier, both on-column and PTV injections require the use of a retention gap or guard column to reduce column deterioration and to aid in focusing the initial band of analytes. Most guard columns are either composed of untreated fused silica with active silanol groups or deactivated fused silica that ranges in polarity. Björklund et al. [17] recommends using a Siltek deactivated guard column, which the authors found had the best precision (measured by peak response) and displayed the least degradation of higher molecular weight congeners. In contrast, a nonpolar fused silica guard column resulted in a 50% reduction in sensitivity for the higher molecular weight PBDE congeners. A variety of different columns have been used for the determination of PBDEs in environmental samples. When choosing a column one must consider the column stationary phase (polarity), column length, film thickness and inner diameter, all of which will influence the response towards PBDEs. Generally speaking, the most sensitive method for measuring the entire range (low to high bromine substitution) of PBDE congeners is found when using short (10–15-m) nonpolar DB columns with thin (0.1-μm) stationary phases [17]. Most GC/MS methods employed for PCB quantification use a 60-m column to increase the resolution power; however, longer columns are not well suited for PBDE analysis, particularly for the higher molecular weight congeners. Longer columns result in longer residence times for the analytes, initiating degradation of the higher brominated congeners (noticeably for heptaBDE, octaBDE, nonaBDE and decaBDE congeners) in the column. Björklund et al. [17] determined that the DB-5MS column (Agilent Technologies, Palo Alto, CA, USA) produced the lowest discrimination and the highest precision compared with five other columns tested (Fig. 2). Columns that contained similar stationary phases but that were produced by two different manufacturers also resulted in different column performance. An HP-1 column from Agilent Technologies significantly degraded nonaBDEs and BDE 209, whereas a comparable DB-1 column from J & W Scientific (Folsom, CA, USA) did not. Narrow-bore columns (inner diameters 0.1 mm or less) have been used recently in combination with PTV injectors, resulting in faster and more efficient separation of congeners [24, 25]. Narrow-bore columns produce extremely narrow peaks, with average peak widths at half peak height of less than 1 s, compared with almost 2 s using columns with 0.25-mm film thickness. Fig. 2The relative response of five selected BDE congeners using different gas chromatography capillary columns. The response for DB-5MS was set to 1. Mean values are plotted (n=5) and the error bars correspond to the standard deviation. (Reprinted from Björklund et al. [17] with permission) Another consideration in choosing a GC column is its resolving power. Korytár et al. [29] recently published a retention time database for 126 PBDE congeners using seven different capillary columns with varying film thicknesses (Table 2). This database is useful for determining the most suitable column for quantitative, congener-specific PBDE analysis. Of the seven columns tested, the DB-XLB column (30 m × 0.25 mm × 0.25 μm, J & W Scientific) was found to have the fewest number of coelutions with other BDE congeners and with other brominated flame retardants, followed closely by the DB-1 column (30 m × 0.25 mm × 0.25 μm, J & W Scientific). However, the DB-XLB column demonstrated greater degradation of the higher molecular weight congeners relative to the DB-1 column. Korytár et al. [29] compared the relative retention times (RRTs) from their study to a GC-RRT model developed by Rayne and Ikonomou [30] which was based on the number of ortho-, meta- and para-bromine substituents, dipole moments and the molecular weight of the compounds. In general the RRTs of lower molecular weight PBDE congeners compared very well between the two studies. In contrast, the higher molecular weight congeners, noticeably the heptaBDE and octaBDE congeners were not tightly correlated, most likely owing to a lack of commercially available standards for heptaBDE and octaBDE congeners when the GC-RRT model was developed. Additional standards are now available and it will be useful to update these types of models in the future. Table 2An assessment of PBDE coelution from seven gas chromatography columns as tested and reported by Korytár et al. [63]ColumnDB-1DB-5HT-5DB-17DB-XLBHT-8CP-Sil 19Dimension (m × mm × μm)30 × 0.25 × 0.2530 × 0.25 × 0.2530 × 0.25 × 0.1030 × 0.25 × 0.2530 × 0.25 × 0.2525 × 0.22 × 0.2517 × 0.15 × 0.30Number of coeluting BDEs62636667566272Number of coelutions with flame retardants24262730222629Co-elution with major BDE congenersMajor BDE 2816, 3316, 3316, 33, 3816, 33, 3816, 33 47 4968, 8068686242, 48, 68, 716851, 75 85155114 99116127 100109101109, 120 138166HBCD166 153HBCD168 154MTBBP-A, BB-153MTBBP-A, BB 153105126BB 153 183BB 169BB 169MTBBP-A dimethylated tetrabromobisphenol-A, HBCD hexabromocyclododecane Chromatographic interferences Coelution of compounds can be an important consideration when using GC/electron capture detection (ECD) and GC/electron capture negative ionization (ECNI) MS techniques to quantify environmental samples, especially as more PBDE congeners become available for analysis. The database of Korytár et al. [29] found that at a minimum, 56 of the 126 PBDE congeners tested were coeluted on the GC columns. The majority of these coelutions were observed for congeners not typically observed in environmental samples; however, a few major congeners (i.e., BDE 28, BDE 49 and BDE 154) can often be composed of different BDE congeners and other brominated flame retardants. Because ECD and ECNI-MS techniques are not as selective as electron ionization (EI) MS for the analysis of lower brominated congeners, coextracted halogenated compounds can sometimes be mistaken for PBDE congeners. Several papers have reported the coelution of 2,2′,4,4′,5,5′-hexabromobiphenyl (BB 153) with BDE 154 and of tetrabromobisphenol-A with BDE 153 [6, 9, 29], typically on 15- and 30-m capillary columns. PCB congeners can also be coeluted if not specifically separated during the extraction process. Alaee et al. [31] reported ten possible interferences between PBDEs and organochlorines. Of particular notice was the interference of BDE 47 with CB-180, and that of BDE 99 with CB-205. The additional mass accuracy of high resolution MS (HRMS) may not be adequate to distinguish certain PCBs and BDEs. Alaee et al. [31] found that the isotopic cluster of [M-Cl2]+ from heptachlorinated biphenyls contains the same mass fragments found in tetrabrominated diphenyl ethers [M-Br2]+ and resolving powers of 25,000 (m/Δm) were required for discriminating the two. Natural brominated compounds have recently been identified in some marine algae, mammals and birds [32–34]. Because GC/ECNI-MS methods rely upon selective ion monitoring (SIM) of Br− ions [79Br and 81Br], other brominated compounds can produce the same fragment ion and confound analysis of PBDEs. Methoxylated PBDEs (MeO-BDEs) are often extracted with PBDEs in environmental samples and can cause interferences when using both GC/EI-MS and GC/ECNI-MS methods. As seen in Fig. 3, the BDEs and MeO-BDEs are eluted very closely together using common techniques and differentiation must be based solely on retention time. Two MeO-BDEs, (6-methoxy-2,2′,4,4′-tetrabromodiphenyl ether and 2′-methoxy-2,3′,4,5′-tetrabromodiphenyl ether) have been identified in several marine species [32, 34] and are eluted between BDE 47 and BDE 100 with identical Br− ion ratios. Other studies have also identified a brominated bipyrrole known as 1,1′-dimethyl-tetrabromo-dichloro-2,2′-bipyrrole (DBP-Br4Cl2) in marine species [35–37]. DBP-Br4Cl2 is also eluted very closely to BDEs and MeO-BDEs using common methods of analysis (Fig. 4). Fig. 3Comparison of elution order for methoxylated BDEs (MeO-BDE) and BDE congeners on a 30-m DB-5MS capillary column using gas chromatography (GC)/electron capture negative ionization (ECNI) mass spectrometry (MS). PDBE polyBDEFig. 4GC/ECNI-MS chromatogram of sea lion blubber containing both PBDEs and a marine natural product, DBP-Br4Cl2 Calibration and quantification standards PBDE calibration standards are commercially available from a few suppliers, including Accustandard (New Haven, CT, USA), Cambridge Isotope Laboratories (Andover, MA, USA), Wellington Laboratories (Guelph, ON, Canada) and Chiron Co. (Trondheim, Norway). All of these suppliers provide the suite of major PBDE congeners typically identified in environmental samples. To the author’s knowledge, about 160 of the 209 possible BDE congeners are currently available commercially. There are still many hexaBDE, heptaBDE and octaBDE congeners that are not available and it can sometimes be difficult to identify unknown peaks in chromatograms that may be those of BDE congeners. For example, octaBDE congeners have been identified as degradation products of BDE 209 through apparent debromination mechanisms [38–40]; however, the bromine substitution of these congeners is unclear owing to a lack of commercially available octaBDE congeners for comparison. Hopefully in the future, more standards will be available for comparison and will provide more information on the fate of higher brominated congeners. Another facet to consider in measuring PBDEs is the choice of an appropriate internal quantification standard. Ideally, isotopically labeled (13C) PBDE standards would be the best choice for quantifying PBDEs. Unfortunately, for some detection methods (see later), 13C PBDEs cannot be distinguished from native PBDEs and cannot be used. Several studies have used alternative internal standards such as 13C-labeled bromobiphenyls and chlorinated diphenyl ethers [41], polychlorinated biphenyls [42], or unlabeled BDE congeners [5, 20] that are not typically identified in samples such as BDE 15 and BDE 75. Chiron Co. recently announced the availability of fluorinated BDEs for use as internal standards. For these standards one fluorine atom is substituted for hydrogen on a BDE congener, resulting in a slightly different vapor pressure. These fluorinated BDEs fragment in a manner similar to that of native BDEs, but have slightly different retention times from their nonfluorinated analogues (Fig. 5), making them very useful as internal standards in GC/ECNI-MS methods when 13C-labeled BDE congeners cannot be used. Fig. 5GC/ECNI-MS chromatogram (a) illustrating the elution order of 6-fluro-BDE 47 relative to the PBDE congeners and its comparable ion fragmentation pattern (b) Detection techniques Determination of PBDEs in environmental samples is sometimes conducted using ECD, but more commonly mass spectrometers are used, the latter being classified into low-resolution (LR) and HR instruments. ECD is advantageous because is relatively inexpensive, user-friendly and sensitive for measurements of halogenated organics; however, ECD is limited in selectivity because detection and identification is based solely on retention time and halogenated interferences can lead to misidentification [31]. Determination of PBDEs using LR instruments is typically made with the instruments operated in either EI-MS or ECNI-MS modes. In contrast, HR instruments are used almost exclusively in EI mode (EI-HRMS). HRMS offers the best selectivity for PBDE measurements, with a mass spectrometric resolution (m/Δm) of approximately 10,000, resulting in fewer coeluting interferences and facilitating the use of isotopically labeled internal standards. However, HR instruments are more expensive relative to LR instruments. In addition, HRMS is labor-intensive, requiring trained personnel to maintain the instruments and keep them actively running. A brief review of LRMS and HRMS techniques are given next. A variety of papers have reported PBDE measurements in environmental samples using both GC/LR-EI-MS [43–45] and GC/LR-ECNI-MS [19, 46, 47]. In general, LR-EI-MS is more selective than LR-ECNI-MS methods because molecular fragments (typically [M-Br2]+) are monitored for each homologue group, in contrast to the bromide ions ([Br−]−, m/z 79 and 81) monitored for all homologue groups in LR-ECNI-MS methods. However, LR-ECNI-MS is a much more sensitive method with lower limits of detection (LOD) relative to LR-EI-MS, even when using SIM mode. The LOD reported for LR-EI-MS range from 0.53 to 32.09 pg, whereas for LR-ECNI-MS the LOD is an order of magnitude lower, ranging from 30 fg to 1.72 pg [48], which is particularly useful for the analysis of low-concentration samples such as human serum and plasma [14, 33, 49]. Additionally, there is a large drop in sensitivity when measuring BDE congeners with more than six bromine atoms using quadrupoles in LR-EI-MS; therefore, one must prioritize selectivity versus sensitivity when choosing a reliable detection method. However, recent work conducted by Ackerman et al. [50] suggests that selectivity can be retained when using LR-ECNI-MS under optimized conditions. The authors reported that optimization of the electron energy, emission current, source temperature and system pressure increased the relative abundance of molecular fragment ions [M-xH-yBr]−, which can be monitored for each homologue group in place of the nonspecific bromide ions. With this method one could use isotopically labeled BDE congeners as internal standards, which cannot be used when monitoring bromide ions (79Br and 81Br) in typical LR-ECNI-MS methods. The exception is BDE 209, in which 13C-labeled BDE 209 can be used in all LR-ECNI-MS applications, since fragmentation produces a higher abundance of the [C6Br5O]− ion relative to bromide [51]. Different reagent gases have also been used to increase PBDE response in LR-ECNI-MS. Typical methods use methane gas as a buffering reagent in the ionization process, but ammonia and isobutane can also be used. Eljarrat et al. [48] found that ammonia and methane gas provided similar LOD for PBDEs, but required significantly different ion source temperatures and system pressure. Ackerman et al. [50] found that isobutane resulted in a 20% increase in the abundance of molecular fragments relative to methane; however, isobutane appeared to contaminate the ion source very quickly, resulting in decreased sensitivity after only 12 h of use and was therefore not recommended for use. HRMS offers both good sensitivity and optimum selectivity. Alaee et al. [52] described a GC/HRMS method for the determination of PBDEs in fish. HRMS instruments have a dual magnetic and electrostatic sector, which increases sensitivity for higher molecular weight analytes such as the higher brominated congeners. This is particularly useful for quantifying heptaBDE through decaBDE congeners with greater confidence. Additionally, HRMS allows one to use isotope dilution with 13C-labeled BDE standards, which are ideal for accurate and precise measurements. In a comparison between GC/HR-EI-MS and GC/LR-ECNI-MS, Thomsen et al. [49] found that both methods provided similar LOD and repeatability. And, as stated previously, HRMS methods will limit the number of potential mass interferences. Therefore, if an instrument and funds are available, HRMS methods provide the best standards for selectivity and sensitivity. Additional ionization methods and detectors have also been used to measure PBDEs in environmental samples. Ikonomou [53] used GC/HRMS with metastable atom bombardment with nitrogen gas as an alternative to electron impact ionization. This method provided less fragmentation of the parent BDE congeners and was useful for identifying the degree of bromination on unknown BDE congeners relative to conventional GC/HR-EI-MS methods. Tandem mass spectrometers using ion traps have also been reported for the analysis of PBDEs [16, 54]. Ion traps offer the advantage of increased selectivity at a low mass resolution because the analytes are fragmented twice. This type of detection minimizes the chance of isobaric interferences and significantly reduces background noise. Ion traps typically use a collision-induced dissociation (CID) cell to fragment a precursor ion, forming a secondary fragment ion. For PBDEs, the precursor ion is typically a molecular ion [M]+or [M-Br2]+ and the secondary fragment formed from the CID is a [M-COBr]+ ion. Wang et al. [54] optimized a GC/ion trap method for 20 BDE congeners and noted that the relative abundance of the fragment ions was dependent upon the number of ortho-substituted bromines on the congener. They also pointed out that coelution could be a potential problem for higher brominated compounds that could produce similar precursor and fragment ions and contribute to additional signals to the MS/MS channel monitored. More work is needed to determine if other brominated compounds would be isobaric using this method. HR time-of-flight (TOF) mass spectrometers have also been used to determine PBDEs in environmental samples [55, 56], with detection limits comparable to those of most other MS methods. TOF methods offer the advantage of acquiring spectral data across a wide mass range without compromising sensitivity, which is often the case with quadrupole mass filters. TOF-MS can be operated in both electron impact and ECNI modes, similar to quadrupole methods. Operation of a TOF mass spectrometer in ECNI mode results in the same type of fragmentation pattern observed in quadrupole MS, and therefore offers little improvement and/or advantage in spectral data because the bromide ions, and not molecular fragments, are found in greatest abundance. HRTOF instruments offer increased mass selectivity and also permit the estimation of elemental composition; however, like most high mass resolution instruments, TOF-MS can be expensive and require high maintenance. Additionally, Cajka et al. [56] found that concentrated samples can result in inaccurate mass determination owing to saturation of the instrument’s time-to-digital converter. Therefore, TOF mass spectrometers have a limited linear range, and dilution and reanalysis would be necessary to quantify concentrated samples. Alternate analytical techniques While a gas chromatograph connected to a mass spectrometer is generally the method of choice for PBDE measurements, alternate methods have been investigated. Despite the limited chromatographic resolving power of LC, methods employing LC/MS and LC/MS-MS offer promise for PBDE determination. Debrauwer et al. [57] recently investigated the use of atmospheric pressure photoionization (APPI) using LC/MS-MS. Most traditional LC/MS-MS methods use electrospray ionization (ESI) or atmospheric chemical ionization; however, PBDEs do not ionize well with either of these two techniques. Using APPI however, PBDEs will ionize in both negative and positive modes, depending on the degree of bromine substitution. Positive ion mode was more sensitive towards diBDE through pentaBDE, whereas negative ion mode was more sensitive towards pentaBDE through decaBDE. APPI appears to be a softer ionization technique relative to EI as M+ ions are the most intense ions produced by the interaction of PBDEs with charged dopants (charged by photons), compared with the [M-Br2]+ ions formed in EI. Multiple reaction monitoring in the MS-MS system follows the M+ to [M-Br2]+ transition. The use of ESI may be limiting for PBDEs, but the analysis of PBDE metabolites can be conducted using ESI-LC/MS methods. Hydroxylated BDEs (OH-BDEs) have been identified in environmental samples in several studies [58–60] and have been observed in serum samples of rats dosed with PBDEs [61], suggesting they are products of PBDE metabolism. A method for measuring OH-BDEs using LC/MS-MS has recently been reported [62]. LC/MS-MS holds much promise for investigating other potential metabolic and breakdown products of PBDEs and other brominated flame retardants in general. The development of comprehensive two dimensional GC (GC × GC) has improved the GC resolving power for organohalogen determination and GC × GC can be used to sidestep some of the coelution problems encountered in standard GC/MS methods. The use of two columns with different separation characteristics increases the chromatographic resolving power and has been proven successful in separating PBDEs from other halogenated compounds. Korytár et al. [63] used a DB-1 column in combination with 65% phenylmethylspolysiloxane (007-65HT) to efficiently separate PBDEs from polychlorinated alkanes. Focant et al. [55] used GC×GC coupled to a TOF mass spectrometer to resolve 58 different halogenated compounds, including PBDEs, PCBs, bromobiphenyls and organochlorine pesticides. Challenges in PBDE analyses Increase in the fate and transport of PBDEs in the environment has led to the development of a number of different analytical methods for measuring this class of persistent halogenated contaminants. An interlaboratory comparison exercise for the determination of PBDEs in marine sediment conducted during 2004–2005 [64] found that laboratory agreement in measurements of PBDEs have improved since the first PBDE intercomparison exercise was conducted in 2001 [5]. Of particular note was the improvement of BDE 209 measurements among laboratories. The development of methods to limit degradation of BDE 209 in the injection port and column of GC systems has drastically improved the accuracy and precision of measurements. With this advance, an increasing number of studies are reporting BDE 209 measurements in environmental samples [65, 66], particularly in human samples [67, 68], suggesting that BDE 209 is more diffuse and ubiquitous in the environment than originally observed. Theoretically, there are 209 different BDE congeners that can potentially be found in the environment. As stated previously, commercial standards are not currently available for all 209 different congeners and thus one of the major challenges in PBDE analysis is the lack of data on the prevalence of higher brominated BDE congeners in the environment. Several laboratory studies have demonstrated that PBDEs, particularly BDE 209, may debrominate both through abiotic [69, 70] and biotic [38, 39, 71–73] pathways, resulting in the formation of pentaBDE through nonaBDE congeners. The lack of standards has made it difficult to determine the products and elucidate the mechanisms of selective bromine loss. Very little is known about the potential for debromination in the ambient environment and few laboratories routinely measure octaBDE and nonaBDE congeners. The identification of octaBDE and nonaBDE congeners in human samples [40, 74] has led to many questions about the origin of these congeners, such as if they are a result of selective uptake of impurities in the technical mixtures, or if they are a result of debromination. Studies have attempted to identify all major and minor BDE congeners present in the commercial mixtures [29, 75]; however, the lack of standards has made congener identification difficult. With the implementation of the European Union’s directive on waste electrical and electronic equipment and on the restriction of the use of certain hazardous substances (including PBDEs) in electrical and electronic equipment (RoHS; 2002/95/EC), fast and reliable methods for determining PBDE content in electrical equipment will be needed to ensure compliance. The use of X-ray fluorescence (XRF) portable analyzers is a promising method that would allow measurement of the total bromine content of a plastic component by scanning its surface with the handheld device. Obviously there will be difficulties in determining which type of brominated flame retardants are present in materials analyzed with this instrument. However, the XRF may be useful for determining if PBDE degradation occurs after exposure to light and it may be possible to determine the percentage of bromine lost through this pathway in an easy and fast method. Future directions in PBDE research may also wish to examine the fate of the bromine atoms that could be lost from PBDEs via the aforementioned degradation/debromination processes. Conclusions In conclusion, a variety of methods are now available to accurately and precisely measure a suite of PBDE congeners in environmental samples. The choice of method selection will involve a compromise between cost, selectivity and sensitivity, particularly since different congeners require different instrumental optimization techniques, especially BDE 209. The Environmental Protection Agency has outlined a protocol for measuring PBDEs (EPA method 1614), but to the author’s knowledge, this method is still in draft form.

Mol_Genet_Genomics-4-1-2413074

Aspergillus niger genome-wide analysis reveals a large number of novel alpha-glucan acting enzymes with unexpected expression profiles

The filamentous ascomycete Aspergillus niger is well known for its ability to produce a large variety of enzymes for the degradation of plant polysaccharide material. A major carbon and energy source for this soil fungus is starch, which can be degraded by the concerted action of α-amylase, glucoamylase and α-glucosidase enzymes, members of the glycoside hydrolase (GH) families 13, 15 and 31, respectively. In this study we have combined analysis of the genome sequence of A. niger CBS 513.88 with microarray experiments to identify novel enzymes from these families and to predict their physiological functions. We have identified 17 previously unknown family GH13, 15 and 31 enzymes in the A. niger genome, all of which have orthologues in other aspergilli. Only two of the newly identified enzymes, a putative α-glucosidase (AgdB) and an α-amylase (AmyC), were predicted to play a role in starch degradation. The expression of the majority of the genes identified was not induced by maltose as carbon source, and not dependent on the presence of AmyR, the transcriptional regulator for starch degrading enzymes. The possible physiological functions of the other predicted family GH13, GH15 and GH31 enzymes, including intracellular enzymes and cell wall associated proteins, in alternative α-glucan modifying processes are discussed. Introduction Aspergillus niger is a saprophytic fungus well known for its production and secretion of a variety of hydrolytic enzymes contributing to its ability to degrade plant polysaccharides such as cellulose, hemicellulose, pectin, starch and inulin (De Vries and Visser 2001; Tsukagoshi et al. 2001; Yuan et al. 2006). Starch is the most abundant storage carbohydrate in the plant kingdom and is present in tubers, seeds and roots of a variety of crop plants including cereals, potatoes and manioc (Peters 2006). Starch is composed of two different molecules: (1) amylose, an unbranched, single chain of α-(1,4)-linked glucose residues and (2) amylopectin, consisting of a α-(1,4)-linked glucose chain with α-(1,6)-branches on every 12–25 glucose residues along the α-(1,4)-linked backbone (Robyt 1998). The degradation of starch is performed by a variety of enzymes, which are divided over three Glycoside Hydrolase (GH) families based on their sequence similarity (http://www.cazy.org) (Coutinho and Henrissat 1999). The first step in starch degradation is the endo-hydrolysis of the long polysaccharide chains into shorter maltooligosaccharides and α-limit dextrins by α-amylases (EC 3.2.1.1). α-Amylases belong to family GH13, a large family containing various hydrolysing and transglycosylating enzymes, mostly acting on α-(1,4) or α-(1,6)-glycosidic bonds. Members of family GH13 have a (β/α)8 barrel structure and can be recognized by four highly conserved amino acid regions containing three catalytic residues (MacGregor et al. 2001; Nakajima et al. 1986). After endo-hydrolysis, subsequent steps in starch degradation involve exo-acting enzymes releasing glucose. This reaction is performed by glucoamylase type enzymes of family GH15 (EC 3.2.1.3), a relatively confined family with regard to enzyme specificity, as all its studied members hydrolyse either α-(1,4) or α-(1,6)-bonds to release β-glucose from the non-reducing end of maltooligosaccharides. Most GH15 enzymes described thus far possess a starch-binding domain (SBD) (Sauer et al. 2000), a discrete C-terminal region of the protein that binds to starch and facilitates hydrolysis (Southall et al. 1999). Additionally, α-(1,4)-glucosidases of family GH31 may release α-glucose from the non-reducing end of starch (EC 3.2.1.20). This family also harbours other enzyme specificities such as α-xylosidase activity. Several A. niger enzymes involved in starch degradation, and their corresponding genes, have been characterized and isolated. A. niger glucoamylase GlaA (family GH15) is an important enzyme for the modification of starch in the food industry (Boel et al. 1984; van Dijck et al. 2003). Additionally, one GH31 α-glucosidase (AglA, renamed AgdA) (Nakamura et al. 1997) has been characterized previously, as well as three family GH13 α-amylases: acid amylase AamA, and the almost identical AmyA and AmyB (Boel et al. 1990; Korman et al. 1990). The transcriptional regulation of the genes encoding starch-degrading enzymes has been studied in several aspergilli (Nakamura et al. 2006). In general, their expression is high on starch and induced by the presence of (iso)maltose (Tsukagoshi et al. 2001; Kato et al. 2002a). The presence of the inducer activates the Zn(II)2Cys6 transcription factor AmyR which binds to CGGN8(C/A)GG sequences in the promoter regions of AmyR target genes thereby activating their transcription (Petersen et al. 1999; Gomi et al. 2000; Tani et al. 2001; Ito et al. 2004). Recent studies have indicated that some GH13 enzymes in fungi may be involved in the synthesis or modification of α-glucan in the fungal cell wall, rather than in starch degradation. The cell wall of aspergilli contains four major classes of polysaccharides: chitin, α-glucan, β-(1,3)-glucan and galactomannan (Fontaine et al. 2000; Beauvais and Latgé 2001). The α-glucan fraction identified in A. niger consists of two types of molecules: a linear polymer with alternating α-(1,3)/(1,4)-glycosidic bonds called nigeran (Barker and Carrington 1953) and pseudonigeran, a linear α-(1,3)-glucan molecule with some (3–10%) α-(1,4)-linkages (Johnston 1965; Horisberger et al. 1972). Synthesis of α-glucan is thought to be carried out by α-glucan synthase enzymes encoded by ags genes. The first putative α-d-glucan synthase encoding gene (ags1) was identified in Schizosaccharomyces pombe (Hochstenbach et al. 1998). The ags1 gene encodes a large, three-domain protein. In addition to the multi-pass transmembrane domain in the C-terminal part of the protein, two predicted catalytic domains are present. The middle domain shows strong similarity to glycogen and starch synthases in Glycosyltransferase family (GT) 5 and is predicted to be involved in the synthesis of α-glucan. The N-terminal part of the protein is similar to α-amylases and belongs to family GH13. This part of the protein is predicted to be localized extracellularly and might be involved in connecting two α-(1,3)-glucan chains (Grün et al. 2005). Apart from the α-glucan synthases, two types of family GH13 enzymes were recently identified in fungi to play a role in fungal cell wall biosynthesis. Marion et al. (2006) provided evidence for the involvement of a putative α-amylase (Amy1p) in the formation of α-(1,3)-glucan in the cell wall of Histoplasma capsulatum. An AMY1 knockout was unable to produce α-(1,3) glucan and showed reduced virulence. In dimorphic fungi like H. capsulatum, cell wall α-glucan is a known virulence factor (Rappleye et al. 2004; Rappleye and Goldman 2006). The second α-amylase-like enzyme Aah3p was first studied in S. pombe (Morita et al. 2006). Disruption of aah3 encoding a GPI-anchored protein resulted in hypersensitivity towards cell wall-degrading enzymes and an aberrant cell shape, indicating that normal cell wall biosynthesis was affected. Disruption of a homologous gene (agtA) in A. niger also affected cell wall stability (van der Kaaij et al. 2007a). We previously surveyed the A. niger genome sequence to identify all GH13, GH15 and GH31 family members present in this important industrial source for amylolytic enzymes (Pel et al. 2007). This resulted in identification of a surprisingly large number of previously unknown enzymes. In this study, we have analysed their phylogeny, the presence of specific protein features and synteny with other Aspergillus species, which allowed the division of the members of each GH family into several groups. Additionally, we studied the transcriptional regulation of the genes encoding these proteins in a wild type A. niger strain, and in a derived amyR deletion strain, during their growth on xylose and maltose. Only few of the identified proteins were induced by maltose. Expression of many of the identified groups of enzymes, including the homologues of both S. pombe Aah3p and H. capsulatum Amy1p, was not induced by maltose and was not dependent on the presence of AmyR. The possible involvement of these enzymes in cell wall α-glucan synthesis and modification is discussed. Material and methods Database mining of A. niger genome and analysis of predicted proteins The full genome sequence of A. niger strain CBS 513.88 has been deposited at the EMBL database with accession numbers AM270980–AM270998 (Pel et al. 2007) and was used for database mining. The nucleotide accession numbers of A. niger genes, as listed in Tables 1 and 2, refer to this database. Hidden Markov Model (HMM) profiles were built with the HMMER package (Durbin and Eddy 1998) (http://hmmer.wustl.edu/) based on the amino acid sequences of known members of GH13, GH15 and GH31. Proteins belonging to these families, originating from the different kingdoms of life, were retrieved from the CAZy website at http://www.cazy.org (Coutinho and Henrissat 1999), and the protein sequences were extracted from the GenBank/GenPept database at http://www.ncbi.nlm.nih.gov/entrez/ and Swiss-Prot database at http://www.expasy.org/sprot/. The A. niger genome was searched with the HMM profiles using the WISE2 package (Birney et al. 2004) (http://www.ebi.ac.uk/Wise2/). Table 1All members of family GH13, GH15 and GH31 identified in the genome sequence of A. niger CBS 513.88 using HMM profiles Accession no.GeneFamilyEnzyme activityFeaturesaAmyR binding motifbProposed biological functionRefcAn11g03340aamAGH13Acid α-amylaseSS Starch degradation1An12g06930amyAGH13α-AmylaseSS+970; +252Starch degradation2An05g02100amyBGH13α-AmylaseSS+252Starch degradation2An04g06930amyCGH13α-AmylaseSS+787; +664; −531Starch degradation3An09g03100agtAGH13α-GlucanotransferaseSS, GPICell wall α-glucan synthesis4An12g02460agtBGH13α-GlucanotransferaseSS, GPI+810Cell wall α-glucan synthesis4An15g07800agtCGH13Putative α-glucanotransferaseSS, GPICell wall α-glucan synthesis4An02g13240agdCGH13Putative α-glucosidase+368UnknownAn13g03710agdDGH13Putative α-glucosidaseUnknownAn01g13610amyDGH13Putative α-amylase+504; −32Cell wall α-glucan synthesisAn09g03110amyEGH13Putative α-amylase−76Cell wall α-glucan synthesisAn01g06120gdbAGH13Glycogen debranching enzyme−487; +393Glycogen metabolismAn14g04190gbeAGH13Glycogen branching enzymeGlycogen metabolismAn04g09890agsAGH13Putative α-glucan synthaseSSCell wall α-glucan synthesis5An15g07810agsBGH13Putative α-glucan synthaseSS+287Cell wall α-glucan synthesis5An12g02450agsCGH13Putative α-glucan synthaseSS−973; +622, −185Cell wall α-glucan synthesis5An02g03260agsDGH13Putative α-glucan synthaseSSCell wall α-glucan synthesis5An09g03070agsEGH13Putative α-glucan synthaseSSCell wall α-glucan synthesis5An03g06550glaAGH15GlucoamylaseSS, SBD−792; −669; +423; −301Starch degradation6An12g03070glaBGH15Putative glucoamylase−878UnknownAn04g06920agdAGH31α-GlucosidaseSS+574; +191, Starch degradation7An01g10930agdBGH31Putative α-glucosidaseSS+904; −334Starch degradationAn09g05880agdEGH31Putative α-glucosidase IISSProtein glycosylationAn18g05620agdFGH31UnknownUnknownAn07g00350agdGGH31UnknownSS+402UnknownAn09g03300axlAGH31Putative α-xylosidaseSS+126Xyloglucan degradationAn01g04880axlBGH31Putative α-xylosidase+430; +138, Xyloglucan degradationThe newly identified proteins are indicated in boldaSS predicted N-terminal Signal Sequence; GPI  predicted Glycosylphosphatidylinositol anchor signal; SBD  predicted starch-binding domainbThe presence of consensus AmyR binding motif (CGGN8(A/C)GG) was analysed in the promoter region up to 1 kb upstream of the start codoncReferences: 1 (Boel et al. 1990); 2 (Korman et al. 1990); 3 (R. M. Van der Kaaij and X. L. Yuan, unpublished). ; 4 (van der Kaaij et al. 2007); 5 (Damveld et al. 2005); 6 (Boel et al. 1984); 7 (Nakamura et al. 1997)Table 2Functionally described family GH13 and GH31 members from other organisms, used for the multiple sequence alignments in Fig. 1Accession no.NameFamilyEnzyme activityFeaturesaBiological functionOrganismRefbBAA78714 AndGbe1GH13Glycogen branching enzymeGlycogen metabolismA. nidulans1BAA34996ScGdb1GH13Glycogen debranching enzymeGlycogen metabolismS. cerevisiae2P19571BsAmyAGH13α-AmylaseSSStarch degradationBacillus sp.3CAA54266BsAglAGH13α-GlucosidaseStarch degradationBacillus sp.4CAA21237 SpAah1GH13UnknownSS, GPIα-Glucan biosynthesisS. pombe5CAA91249SpAah2GH13UnknownSS, GPIα-Glucan biosynthesisS. pombe5CAB40006SpAah3GH13UnknownSS, GPIα-Glucan biosynthesisS. pombe5CAA16864SpAah4GH13UnknownSS, GPIα-Glucan biosynthesisS. pombe5ABK62854HcAmy1GH13Unknownα-Glucan biosynthesisH. capsulatum6ABF50883AN7345.2GH31α/β-GlucosidaseSSStarch/cellulose degradationA. nidulans7ABF50846AN7505.2GH31α-XylosidaseXylan degradationA. nidulans7BAB39856AndAgdBGH31α-GlucosidaseSSStarch degradationA. nidulans8AAU87580 TrAguIIGH31α-Glucosidase IISSProtein glycosylationT. reesei9A45249CAMAL2GH31MaltaseMaltose degradationC. albicans10For each A. niger protein identified, a functionally or biochemically characterized protein with the highest similarity was used in the phylogenetic analysis aSS  predicted N-terminal Signal Sequence; GPI  predicted Glycosylphosphatidylinositol anchor signalbReferences: 1 (Sasangka et al. 2002); 2 (Teste et al. 2000); 3 (Tsukamoto et al. 1988); 4 (Nakao et al. 1994); 5 (Morita et al. 2006); 6 (Marion et al. 2006); 7 (Bauer et al. 2006); 8 (Kato et al. 2002b); 9 (Geysens et al. 2005); 10 (Geber et al. 1992) The presence of signal peptidase cleavage sites, glycosylphosphatidylinositol (GPI-) attachment sites and SBD in the obtained sequences were predicted by Web-based tools at URL: http://www.cbs.dtu.dk/services/SignalP/ (Bendtsen et al. 2004), URL: http://mendel.imp.ac.at/sat/gpi/gpi_server (Eisenhaber et al. 2004), and URL: http://www.ncbi.nlm.nih.gov/BLAST/ (Marchler-Bauer and Bryant 2004), respectively. Multiple sequence alignments of GH13, 15 and 31 family members were performed using DNAMAN version 4.0 (Lynnon BioSoft, Canada). The alignments were based on the full length of the predicted proteins, except in case of predicted α-glucan synthases for which only the N-terminal part, encoding the family GH13 domain, was used for the alignment. The phylogenetic relationship was calculated by using Optimal Alignment (Thompson et al. 1994) with gap opening penalty and gap extension penalty of 10 and 0.05, respectively. A bootstrapped test of phylogeny was performed by the Neighbour-Joining method using 1,000 replicates. Wherever possible, one protein with described activity was included for each of the groups identified based on phylogenetic analysis. Strains and transformations The wild type A. niger strain used in this study is N402, a cpsA1 derivative of A. niger van Tieghem (CBS 120.49, ATCC 9029) (Bos et al. 1988). Strain AB4.1 is a pyrG negative derivative of N402 (van Hartingsveldt et al. 1987) and was used to construct the amyR disruption strain. A. niger strains were grown in Aspergillus minimal medium (MM) (Bennett and Lasure 1991), or Aspergillus complete medium (CM) consisting of MM with the addition of 0.5% (w/v) yeast extract and 0.1% (w/v) casamino acids. Growth medium was supplemented with 10 mM uridine (Serva, Germany) when required. Transformation of A. niger AB4.1 was performed as described earlier (Punt and van den Hondel 1992) using lysing enzymes (L1412, Sigma, USA) for protoplastation. The bacterial strain used for transformation and amplification of recombinant DNA was Escherichia coli XL1-Blue (Stratagene, USA). Transformation of XL1-Blue was performed according to the heat shock protocol (Inoue et al. 1990). Disruption of the maltose utilization activator amyR in A. niger Plasmid pJG01 containing the A. nigeramyR gene as a 4.3 kb NsiI fragment in pGEM11 was kindly provided by P. van Kuyk (Wageningen University, the Netherlands) and was used to disrupt the amyR gene. The construction of the amyR deletion cassette was performed as follows. The BamHI–EcoRI fragment and NsiI–SalI fragment flanking the amyR ORF at the 5′ and 3′ region, respectively, were isolated from pJG01. The isolated NsiI–SalI fragment was cloned into pUC19 to obtain plasmid pAmyRF3. Subsequently, a BamHI–SalI fragment carrying the A. oryzaepyrG gene, obtained from plasmid pAO4-13 (de Ruiter-Jacobs et al. 1989) was inserted into pAmyRF3 which resulted in plasmid pAmyRF3-pyrG. The BamHI–EcoRI fragment isolated from pJG10 was ligated into pAmyRF3-pyrG resulting in the amyR deletion plasmid (pΔamyR). Prior to transformation to AB4.1, pΔamyR was linearized with EcoRI. Uridine prototrophic transformants were selected by their ability to grow on MM without uridine. After two rounds of purification, transformants were tested for their ability to grow on starch. Approximately 10% of the pyrG+ transformants showed defective growth on MM agar plates containing starch as sole carbon source. Six independent putative amyR deletion strains (YvdM1.1-1.6) with identical phenotypes were obtained. Southern blot analysis confirmed proper deletion and a single integration of the amyR disruption cassette at the amyR locus. Strain YvdM1.1 was used for further analysis and we will refer to this strain as the ΔamyR strain in the remaining of this paper. Culture conditions, RNA preparation, microarray experiments and data analysis RNA extracted from the A. niger ΔamyR strain and its parental strain (N402) grown on different carbon sources were used for microarray experiments using custom-made ‘dsmM_ANIGERa_coll’ Affymetrix GeneChip® Microarrays kindly provided by DSM Food Specialties (Delft, The Netherlands). All experiments for each growth condition (culturing the mycelia, RNA extractions and microarray hybridizations) were performed twice as independent biological experiments. Aspergillus niger spores (2 × 106 spores ml−1) were inoculated in 250 ml MM supplemented with 2% (w/v) xylose (Sigma) and 0.1% (w/v) casamino acids and grown for 18 h at 30°C on a rotary shaker at 300 rpm. The mycelium was harvested by suction over a nylon membrane and washed with MM without carbon source. Aliquots of 1.6 g wet weight of mycelium were transferred to 300 ml Erlenmeyer flasks containing 70 ml MM supplemented with 1% (w/v) carbon source [maltose (Sigma) or xylose] and incubated at 30°C for a further 2 or 8 h. The pH of all cultures grown for 2 h was equal to the pH at the time of transfer (pH 6.2). Cultures grown for 8 h were buffered at pH 4 by the addition of 100 mM of citric acid/sodium citrate to allow comparison between the N402 and the ΔamyR strain at this time point. The mycelium was harvested over Miracloth filter, frozen in liquid nitrogen and stored at −80°C. Total RNA was isolated from mycelia using TRIzol reagent (Invitrogen) and RNA quality was verified by analyzing aliquots with glyoxal/DMSO gel electrophoresis and Agilent Bioanalyzer “Lab on chip” system (Agilent Technologies, USA). Processing, labeling and hybridization of cRNA to A. niger Affymetrix GeneChips were performed according to the corresponding Affymetrix protocols for “Eukaryotic Target Preparation” and “Eukaryotic Target hybridization”. For probe array washing and staining, the protocol “Antibody Amplification for Eukaryotic Targets” was followed. Hybridized probe array slides were scanned with Agilent technologies G2500A Gene Array Scanner at a 3 μm resolution and a wavelength of 570 nm. Affymetrix Microarray Suite software MAS5.0 was used to calculate the signal and P-values and to set the algorithm’s absolute call flag, which indicates the reliability of the data points according to P (present), M (marginal) and A (absent). The data on each chip were globally scaled to an arbitrary target gene intensity of 500. The complete microarray data were deposited into ArrayExpress with an accession E-TABM-324 at http://www.ebi.ac.uk/miamexpress. The pre-scaled data from each hybridization experiment was then normalized for statistical analysis using Genespring 7.0 software (Silicon Genetics, USA). The per chip normalization was performed to ensure that the overall characteristic of the expression distribution such as median should be the same for all the chips. For the genome-wide analysis, we focused on maltose-induced genes and therefore a pre-filtering of data was performed to select for genes whose detection calls are present in both maltose duplicate samples in the wild-type strain (N402). The selected dataset was used to perform a one-way ANOVA analysis under the test type of “parametric test, don’t assume variances equal”. Fold changes in expression between two different conditions were then computed for genes with P < 0.08 based on one-way ANOVA analysis. Results Identification of glycoside hydrolase family 13, 15 and 31 genes in the A. niger CBS 513.88 genome sequence α-Amylases, glucoamylases and α-glucosidases, members of families GH13, 15 and 31, respectively, are the three main types of enzymes involved in breakdown of starch by aspergilli (Tsukagoshi et al. 2001). To identify all genes encoding enzymes that might play a role in starch utilization, or other α-glucan modifying processes in A. niger, the genome of A. niger CBS 513.88 was searched with HMM profiles based on known enzymes from families GH13, 15 and 31. This resulted in the retrieval of a total of 27 protein sequences including 17 previously unknown proteins (Pel et al. 2007), as listed in Table 1. The predicted proteins were annotated based on their similarity to known enzymes. Gene names were assigned based on this annotation and do not necessarily match the activity of the enzymes, as this is often unknown. Two approaches were combined to predict putative functions in cellular processes for this surprisingly large number of newly identified proteins. First, phylogenetic trees were constructed using the GH13, GH15 and GH31 family members identified in the A. niger genome, as well as functionally characterized proteins from other organisms with similarity to the identified A. niger proteins (Fig. 1). Second, using DNA microarrays, the expression of all the A. niger genes encoding GH13, GH15 and GH31 enzymes was examined in both the A. niger wild type strain N402 and the ΔamyR strain derived, after growth on different carbon sources (Fig. 2; Table S1). Both the N402 and the ΔamyR strains were pregrown in xylose for 18 h, and mycelia were transferred to either xylose or maltose media and grown further for 2 or 8 h. Expression levels were determined based on geometric mean data of biological duplicate samples. We will discuss each enzyme family in detail and combine the findings in A. niger with the predicted proteins present in the genomes of A. fumigatus (Nierman et al. 2005), A. nidulans (Galagan et al. 2005) and A. oryzae (Machida et al. 2005). Fig. 1Bootstrapped phylogenetic tree of A. niger GH13 (a) and GH31 (b) enzymes and several closest homologues from other species. Newly identified proteins in the genome of A. niger are shown in bold. A description of each protein is listed in Tables 1 and 2. Bootstrap values are indicated on the node of each branch. The tree was created with DNAMAN 4.0 using gap and extension penalties of 10 and 0.5, respectively. The scale bar corresponds to a genetic distance of 0.05 substitution per positionFig. 2Expression profiles of A. niger family GH13, 15 and 31 enzymes. Accession numbers of the gene names are given in Tables 1 and 2. Strain and time points after transfer from the preculture are indicated on the right-hand side. The numeric values and Present/Absent calls from the expression data are provided as Supplementary Tables 1, 2 Identification and transcriptional regulation of GH13 family members The HMMer search for family GH13 enzymes in the A. niger genome resulted in the identification of 18 protein sequences of which 10 had not been identified previously (Table 1). Table 3 displays the four conserved regions typical for family GH13 proteins as identified in these enzymes. A phylogenetic tree was produced combining the A. niger family GH13 enzymes with several functionally characterized GH13 family proteins from other organisms (Fig. 1). The combination of this phylogenetic analysis with a functional annotation of the proteins revealed six recognizable subgroups. Table 3Alignment of the four conserved regions of all family GH13 enzymes identified in A. niger, as well as in four Aah proteins from S. pombe and Amy1p from H. capsulatumThe seven residues generally conserved in family GH13 are indicated in bold and the three catalytic residues are additionally underlined. The group to which the proteins are assigned, as described in this paper, is indicated Group I consists of four extracellular α-amylases. Three of these are the previously characterized extracellular α-amylases acid-amylase (AamA) and α-amylases AmyA and AmyB (Korman et al. 1990). One new extracellular α-amylase was identified and named AmyC. This protein displays high similarity with the known A. niger α-amylases (74% identity to AmyA and AmyB, 65% identity to AamA). The amyC gene is located in a gene cluster also containing an α-glucosidase gene (agdA) and the amyR gene encoding the AmyR transcription factor. Expression of aamA in A. niger N402 was not detectable in xylose media, but was strongly induced in the presence of maltose (Fig. 2). The expression of aamA was reduced to a non-detectable level in the ΔamyR strain. Expression of amyA and amyB was not detected in A. niger N402 in any of the conditions tested (Fig. 2) (see Discussion). The expression of the newly identified amyC gene was relatively low compared to the aamA gene. At 2 h after transfer from the preculture, the expression level of amyC was independent of the carbon source, while after 8 h the expression level was reduced 3-fold on xylose compared to maltose. Additionally, the expression on maltose was reduced 2- to 3-fold in the ΔamyR strain (Table S1). The presence of three putative AmyR-binding elements in the promoter region of amyC further suggests that its expression is controlled by AmyR (Table 1). The A. niger acid amylase (encoded by aamA) in the CBS 513.88 strain does not contain a predicted SBD, whereas its homologues in A. nidulans, A. fumigatus and other aspergilli contain a full-length SBD (Table S2). In addition, the presence of a functional SBD in the AamA protein of A. niger N402 was suggested because of the purification of AamA via its SBD from culture fluid of a N402 ΔglaA strain, and subsequent demonstration of the SBD with specific antibodies (M. F. Coeffet-Le Gal and D. Archer, personal communication, University of Nottingham, UK). We therefore PCR amplified the aamA gene and its 3′ flanking regions using N402 genomic DNA as a template, determined its DNA sequence and compared it to the aamA gene and its 3′ flanking region from CBS 513.88. The comparison revealed that the aamA gene of A. niger strain N402 does include an SBD and that the CBS513.88 strain harbours a deletion of 230 nucleotides right after the part encoding the (β/α)8 barrel, causing a frame shift and the introduction of a stop codon resulting in a truncated protein (Fig. S2). After the deletion, the DNA sequence continues by encoding part of a predicted SBD, but this part is not translated (Fig. S2). The SBD in AamA found in the A. niger N402 strain is also predicted to be present in A. niger strain ATCC 1015 (Baker 2006). Group II contains three putative GPI-anchored enzymes, recently identified as α-glucanotransferases, and named AgtA, AgtB and AgtC, respectively (van der Kaaij et al. 2007a). This subgroup of proteins is characterized by the presence of two hydrophobic signal sequences. The N-terminal signal sequence is predicted to serve for translocation to the endoplasmic reticulum (ER), whereas the C-terminal sequence is predicted to be replaced by a preassembled glycosylphosphatidylinositol (GPI) anchor in the ER (Orlean 1997). The three enzymes cluster together with the α-amylases in the phylogenetic tree (Fig. 1a), but can be distinguished from the α-amylases by their catalytic domains which are clearly different from the consensus sequence for the α-amylase family. In all three proteins, one or two highly conserved histidines in conserved regions I and IV are replaced by other hydrophilic residues (Table 3). Family GH13 members without these histidine residues, which are part of the active site, are very rare (Uitdehaag et al. 1999). Mutation of these residues generally results in reduced activity or altered reaction specificity of the enzymes (Chang et al. 2003; Leemhuis et al. 2004). Interestingly, both the conserved His-residues in Regions I and IV are also missing in all (putative) α-glucan synthases (Table 3). Other aspergillus genomes harbour two or three Agt homologues (Fig. S1A; Table S2), all sharing the aberrant conserved regions and predicted GPI-anchoring. In the A. niger genome, both agtB and agtC are located next to genes encoding putative α-glucan synthases, and this arrangement of genes is conserved in other aspergilli. The agtA gene is constitutively expressed in both the wild type strain N402 and the ΔamyR strain under all growth conditions examined (Fig. 2). Expression of agtB was only detected 8 h after transfer, regardless of the carbon source and independent of AmyR, while expression of agtC was not detected. Group III consists of two putative, intracellular α-glucosidases, named AgdC (An02g13240) and AgdD (An13g03710). The protein sequences contained all residues commonly conserved in the α-amylase superfamily (Table 3). The predicted intracellular proteins lack clear similarity to any previously characterized fungal protein, although similar enzymes are predicted in A. oryzae, A. nidulans and A. fumigatus (Fig. S1A). Their most related functionally characterized homologue is an α-glucosidase from Bacillus sp. SAM1606 (Nakao et al. 1994). Expression of agdC was low and not induced on maltose, and expression of agdD was not observed (Fig. 2). Group IV contains two putative intracellular α-amylases, named AmyD (An01g13610) and AmyE (An09g03110), which share 55% identity. AmyD was recently characterized as an α-amylase with low-hydrolyzing activity on starch and related substrates (Van der Kaaij et al. 2007b). AmyD and AmyE are similar to a recently identified protein Amy1p from H. capsulatum, which was shown to be involved in cell wall α-glucan synthesis. The latter protein has not been characterized biochemically. Functionally characterized enzymes with similarity to this cluster therefore included only bacterial enzymes, of which maltohexaose-forming α-amylase of alkalophilic Bacillus sp. #707 (Tsukamoto et al. 1988) had the highest similarity. No significant expression of amyD or amyE was detected in our experiments (Fig. 2). Predicted enzymes highly similar to A. niger AmyD and AmyE were also present in other Aspergillus species (Fig. S1A; Table S2). Like the A. niger amyE gene, the orthologues in A. nidulans (AN3309.3) and in A. oryzae (AO003001497) are clustered in the genome with genes encoding α-glucanotransferases and α-glucan synthases (Table S2). The two A. niger proteins in Group V could be reliably annotated as enzymes involved in glycogen metabolism: glycogen branching enzyme (GbeA) and glycogen debranching enzyme (GdbA). A homologue for each of these enzymes is present in the other Aspergillus genome sequences. Transcriptional analysis in A. niger showed that both genes were expressed both on xylose and maltose, and that their expression was unaffected in the ΔamyR strain (Fig. 2). Group VI contains the five predicted α-glucan synthase genes (Damveld et al. 2005). The derived proteins are highly similar (66–83%) to each other, and all contain the two catalytic domains (GT5 and GH13) characteristic for these proteins. Both agsB and agsC are clustered in the genome with genes encoding the aforementioned α-glucanotransferases agtC and agtB, respectively. In both cases, the direction of transcription of the pair of genes is such that they can be transcribed from their intergenic region. Expression analysis in our microarray data collection, which includes data from various time points on several carbon sources (glucose, maltose, xylose, inulin, sucrose) indicate that agsC and agtB are co-expressed at a later growth stage, independent of the carbon source (data not shown). Expression of both agsB and its neighbouring gene agtC was not detected in any of the growth conditions. The expression levels of agsA and agsD were very low, or not detectable. AgsE was highly expressed in all experiments independent of AmyR (Fig. 2). As expected, several proteins with high similarity to the A. niger α-glucan synthases are predicted from the other Aspergillus genomes, although the number of homologues present is highest in A. niger (Fig. S1A; Table S2). Identification and transcriptional regulation of GH15 family members An HMMer search for GH15 family members in the A. niger genome returned two predicted proteins: the previously described glucoamylase GlaA (Boel et al. 1984) and an unknown predicted protein named GlaB (An12g03070). The GlaB protein lacks both an N-terminal signal sequence and an SBD, and displays a low similarity of 26% with GlaA. These two types of family GH15 enzymes are also recognized in other aspergilli studied (Fig. S1B). All predicted proteins similar to GlaB lack both a signal for secretion and an SBD, features typically present in the fungal GH15 enzymes described to date. A. niger glaA was expressed on xylose and strongly induced on maltose, as described previously (Fowler et al. 1990). The induction of glaA was AmyR dependent. Expression of glaB was not detected in any of the conditions tested (Fig. 2). Identification and transcriptional regulation of GH31 family members HMMer searches in the genome of A. niger revealed the presence of seven GH31 family members, of which only one was previously identified as an α-glucosidase (aglA) (Nakamura et al. 1997). We propose to name all (putative) α-glucosidases in A. niger Agd enzymes, similar to the nomenclature in A. nidulans, and to rename AglA as AgdA, to prevent confusion with α-galactosidases (den Herder et al. 1992). The phylogenetic tree (Fig. 1b) shows the presence of (at least) four subgroups within family GH31. In the group of the α-glucosidases, AgdA clusters with AgdB, a predicted extracellular protein. AgdB has some similarity to two A. nidulans enzymes: AN7345.2 (62% identity) with both α- and β-glucosidase activity (Bauer et al. 2006) and α-glucosidase B (AndAgdB/ AN8953.3, 53% identity), with strong transglycosylation activity (Kato et al. 2002b). However, none of the true orthologues of AgdB has been characterized (Fig. S1C). Transcript analysis revealed that agdB was regulated similar to agdA, as the strong induction of both genes in the presence of maltose was dependent on the presence of amyR. However, where most AmyR-regulated genes (aamA, glaA and agdA) reached their highest level of induction after 8 h growth on maltose, the expression level of agdB was decreased after 8 h compared to 2 h (Fig. 2). From the remaining five GH31 family members, AgdE (An09g05880) shows similarity to Trichoderma reesei glucosidase II (TrAguII) (Geysens et al. 2005). This type of α-glucosidases is located in the ER where it is involved in the trimming of α-(1,3)-linked glucose residues from N-glycan core structure Glc3Man9GlcNAc2, which may be attached to proteins designated to be secreted (Geysens et al. 2005). AgdE contains a predicted signal sequence which might serve to direct the protein to the ER. The agdE gene was constitutively expressed in all conditions tested (Fig. 2), consistent with its predicted function as an α-glucosidase II. Two additional GH31 family members, AgdF (An18g05620) and AgdG (An07g0350), lack similarity to any functionally described proteins. Expression of both genes was not detected in any of the tested growth conditions. The two final family GH31 members, named AxlA and AxlB, are highly similar to AN7505.2 from A. nidulans, which was recently characterized as an α-xylosidase (Bauer et al. 2006). Gene axlA was highly expressed in the presence of xylose, whereas no expression was detected in maltose grown cultures (Fig. 2). The high expression of axlA on xylose further supports its putative function as a xylanolytic enzyme. The gene encoding the putatively intracellular AxlB was expressed at a very low level independent of the carbon source (Fig. 2). A BLASTP search in the genomes of A. fumigatus, A. nidulans and A. oryzae for family GH31 enzymes resulted in a similar collection of enzymes as identified in A. niger (Fig. S1C). Several clusters of orthologous proteins are distinguishable, but the assignment of an enzymatic activity to these clusters is not yet possible due to a lack of well studied homologues for these enzymes, thus requiring biochemical studies in our future work. Genome-wide analysis of AmyR dependent maltose induced genes using microarrays The expression analysis of genes encoding family GH13, GH15 and GH31 enzymes in the A. niger genome revealed that the expression of only a limited number of genes was induced by maltose in an AmyR dependent way. In fact, only four genes (aamA, glaA, agdA, and agdB) showed the predicted expression pattern for genes encoding enzymes involved in the breakdown of starch (Fig. 2). To identify additional genes with a possible role in starch metabolism, the expression of all 14,509 predicted open reading frames on the Affymetrix microarray chips was analysed. Comparison of the transcriptome of the wild type strain (N402), grown for 2 h after transfer on either xylose or maltose, identified 634 genes that were significantly induced by maltose (One-way ANOVA analysis P < 0.08 and >2-fold change in expression level). A set of 12 genes was expressed >2-fold higher in N402 compared to the ΔamyR strain (One-way ANOVA analysis P < 0.08) after the transfer of these strains from xylose to maltose. Combining the two gene sets resulted in a collection of six genes that were >2-fold induced on maltose in an AmyR-dependent manner (Fig. 3, Tables 4, 5). Five of these were assigned to the category of carbohydrate transport and metabolism according to FunCat (Ruepp et al. 2004), including the three genes encoding known extracellular starch degrading enzymes (aamA, glaA and agdA) and a putative α-glucosidase (agdB) (Table 4). Gene An15g03940, encoding a protein with high similarity (68%) to a Candida intermedia glucose/xylose symporter (Leandro et al. 2006), was 2.3-fold induced by maltose; this induction was not observed in the ΔamyR strain. The sixth induced gene (An11g02550) encodes a protein highly similar (72%) to Kluyveromyces lactis phosphoenolpyruvate carboxykinase (Kitamoto et al. 1998) which functions in gluconeogenesis by catalyzing the conversion of oxaloacetate into phosphoenolpyruvate. It should be noted that the pH of the growth medium remained 6.2 during the 2 h growth period after transfer. Fig. 3Results of microarray analysis for maltose induced and AmyR dependent genes. Venn-diagram showing the number of genes induced on maltose compared to xylose in A. niger N402, and the number of genes induced in N402 compared to the ΔamyR deletion strain. The number of genes both induced by maltose and dependent on AmyR is indicated in bold. The maltose induced and AmyR dependent genes which are present in both 2 and 8 h after transfer from a preculture are shown in the boxTable 4Overview of six maltose induced and AmyR dependent genes in A. niger at 2 h (pH 6.2) after transfer from precultureORF no.aN402 maltosebCallcN402 xyloseCallΔamyR maltoseCallFold inductionP-valuePutative functionMaltose vs xyloseN402 vs ΔamyRCarbohydrate transport and metabolismAn11g033403.15 ± 0.12P0.08 ± 0.04A0.13 ± 0.09A38.624.20.080Acid alpha-amylase AamAAn04g0692085.96 ± 22.54P6.24 ± 0.98P5.03 ± 0.03P13.817.10.064Extracellular alpha-glucosidase AgdAAn01g1093096.50 ± 31.50P10.47 ± 2.59P8.35 ± 2.13P9.211.50.062Extracellular alpha-glucosidase AgdBAn03g06550117.80 ± 43.16P13.78 ± 4.14P5.85 ± 0.47P8.520.10.072Glucoamylase GlaA An15g03940152.30 ± 19.00P65.74 ± 3.04P70.59 ± 2.88P2.32.20.074Putative monosaccharide transporterEnergyAn11g0255058.87 ± 3.86P10.13 ± 1.41P25.27 ± 5.35P5.82.30.033Putative phosphoenolpyruvate carboxykinase aGene name in bold indicates that the gene is >2-fold induced on maltose in an AmyR dependant way after both 2 and 8 hbThe expression level was based on the geometric mean value of the duplicate samples, and the deviation values between the duplicate samples are indicated. The P-value was based on 1-way ANOVA analysiscP, M or A represent detection calls for present, marginal or absent, respectively. Genes were divided over different functional groups according to FunCat (Ruepp et al. 2004)Table 5Overview of the 18 maltose induced and AmyR dependent genes in A. niger at 8 h (pH4.0) after transfer from precultureORF no.aN402 maltosebCallcN402 xyloseCallΔamyR maltoseCallFold inductionP-valuePutative functionMaltose vs xyloseN402 vs ΔamyRCarbohydrate transport and metabolismAn11g0334065.26 ± 7.48P0.58 ± 0.57A0.08 ± 0.01A113.0808.40.004Acid alpha-amylase AamAAn01g1093049.46 ± 3.42P2.37 ± 0.08P6.84 ± 1.99P20.97.20.006Extracellular alpha-glucosidase AgdBAn04g06920179.10 ± 13.90P9.89 ± 3.63P3.36 ± 0.46P17.651.70.012Extracellular alpha-glucosidase AgdAAn02g0354062.79 ± 8.36P8.81 ± 0.61P0.96 ± 0.82P, A6.761.90.012Putative hexose transport protein MstCAn15g03940144.40 ± 6.80P35.93 ± 2.72P3.04 ± 0.03P, M4.047.60.003Putative monosaccharide transporterAn03g06550239.30 ± 2.05P83.03 ± 4.35P19.03 ± 1.75P2.912.60.012Glucoamylase GlaA An04g069304.36 ± 0.50P1.50 ± 0.16P1.40 ± 0.03P2.93.10.062Extracellular alpha-amylase AmyCAn09g0481018.47 ± 0.44P6.46 ± 0.35P2.52 ± 0.02P2.97.40.003Putative hexose transporterAn12g07450137.60 ± 13.00P51.57 ± 11.82P52.50 ± 6.31P2.72.60.064Sugar/H+ symporter MstAEnergyAn03g062704.72 ± 0.29P1.59 ± 0.14A0.20 ± 0.25A3.325.60.035Putative isoamyl alcohol oxidase An16g060105.18 ± 1.55P1.80 ± 0.39P0.22 ± 0.02A2.923.80.032Putative phosphoglycerate mutaseAmino acid transport and metabolismAn03g002808.35 ± 0.90P3.57 ± 0.74P, A0.12 ± 0.06A2.368.00.050Similarity to tyrosinase protein Cell rescue, defence and virulence An14g057303.99 ± 0.59P0.23 ± 0.03A1.37 ± 0.73P, A18.83.20.037Similarity to integral membrane protein An03g002905.96 ± 0.89P1.68 ± 0.47P0.64 ± 0.21P3.59.30.068Similarity to integral membrane protein An06g004908.14 ± 0.89P3.81 ± 0.30P, A2.23 ± 0.44A2.13.70.066Similarity to integral membrane proteinProtein fateAn18g042605.44 ± 0.40P2.57 ± 0.07P1.77 ± 0.28M, A2.13.10.056Similarity to UDP-galactose transporterUnclassifiedAn16g0129015.04 ± 0.28P5.22 ± 1.69P, A1.97 ± 0.26A2.66.90.027Unknown An09g0613012.62 ± 2.51P5.41 ± 0.93P2.69 ± 0.30P, A2.34.70.059Unknowna,b,cSee footnotes of Table 4 A similar comparative analysis was performed for samples taken for cultures that had grown for 8 h after the transfer. To prevent differences in pH between the N402 and the ΔamyR strain after 8 h of growth, the pH of the medium was buffered to 4.0 using 100 mM of citric acid/sodium citrate to allow comparison between the N402 and the ΔamyR strain at this time point, as the medium of the ΔamyR strain did not acidify as quickly as the medium of the N402 strain. A total of 28 genes were significantly induced by maltose in comparison with xylose, and 161 genes were 2-fold higher expressed in N402 compared with the ΔamyR strain (one-way ANOVA analysis P < 0.08). By combining the two sets, we identified 18 genes which were induced by maltose and whose induction was AmyR dependent (Fig. 3, Table 5). Nine of these genes encode proteins involved in carbohydrate transport and metabolism, from which five were also identified as differentially expressed after 2 h (aamA, glaA, agdA, agdB, and An15g03940). Additionally identified genes included amyC, encoding an extracellular α-amylase, three genes encoding putative sugar transporters, and several other genes belonging to various functional categories (Tables 4, 5). At this stage we cannot exclude that additional overlapping genes have escaped our attention, because of the different pH values of the medium after transfer, which also could affect gene expression. The results from the microarray experiments were validated using Northern blot analysis for a selected number of amylolytic genes. These genes include glaA, aamA, agdB and amyC. As shown in supplementary Fig. 3, the results of the Northern hybridizations are in good agreement with the microarray data (Supplementary Table 1). The Northern analysis also revealed an additional mRNA of a larger size for the amyC gene, 8 h after transfer. The detection of two different-sized mRNAs suggests two different mRNA start sites or different polyadenylation sites, but the exact sequence of the different mRNAs and possible consequences for the gene model have not been addressed so far. Interestingly, the transcription factor gene amyR was also induced 2 and 8 h after transfer to maltose compared to the transfer to xylose (2.6-fold, P-value 0.014; 2.8-fold, P-value 0.027, respectively), indicating transcriptional regulation of the amyR gene itself. As the AmyR transcription factor is per se missing in the deletion strain, it is not appropriate to include the amyR gene in the group of maltose induced, AmyR dependent genes. However, the 1-kb promoter region of the amyR gene contains two AmyR binding motifs, allowing for the possibility that AmyR can induce its own expression. Discussion The full inventory of glycoside hydrolases belonging to families GH13, GH15 and GH31 in A. niger strain CBS 513.88 has recently been described (Pel et al. 2007). Members of these protein families in aspergilli, including A. niger, have been studied extensively mainly because of their industrial relevance. Nevertheless, several groups of new alpha-glucan acting enzymes that had not been identified previously were identified. These novel enzymes are conserved among several Aspergillus species as well as other Ascomycetes, indicating that they may play an important role in fungal metabolism. In the present study, we have combined transcriptional analysis and more detailed phylogenetic analysis to further understand their function. The GH13 family in A. niger contains six separate groups of amylase-type enzymes (Fig. 1), of which three groups had not been described thus far. The best-described group comprises the extracellular α-amylases, and is now extended with AmyC in addition to the three known α-amylases. Overexpression of the amyC gene in A. niger resulted in increased levels of endo-α-amylase activity in the medium, indicating that this gene indeed encodes an extracellular α-amylase (R. M. Van der Kaaij and X. L. Yuan, unpublished results). The relatively low expression of amyC compared to other starch degrading enzymes may explain why this protein has not been identified previously. The localization of the amyC gene in the genome is noteworthy, as it is part of a small cluster of amylolytic genes (with agdA) and their transcriptional regulator gene amyR. A similar organization is observed in the genomes of other aspergilli such as A. nidulans and A. oryzae RIB40 (Gomi et al. 2000). In A. fumigatus the same cluster is extended with a glucoamylase (similar to GlaA). Gene clusters of transcriptionally co-regulated genes in filamentous fungi are often involved in the same process, e.g. secondary metabolite production (Woloshuk et al. 1995) or catabolism of amino acids (Hull et al. 1989). A possible function of AmyC might be to act as a scouting enzyme for the presence of starch, resulting in the subsequent activation of AmyR by starch-derived molecules (maltose or isomaltose). Alternatively, AmyC might be regulated by another system additional to AmyR, e.g. pH-regulated expression, or its expression might be upregulated locally, e.g. in hyphal tips. The lack of detectable expression of amyA and amyB in A. niger N402 is puzzling. Both genes were expressed in A. niger strain CBS 513.88 in a fed-batch fermentation using glucose as a carbon source (Pel et al. 2007), but batch cultivation of A. niger N402 on glucose did not result in detectable expression in microarray studies (E. Martens, H. Kools, P. Schaap, personal communication). These results indicate a difference between both strains with regard to either the presence of amyA/B in A. niger strain N402 or in the transcriptional regulation of amyA/B. In the recently released genome sequence of A. niger strain ATCC 1015 from the Joint Genome Institute (Baker, 2006) we could not identify orthologues of the AmyA/AmyB proteins suggesting that these genes might be actually absent from the N402 strain. The absence, or lack of expression of amyA/amyB, is consistent with the observation that the A. niger MGG029ΔaamA strain (N402 background) with gene disruptions for both aamA and glaA grows very poorly on starch (Weenink et al. 2006), confirming that AmyA and AmyB do not contribute significantly to the amylolytic potential of N402. The microarray analysis revealed that in addition to the amyA/B genes, five more amylolytic genes (agtC, amyD, agdD, agdG and glaB) were not detected (Absent calls) in any of the conditions tested. However, orthologous genes were identified in the genome of A. niger strain ATCC1015, suggesting that these genes are not absent from the N402 genome, but rather not expressed under the tested conditions. In this study we have identified three new groups within the GH13 family that had not been described before in aspergilli: α-amylase-like proteins predicted to be GPI-anchored enzymes (group II) (subfamily GH13_1, as defined by Stam et al. 2006), intracellular α-glucosidases (group III) (unknown subfamily) and intracellular α-amylases (group IV) (subfamily GH13_5). Detailed biochemical studies of two GPI-anchored proteins revealed that they are α-glucanotransferases, acting mainly on maltooligosaccharides (van der Kaaij et al. 2007a). The expression of these genes was not regulated in response to maltose metabolism or AmyR. The Agt enzymes are homologues of a group of four GPI-anchored amylase-like proteins recently identified in S. pombe (Aah1-4p), which have similarly aberrant conserved regions (Table 3). A deletion of one of these homologues resulted in aberrant morphology of the cell and an increased sensitivity towards cell wall degrading enzymes (Morita et al. 2006). A similar phenotype was observed for an A. niger knockout of agtA (van der Kaaij et al. 2007a). Such a phenotype can be explained if α-glucan synthesis is affected. The clustering of agt genes with genes encoding α-glucan synthases in all four Aspergillus species studied, and the expression pattern of the A. niger agt genes both support the hypothesis that Agt enzymes play a role in α-glucan synthesis. Aspergillus niger contains two putative intracellular α-amylases, AmyD and AmyE, that belong to the recently identified subfamily GH13_5 (Stam et al. 2006). A homologous gene in H. capsulatum, AMY1, was shown to be essential for the formation of α-(1,3)-glucan in the cell wall (Marion et al. 2006). The possibility of a role for subfamily GH13_5 enzymes in the cell wall of Aspergilli is strengthened by their location in the genome: A. niger, A. nidulans and A. oryzae, all contain a cluster of genes, containing a homologue of AMY1 as well as ags and agt genes (Table S2). Both the GPI-anchored α-glucanotransferases and the family GH13_5 α-amylase-like proteins are only present in fungi with cell wall α-glucan and not in the true yeasts, which lack this type of cell wall glucans. However, a study with heterologously expressed AmyD did not confirm a role for this enzyme in α-(1,3)-glucan formation, as the enzyme only showed a (low) hydrolyzing activity on substrates with α-(1,4) glycosidic bonds in vitro (van der Kaaij et al. 2007b). The expression levels of amyD and amyE were below detection level, and thus could not confirm nor refute the possible involvement of these proteins in cell wall α-glucan synthesis in A. niger. At this moment, no function can be assigned to the predicted intracellular α-glucosidases AgdC and AgdD, and their homologues identified in other fungi. The enzymes show similarity to a Bacillus α-glucosidase as well as to a maltase from C. albicans, and might therefore be involved in the intracellular hydrolysis of maltose. Alternatively, these enzymes could play a role in trehalose metabolism, as they show some similarity to bacterial trehalose-6-phosphate hydrolases. Trehalose is known to be a common reserve carbohydrate in fungi including A. niger (Wolschek and Kubicek 1997; Arguelles 2000). A surprising finding from the genome mining of A. niger was the high number of GH enzymes with a predicted intracellular location. This group included not only the starch-acting enzymes described here, but also other predicted intracellular glycoside hydrolases such as β-glucosidase, chitinase, β-mannosidase, β-xylosidase, rhamnosidase and invertase, present in various Aspergillus genomes (Pel et al. 2007; Goosen et al. 2007). Most of these proteins were predicted to be exo-acting, i.e. releasing terminal mono- or disaccharide residues from the non-reducing end of the substrate. In A. niger, the genes encoding these proteins are expressed at a relatively low level compared to the extracellular starch degrading enzymes, sometimes even below the detection limit. In a recent publication, expression of intracellular chitinases in A. nidulans was found to be induced during the autolysis phase of the culture (Yamazaki et al. 2007). A role for these and other predicted intracellular proteins during the autolysis phase would explain their low expression levels observed in A. niger during vegetative growth, as well as their lack of secretion signals. The genome-wide expression analysis identified only five genes that were >2-fold induced by maltose in an AmyR dependent way, 2 and 8 h after the transfer to maltose. Among them are genes encoding known starch degrading enzymes (aamA, glaA), two (putative) α-glucosidases (agdA, agdB), and a putative sugar transporter protein (An15g03940). As described in the Results section, the samples taken after 2 and 8 h after transfer were grown at different pH values (pH 6.2 and 4.0, respectively), which might influence our results and lead to an underestimation of the number of genes that are induced by maltose in an AmyR dependant way at both 2 and 8 h as additional pH-controlled gene expression might also occur. To fully explore this, both strains should be cultivated at controlled conditions at identical pH. The specificity of the putative sugar transporter encoded by the An15g03940 gene has not been examined, but its expression profile suggests that it might function as a glucose or a maltose transporter. Three additional sugar transporters were expressed higher on maltose than on xylose after 8 h (Table 5). One of them, An12g07450/MstA has been functionally characterized as a high-affinity glucose transporter (Vankuyk et al. 2004) which is in-line with its expression profile. The two additional sugar transporters encoding genes (An02g03540/mstC and An09g04810) showed an interesting expression pattern, with induced expression on maltose but also relatively high expression levels on xylose (Table 5), suggesting that these transporters might have a broad sugar transporting activity. In the ΔamyR strain both their expression levels were strongly reduced, also relative to the xylose conditions, indicating that the deletion of AmyR might have had an indirect effect. A possible explanation is that disruption of the AmyR transcription factor resulted in low levels of extracellular enzymes converting maltose to glucose, and consequently stress due to low glucose levels. The low availability of glucose might be a signal to downregulate mstC and An09g04810 if these genes encode low-affinity sugar transporters. To further address these observations additional array analysis should be performed using the ΔamyR strain grown on xylose. Interestingly, the amyR gene itself was also induced by the presence of maltose suggesting that its regulation takes place, at least partly, at the transcriptional level. Furthermore, conserved AmyR binding motifs are present in the promoters of all co-regulated genes, including the 1-kb amyR promoter region, with the exception of aamA (Table 1). Possibly the aamA promoter contains a currently unknown AmyR binding motif. Alternatively, an AmyR binding site might be present in the promoter region of aamA in the N402 background. The results presented in this paper suggest that A. niger can metabolize maltose by inducing the expression of a limited amount of enzymes and sugar transporters. Other members of the GH13, GH15 and GH31 protein families might function in starch metabolism in different conditions (solid state growth, or influenced by pH, nitrogen metabolism, etc.), or they may be involved in other processes such as the synthesis of cell wall α-glucan, or the glycosylation of proteins. To determine the exact function of these enzymes will require further biochemical characterization in combination with the detailed analysis of gene deletion mutants. Electronic supplementary material Below is the link to the electronic supplementary material. Supplementary material (DOC 581 kb)

Ann_Hematol-3-1-2082654

The diagnosis of BCR/ABL-negative chronic myeloproliferative diseases (CMPD): a comprehensive approach based on morphology, cytogenetics, and molecular markers

Recent years showed significant progress in the molecular characterization of the chronic myeloproliferative disorders (CMPD) which are classified according to the WHO classification of 2001 as polycythemia vera (PV), chronic idiopathic myelofibrosis (CIMF), essential thrombocythemia (ET), CMPD/unclassifiable (CMPD-U), chronic neutrophilic leukemia, and chronic eosinophilic leukemia (CEL)/hypereosinophilic syndrome, all to be delineated from BCR/ABL-positive chronic myeloid leukemia (CML). After 2001, the detection of the high frequency of the JAK2V617F mutation in PV, CIMF, and ET, and of the FIP1L1–PDGFRA fusion gene in CEL further added important information in the diagnosis of CMPD. These findings also enhanced the importance of tyrosine kinase mutations in CMPD and paved the way to a more detailed classification and to an improved definition of prognosis using also novel minimal residual disease (MRD) markers. Simultaneously, the broadening of therapeutic strategies in the CMPD, e.g., due to reduced intensity conditioning in allogeneic hematopoietic stem cell transplantation and the introduction of tyrosine kinase inhibitors in CML, in CEL, and in other ABL and PDGRFB rearrangements, increased the demands to diagnostics. Therefore, today, a multimodal diagnostic approach combining cytomorphology, cytogenetics, and individual molecular methods is needed in BCR/ABL-negative CMPD. A stringent diagnostic algorithm for characterization, choice of treatment, and monitoring of MRD will be proposed in this review. Introduction Chronic myeloproliferative disorders (CMPD) are clonal stem cell disorders encompassing a very heterogeneous complex of different entities which are defined by distinct clinical and cytomorphological phenotypes and, in some part, known genetic features. They are characterized by increased and effective proliferation of one to three hematopoietic cell lineages in the bone marrow associated to increased peripheral blood parameters. The recent detection of the high incidence of the JAK2 mutations in polycythemia vera (PV), chronic idiopathic myelofibrosis (CIMF), and essential thrombocythemia (ET) [1–5], the detection of the FIP1L1–PDGFRA gene fusion in chronic eosinophlilic leukemia (CEL) [6, 7], and the introduction of tyrosine kinase inhibitors such as imatinib in chronic myeloid leukemia (CML) or hypereosinophilic syndrome (HES)/CEL shed new interest on molecular diagnostics and detection of minimal residual disease (MRD) in CMPD. Today, CMPD are primarily separated in CML as defined by the Philadelphia translocation t(9;22)(q34;q11)/BCR–ABL and in all other so-called BCR/ABL-negative CMPD. According to the WHO proposal of 2001 [8], the latter are subdivided into PV, CIMF, ET, and unclassified CMPD (CMPD-U). Some very rare disorders as chronic neutrophilic leukemia (CNL), HES, and CEL are additionally included. This broad spectrum becomes even more heterogeneous due to the continuous progress of stages, as all CMPD have the potential of clonal evolution and stepwise progression. They often terminate in bone marrow failure due to myelofibrosis or ineffective hematopoiesis or in acceleration and finally transformation to blast crisis. Differential diagnosis in CMPD is further hampered by the biologically given overlap of the diverse morphologic phenotypes and the sometimes close relationship to reactive conditions, and even show overlaps to myelodysplastic disorders. The WHO, thus, created a category of disorders combining myeloproliferative and myelodysplastic features in which chronic myelomonocytic leukemia (CMML), unclassified myelodysplastic/myeloproliferative disorder (MDS/MPD, U), and some very rare disorders as juvenile myelomonocytic leukemia (JMML) were incorporated [8, 9]. It is well known that PV has a median survival of 10 years, ET of 10–15 years, but CIMF of only 4 years. However, the clinical course in CMPD ranges from a few months with rapid leukemic transformation to several decades. These uncertainties in prognosis and the similarities in the clinical and morphological phenotypes at diagnosis plead for inclusion of other than clinical and morphologic parameters only into classification. Although chromosomal abnormalities are not specific for distinct CMPD, they contribute to the definition of the prognosis and to the classification of the CMPD. However, aberrant karyotypes are detectable in only 5–45% of all BCR/ABL-negative CMPD, depending on the specific subtype. Routinely applied methods further include by now polymerase chain reaction (PCR) screening for the V617F mutation in the JAK2 non-receptor tyrosine kinase [1–5] and will probably soon be included as diagnostic criteria in a revised WHO classification of the CMPD [10]. This novel marker is highly utile for the confirmation of a BCR/ABL-negative CMPD and might contribute to the definition of the prognosis and even for MRD strategies in the future [11]. Molecular methods in the CMPD further encompass PCR analyses of some rare gene fusions, e.g., in HES/CEL [6, 7] or in the 8p11 syndrome [12]. Adaptation of these extensive diagnostic procedures in CMPD to the laboratories’ resources becomes a major challenge. Major goals are standardization of diagnostic workflow, hierarchical order of methods, and combination of single results. This review intends to propose specific diagnostic algorithms for scenarios in BCR/ABL-negative CMPD. Preanalytic conditions To achieve optimal conditions in the diagnostic procedures, a standardized preparation of the samples and optimal conditions for transport are essential in the CMPD: Cytomorphology requires 3 ml bone marrow and 2 ml peripheral blood anticoagulated with ethylenediaminetetraacetic acid (EDTA), being aware that cytomorphology is hampered by heparine. Cytogenetics, in contrast, requires 5–10 ml heparinized bone marrow and 10–20 ml heparinized peripheral blood, as cultivation of metaphases is nearly inhibited by EDTA which induces apoptosis of cells. Multiparameter flow cytometry and all molecular genetic methods can be performed either on EDTA or heparinized material. Trephine biopsies should be performed for histomorphology and immunohistochemistry and allow cytomorphological evaluation by smears from the trephine cylinder in case of a dry tap. In the latter case, also for cytogenetics, a trephine cylinder can be transferred to isotone saline solution plus heparine, which, in many cases, makes metaphases after cultivation in cytogenetic medium possible. Cytomorphology in CMPD Differential diagnosis in the CMPD should always include investigation of peripheral blood smears, bone marrow aspirates, and trephine biopsies in parallel. Smears from peripheral blood and bone marrow are stained according to May Grunwald Giemsa. This may be completed by other stainings: Myeloperoxidase (MPO) reaction and non-specific-esterase (NSE) should be performed in blast crisis after CMPD and are warranted in cases of CMML. Iron staining may be performed additionally, but is of minor importance for the differential diagnostics, as in an early PV, iron will not always be absent. MPO and iron staining are further helpful in all cases with an overlap between the CMPD and MDS for detection of MPO deficiency and ringed sideroblasts. Cases with suspicious HES or CEL should, in addition, undergo toluidine blue staining for detection of mast cells which are frequently increased in CEL with the FIP1L1–PDGFRA gene fusion. In PV, cytomorphology shows increased cellularity with trilineage cell proliferation. Stainable iron is completely missing in many cases. PV is further characterized by elevated blood counts in either cell line (Hb > 18.5 g/dl in men, >16.5 g/dl in women, platelets ≥400 × 109/l, WBC ≥12 × 109/l). In addition, other criteria as reduced serum erythropoietin levels below normal ranges [10, 13] or in vitro formation of endogenous erythroid colonies are included in the classification criteria [8]. In CIMF, bone marrow cytology is often hampered by myelofibrosis, and the peripheral blood shows the characteristical poikilocytosis and leukoerythroblastosis. However, these findings are not specific but occur as well in secondary myelofibrosis following other CMPD. ET is characterized by proliferation of the megakaryocytic lineage with clusters of enlarged mostly mature megakaryocytes in normocellular bone marrow. Granulopoiesis and erythropoiesis present as normal. According to the WHO [8], a diagnosis of ET requires a sustained peripheral platelet count ≥600 × 109/l, whereas reactive thrombocytosis must be excluded. Recently, an international expert panel recommended to lower the threshold for the diagnosis of ET to 450 × 109/l [10]. All CMPD, but especially CML, may show the so-called pseudo-Gaucher cells in the bone marrow, which represent glycolipide storing histiocytes resulting from increased cell proliferation [14]. Another specific cytomorphological subentity is represented by refractory anemia with ringed sideroblasts associated with marked thrombocytosis >500 × 109/l (RARS-T). This rare subtype may be included within the category of overlapping myelodysplastic and myeloproliferative diseases in the upcoming WHO classification [15–17]. Histomorphology in CMPD Bone marrow histology has a central role in the diagnosis of CMPD. Staining is performed according to Giemsa, PAS, and chloroacetate esterase. Gomori silver impregnation allows assessment of reticulin fibers to quantify bone marrow connective tissue. Thus, a diagnosis of CIMF requires at least a bone marrow histology, as bone marrow aspirates are hampered in most cases. Histology shows variable reticulin or collagen fibrosis, osteosclerosis, and sometimes even decreased cellularity. Sinuses are dilated with intraluminal hematopoiesis and erythrocyte extravasates. Megakaryopoiesis is increased with signs of dysplasia [8, 18]. PV demonstrates increased megakaryopoiesis, increased granulopoiesis, and erythropoiesis without storage iron, sinusoid hyperplasia, and variable myelofibrosis in combination with osteopenia. These characteristics allow discrimination from reactive erythrocytosis [19]. In more detail, differentiation from cases of secondary polycythemia includes the evaluation of megakaryopoiesis which shows clustering and pleomorphous appearance with very small and giant megakaryocytes with the characteristical staghorn-like nuclei neighboring each other [19, 20]. ET is characterized by clusters of enlarged mature megakaryocytes close to the sinusoids, whereas erythropoiesis and granulopoiesis are normal [10, 18]. A specific problem is provided by the differentiation between ET and prefibrotic CIMF, as both might be characterized by thrombocytosis in initial stages, and reticulin fibrosis might be minimal or absent in prefibrotic CIMF. However, prefibrotic CIMF is characterized by marked hypercellularity, left-shifted increased granulopoiesis, and a particular megakaryocyte morphology with nuclear features, whereas ET shows hyperlobulated and mature-appearing megakaryocytes [10, 21]. Cytogenetics in BCR/ABL-negative CMPD The frequency of clonal karyotype anomalies varies considerably between the different BCR/ABL-negative CMPD entities. The presence of karyotype abnormalities at diagnosis per se seems to be prognostically negative [9]. CIMF has the highest karyotype aberration rate with 33–43% of all cases, followed by PV in 33–35%, whereas in ET, clonal abnormalities are extremely rare (<5%) [22–25]. In CMPD-U, aberrant karyotypes were reported in ∼20% [22], but definition of the true incidence is difficult due to the heterogeneity of subtypes in this category. Chromosomal changes in the CMPD are not specific, but their presence at least confirms the diagnosis of a malignant hematopoietic disorder and contributes additional aspects to differential diagnosis. This can be exemplified in the 9p-aberrations which are closely associated to PV and to CIMF. In addition, translocations involving ABL, PDGFRA, PDGFRB or other tyrosine kinases can be detected by chromosome banding analyses, allowing the identification of patients who probably benefit from treatment with tyrosine kinase inhibitors. Thus, chromosome banding analyses contribute a lot at diagnosis in many in cases with a suspicious or proven CMPD. However, they do not lead to important information for clinically clearly proven cases of ET. Cytogenetics may also be needed for the follow-up of the CMPD, as leukemic transformation is characterized in many cases by clonal evolution to more complex karyotypes resulting in higher rates of chromosomal abnormalities of ≥90% [9, 23, 26]. Interphase (IP-), metaphase (HMF-), and 24-color fluorescence in situ hybridization (FISH) may further confirm and clarify the results of the chromosome banding analyses. IP-FISH probes can be used for future MRD studies. Nearly all typically observed aberrations—e.g., +8, +9, gain of 9p, or del(20q)—can be monitored. Trisomy 8 is the most frequent aberration in the CMPD being detected in ∼20% of all cytogenetic aberrant PV cases and in ∼10% in chromosomally aberrant CIMF—mostly as sole abnormality or in combination with +9. This is followed by trisomy 9 in ∼10% of all cytogenetically aberrant cases. Partial trisomies of 9p are equally frequent with a special association to PV [27–30]. Other recurrent aberrations are deletions of 13q and 20q and partial trisomies of 1q [9, 22, 31], whereas +19, +21, −7, −Y, del(12p), and i(17q) are less frequent. Chromosomal changes show a characteristic distribution within the diverse CMPD. In detail, PV shows, as the most frequent changes, +9, followed by +8 and by del(20q) [23, 26]. CIMF has a more heterogeneous pattern with deletions of 13q and of 20q both in ∼9% of all cases [9, 25], structural abnormalities of 1q and 5q, and chromosome 7 abnormalities [23, 31]. In ET, chromosomal abnormalities are found in <5% of cases only, mostly represented by numerical gain of chromosome 9. Table 1 presents an overview on recurrent cytogenetic and molecular markers in the CMPD. Table 1Chromosomal and molecular markers in CMPD [8, 9, 22, 23, 52, 69] Karyotype abnormalitiesMolecular markersMolecular MRD markersCMLt(9;22)(q34;q11), in all cases BCR–ABLBCR/ABL+PVIn some cases: +8, +9, del(20q), del(13q), del(1p)JAK2V617F+JAK2 exon 12 in V617F-negative casesCIMFIn some cases: del(13q), del(20q), +8, +9, partial trisomy 1qJAK2V617F+MPLW515ETIn rare cases +8, +9, del(13q), JAK2V617F+MPLW515CMPD-UIn some cases +8, +9, del(20q)JAK2V617F+In rare cases 8p11 translocationsFGFR1 rearrangementsCEL/HESin some cases: +8, i(17q),PDGFRA/FIP1L1 in CEL+CNLin some cases: +8, +9, del(20q)−−CMML−7, +8, del(20q)NRAS in some cases+ Balanced translocations as revealed by cytogenetics are rare in the CMPD. Many of these lead to the disruption of genes encoding tyrosine kinases. The breakpoints cluster in two regions at 5q31-33 and 8p11 which target the platelet-derived growth factor receptor beta [e.g., in the t(5;12)(q31q33;p12)/ETV6-PDGFRB)] and the fibroblast growth factor receptor 1 kinase [e.g., in t(8;13)(p11;q12)/FGFR1-ZNF198)]. Further, the ABL non-receptor tyrosine kinase might be involved in these rare rearrangements as in the t(9;12)(q34;p13)/ETV6-ABL [32]. The 8p11 myeloproliferative syndrome shows a specific profile outlined by frequent association to Non-Hodgkin’s lymphoma, high leukemic transformation rates, eosinophilia, and CML-like findings in bone marrow cytomorphology. It is most frequently caused by the t(8;13)(p11;q12)/FGFR1-ZNF198, but many other variants all involving 8p11/FGFR1 have been described. Bone marrow cytomorphology shows CML-like findings and eosinophilia [12]. As patients with PDFRB and ABL rearrangements are all candidates for tyrosine kinase inhibitor treatment, detection of these rare rearrangements by cytogenetics in combination with FISH and PCR is obligatory. For an overview on these reciprocal gene fusions, we refer to Cross and Reiter [32]. Molecular mutations in BCR/ABL-negative CMPD As published in 2005 by several study groups, a high proportion of patients with BCR/ABL-negative CMPD have a somatic point mutation in the JAK2 gene on 9p24 (V617F) which codes for the JAK2 kinase. In detail, the mutation was found in 80–97% of all patients with PV, in >50% of all patients with CIMF, and in 40–57% in ET [1–5]. Janus kinases are non-receptor TKs which regulate the phosphorylation of several signaling pathways, e.g., JAK/STAT, whose activity is increased by the JAK2 mutation [3, 5]. Interestingly, JAK2V617F-positive ET cases were found to show considerable clinical similarities to PV. This pleads for common pathogenetic pathways in part of the ET cases and in PV [33]. It has to be expected that the JAK2V617F mutation will soon be included as a major criterion for PV diagnosis in a revised WHO classification, as nearly all cases were found to be positive [10]. According to this proposal, also in ET or in CIMF, the respective mutation will serve likewise to any other clonal marker as criterion for the diagnosis of ET or CIMF, which further emphasizes its value for diagnostics in the CMPD. A positive mutation status seemed correlated with further advanced stages. Homozygous JAK2 mutations are more frequent in PV and CIMF than in ET [26] and are associated with a longer history of disease than heterozygous mutations [34]. Thus, a homozygous mutation status correlates with a more aggressive course and might indicate an inferior outcome. Different assays were developed for JAK2 mutational analyses, e.g., allele specific PCR, real-time PCR, or pyrosequencing, which is able to convey information on the frequency of mutated alleles [35]. Kroger et al. [11] showed that quantitative assessment of the JAK2 mutation with real-time PCR after allogeneic hematopoietic stem cell transplantation was valid as minimal residual disease parameter allowing the adaptation of adoptive immunotherapy accordingly. Thus, determination of the JAK2V617F mutation status is highly valuable for all cases with a suspicious or proven BCR/ABL-negative CMPD and even contributes to determination of the prognosis and to MRD strategies. The role of the JAK2V617F mutation is not limited to the “classical” CMPD but was detected in other myeloid malignancies as well, e.g., in 3–10% of all MDS cases [1, 36, 37] or in 50–90% in RARS-T representing an ambiguous subentity with overlapping myeloproliferative and myelodysplastic features [17, 38]. Interestingly, the JAK2V617F mutation was as well found in 20–30% of patients with abdominal vein thromboses and in 5% of patients with cerebral vein thromboses without signs of an overt hematologic disorder [39, 40]. Since the detection of the JAK2617F, the panel of known activating mutations in the CMPD is continuously increasing: PV patients who were JAK2V617F-negative were detected to carry somatic gain-of-function mutations within exon 12 of the JAK2 gene in 40% of cases in a recent study. This mutation subtype was shown to stimulate erythroid proliferation in in vitro experiments [40, 41]. Further on, ∼5% of CIMF and ∼1% of all ET cases show somatic mutations in codon 515 within the transmembrane domain of the MPL gene which encodes the thrombopoietin receptor. The respective point mutations lead to single amino exchanges (W515L and W515K) and induce constitutive cytokine-independent activation of the JAK-STAT pathway as gain-of-function-mutations likewise to the V617F [42–44]. Chronic neutrophilic leukemia CNL is a very rare CMPD defined by persistent leukocytosis in pB ≥25 × 109/l, segmented neutrophils and bands >80% of WBC, immature WBC <10%, and myeloblasts <1% [8]. Survival is extremely heterogeneous and was reported from a few months to 20 years. Cytogenetic aberrations are rare and include +8, +9, del(20q), and del(11q) [8, 45, 46]. The JAK2 mutation was described in few cases of CNL [4, 22, 37, 45–48], but determination of the true incidence is extremely difficult due to the rare occurrence. Some CNL cases were identified to bear rare BCR–ABL fusion transcripts with a breakpoint between exons c3 and c4 of the BCR gene leading to a 230-kDa fusion protein [49, 50]. Hypereosinophilic syndrome/chronic eosinophilic leukemia Persisting hypereosinophilia is, in most cases, reactive and is only rarely caused by a malignant disorder such as HES or CEL or other eosinophilia-associated CMPD [6, 7, 51–53]. HES is defined by persistent eosinophilia ≥1.5 × 109/l in peripheral blood >6 months and an increased number of bone marrow eosinophils of unknown origin; this is accompanied by organ involvement and dysfunction. Classification as HES requires exclusion of all other causes and failure of detection of the underlying genetic defect by cytogenetic and/or molecular screening. A diagnosis of CEL requires >2% blasts in peripheral blood and >5–19% bone marrow blasts or evidence of clonality [8, 54]. The most frequent aberration in CEL is the FIP1L1–PDGFRA fusion which results from a cryptic interstitial deletion on chromosome 4q12. Whereas chromosome banding analyses fail to detect the respective cytogenetic correlate, it is revealed by IP-FISH with differently marked probes for CHIC2, FIP1L1, and PDGFRA, and with reverse transcription (RT)-PCR for FIP1L1–PDGFRA [55, 56]. Some more rare TK gene fusions were detected in eosinophilia-associated CMPD, which involve the tyrosine receptor kinases PDGFRA on 4q12, PDGFRB on 5q31, FGFR1 on 8p11, and the non-receptor kinase JAK2 on 9p24 [9, 57]. The beneficial response of patients with PDGFRA and PDGFRB rearrangements to imatinib makes the detection of these rearrangements obligatory [6, 7, 53, 58]. Therefore, a combination of cytomorphology, cytogenetics, IP-FISH, and RT-PCR based on the patient’s history provides the basis for optimized diagnosis in eosinophilia-associated CMPD followed by targeted therapy [32, 59, 60]. Chronic myelomonocytic leukemia Due to its ambiguous character, CMML was incorporated by the WHO into a category which overlaps between myelodysplastic and myeloproliferative disorders [8] and was defined by peripheral monocytes >1 × 109/l and by dysgranulopoiesis >10%. Blasts and promonocytes were defined by <20% of WBC and by <20% of all nucleated cells in bone marrow. NSE was strongly recommended for bone marrow examination. Clonal karyotype abnormalities occur in 25–35% of all cases. Most frequent are changes of chromosome 7, trisomy 8, and complex aberrant karyotype which is defined by three or more chromosomal aberrations [9, 22, 61]. Mutations of the NRAS protooncogene are the most frequent so far identified molecular markers with variable incidences of 10–66% in this entity. Although there was a wide range in these studies (probably due to the limited samples size in the different studies), this high incidences suggest parallels to MDS or AML [61–63]. The JAK2 mutation was detected in 3–13% of all cases with CMML [1, 4, 37], which illustrates the vicinity to the CMPD in another part of CMML cases. This molecular heterogeneity correlates with the clinical and morphological diversity of CMML and supports the ambiguous position as suggested in the WHO classification. Remission criteria in the CMPD New therapeutic strategies including allogeneic stem cell transplantation as potentially curative option or targeted therapies in the CMPD implicate the need for more differentiated and sensitive criteria of remission. For CIMF, an international working group formulated new consensus criteria for response to treatment [64]. These criteria are based on a combination of clinical and laboratory parameters and include cyto- and histomorphological, cytogenetic, and molecular findings. Thus, highest so far applicable sensitivity is achieved. Clinical criteria include disappearance of clinical symptoms as palpable hepatosplenomegaly. The laboratory and cytomorphological criteria of complete remission imply normalization of trilineage peripheral blood count and the absence of blasts, immature progenitor cells, or nucleated erythroid precursors in peripheral blood. Bone marrow histologic remission criteria apply to cellularity, myeloblast percentage, and osteomyelofibrosis. Finally, cytogenetic response criteria discriminate major cytogenetic response, meaning absence of chromosomal abnormalities in cases with a preexisting aberration from minor cytogenetic response requiring a ≥50% reduction of abnormal metaphases. As the most sensitive criterion, major molecular response defines absence of a specific disease-associated mutation in previously positive cases [64]. Approach to a diagnostic algorithm in the CMPD A diagnostic algorithm for all cases with diagnosis or suspicion of CMPD (Fig. 1) should start with the cytomorphologic evaluation of peripheral blood and bone marrow. This allows, in many, cases a differentiation between CML and a BCR/ABL-negative CMPD. In parallel, all cases should be evaluated by histomorphology. Fig. 1Proposal for a diagnostic algorithm in BCR/ABL-negative CMPD. HES Idiopathic hypereosinophilic syndrome, CEL chronic eosinophilic leukemia, PV polycythemia vera, CIMF chronic idiopathic myelofibrosis, CMPD-U unclassifiable CMPD, ET essential thrombocytosis, RARS-T refractory anemia with ringed sideroblasts, CMML chronic myelomonocytic leukemia, MFC multiparameter flow cytometry, RT-PCR reverse transcription polymerase chain reaction. Dashed line may add information in difficult cases, but not obligatory Chromosome banding analyses might gain important information in PV, CIMF, and also in CMPD-U, as chromosomal aberration rates of ∼20–45% were reported [22]. According to the recent guidelines of a British Committee, cytogenetics are considered as “stage 2 investigations” in case of PV and erythrocytosis which should be performed in dependence on the results of clinical evaluation, blood count, and the JAK2 mutation as well as other laboratory parameters such as serum ferritin which are obligatory as “stage 1 investigations” [65]. In contrast, cytogenetics can be neglected in clinically clear cases of ET due to the extremely low incidence of aberrant karyotypes. In cases in which the discrimination of a CMPD from a reactive disorder is not possible according to clinical, laboratory, and cyto-/histomorphological aspects, cytogenetics might contribute to differential diagnosis, as, in some cases, clonal abnormalities confirm the diagnosis of a hematologic malignancy. CMML should as well undergo cytogenetic analyses; in addition, molecular screening for NRAS mutations might be discussed due to their frequent occurrence and new drugs that may come up. This also includes the investigation of PDGF receptors expression. The chromosomal aberrations as revealed by chromosome banding analyses—e.g., +8, +9, del(20q)—can be verified by FISH techniques. This further allows the selection of IP probes for distinct numerical and structural aberrations to provide parameters for MRD diagnostics. Today, the backbone in all cases of suspicious or proven CMPD should be screening for the JAK2V617F mutation status by PCR, e.g., by a melting curve light cycler assay [66]. According to the proposals for a revised WHO classification, evidence of the JAK2V617F mutation (localized in exon 14 of the JAK2 gene) or a functionally similar mutation will be required as major criterion for the diagnosis of PV. This might justify additional screening for JAK2 mutations in exon 12 in cases which are highly suspicious for PV from morphological aspects but are negative for the JAK2V617F [10]. Finally, all cases with a suspected or proven CMPD should be evaluated by IP-FISH or PCR for BCR–ABL to exclude a diagnosis of CML due to the therapeutic consequences. This is even more emphasized by single cases showing a coincidence of the BCR–ABL fusion and the JAK2V617F mutation where the cytomorphological features of the CMPD can mask the CML aspect [67]. In cases with diagnosis or suspicion of HES/CEL, cytogenetic analysis is informative only in very few cases and may be omitted, whereas molecular diagnostics in search of the FIP1L1–PDGFRA gene fusion by IP-FISH and/or RT-PCR is obligatory. Conclusions For many years, diagnostics in the BCR/ABL-negative CMPD were mainly based on clinical symptoms, cytomorphology, and histomorphological findings. This has dramatically changed in recent years. The WHO (2001) [8] included cytogenetic aspects directly and indirectly in their classification system. This can be exemplified in ET which is excluded by evidence of a del(5q) or an inv(3)(q21q26), as both the 5q- syndrome in MDS or AML with the respective inversion 3 can show microkaryocytes and high thrombocytes. Clonal chromosomal abnormalities other than the Philadelphia translocation support the diagnosis of CMPD and allow to discriminate this from reactive conditions. Additionally, the spectrum of molecular mutations considerably increased. Most molecular events target receptor TK (such as FGFR1, PDGFRA, and PDGFRA) and non-receptor-TK (such as ABL or JAK2). Especially, the detection of the JAK2 mutation in the majority of patients with a CMPD has revolutionized diagnostics in the CMPD [13] and allows, in many cases, now a rapid and clear discrimination of the CMPD from reactive conditions. It further may abolish the measurement of the PRV1 expression in cases with suspected PV. JAK2 mutated cases seem to be associated with higher complication rates and an increased need for therapeutic interventions when compared to wild-type patients [3]. This might play a role for the choice of therapy in the future. Finally, JAK2 might represent a target for specific compounds with anti-tyrosine kinase activity [2, 34]. The recent description of JAK2 mutations in exon 12 in V617F-negative PV cases [40, 41] and of the W515 mutations of the MPL gene in a low frequency in ET and CIMF [42–44] illustrate that we have to assume a complex network of activating mutations in the CMPD of which only parts are so far identified. Thus, a new classification of the CMPD according to the molecular substrate, e.g., the JAK2 mutation, likewise to the definition of CML, will be more appropriate [10, 68]. The inclusion of JAK2 mutation analysis as a major criterion for the PV diagnosis within the current and upcoming World Health Organization (WHO) diagnostic criteria was suggested [10]. The detection of the FIP1L1–PDGFRA fusion in CEL/HES in association to the good response to imatinib [6, 52] and the increasing detection of rare fusion transcript in the BCR/ABL-negative CMPD [32] contributed as well significantly to an improved molecular classification in CMPD. MRD strategies are so far poorly established in the Philadelphia negative CMPD when compared to the acute leukemias or to CML. This might change in the near future, as it was already shown in a post-transplantation setting that quantitative assessment of the JAK2 mutation qualifies as MRD marker [11]. In CML, the definition of remission criteria allowed an international standardization in clinical studies and was helpful for clinical routine. This approach is increasingly important also for the BCR/ABL-negative CMPD as realized by an international working group proposing cytogenetic and molecular response criteria in CIMF [64]. In conclusion, diagnostics in the BCR/ABL-negative CMPD have abandoned the former perception which classified these complex disorders mainly on clinical and morphological aspects and are on the way to a comprehensive approach focussing increasingly on cyto- and molecular genetic aspects. Individual treatment is already available or will hopefully follow.

World_J_Urol-3-1-1913182

Overdiagnosis and overtreatment of early detected prostate cancer

Early detection of prostate cancer is associated with the diagnosis of a considerable proportion of cancers that are indolent, and that will hardly ever become symptomatic during lifetime. Such overdiagnosis should be avoided in all forms of screening because of potential adverse psychological and somatic side effects. The main threat of overdiagnosis is overtreatment of indolent disease. Men with prostate cancer that is likely to be indolent may be offered active surveillance. Evaluation of active surveillance studies and validation of new biological parameters for risk assessment are expected. Introduction What is the rationale for screening? Screening for diseases, especially cancer, has become part of modern medicine. Screening for breast, cervical and colorectal cancer is already normal practice in some countries, and will probably become routine in other countries in the future. Screening for prostate, melanoma and lung cancer are subject to ongoing studies [1–5]. The rationale behind screening is simple: to detect cancers at an early stage, when they are still curable. Screening is currently performed using one of the three methods: mass screening (i.e. large scale screening of an entire population), selective screening (i.e. screening of high-risk populations) or opportunistic screening (e.g. incorporated as part of a medical consultation). Diagnostic testing differs from screening because it attempts to identify the disease in the presence of symptoms, while screening is offered to symptom-free individuals. In any population screened for cancer, four basic groups of patients exist: those diagnosed with cancer who would not have developed cancer symptoms during their lifetime (overdiagnosis); those diagnosed with cancer at an early stage that might otherwise have led to symptoms and/or the need for more aggressive curative treatment; those diagnosed with cancer at a curable stage with aggressive disease that might otherwise have progressed to metastatic disease at the time of diagnosis; and those whose cancer is diagnosed by screening at the same stage as it would have been diagnosed through clinical routines, and that involves cancers that are too late for curative therapy. Ideally, screening should reduce the number of patients in the fourth group (that cannot be cured), and increase those in the second and third group. The window of opportunity for decreasing cancer-mortality by screening for cancer lies with the second and third group. Randomized clinical trials, considered the gold standard for the evaluation of a screening test, have to show how sizeable the window of opportunity is. The difference between the first group and the second is however not always clear at the time of initial diagnosis. Any screening procedure carries a risk of overdiagnosis and overtreatment, which should be balanced against the benefits for those in which the cancers are diagnosed at a curative stage. Whether this balance is justifiable depends on more than mortality differences of randomized study groups only, but also on quality of life issues measured against the cultural background of the population studied. Incidence Does screening influence prostate cancer incidence? Since the potential value of PSA for the early detection of prostate cancer was described in the early 1990s, both prostate cancer incidence and mortality rates have changed profoundly [6]. Between 1989 and 2003, for example, the age-standardized incidence rate of prostate cancer increased by 48.4% in The Netherlands (reaching an incidence of 93.2 cases per 100,000 men). Based on rates from 2001 to 2003, 17.1% of U.S. men born today will be diagnosed with cancer of the prostate at some time during their lifetime [http://www.cancer.gov]. It is now the most frequently diagnosed non-cutaneous cancer, with 225,000 new cases reported each year in Europe alone [7]. This increase of incidence suggests that this is due to the detection of cancers in the first three groups described above. This is supported by the reports on autopsy studies. These autopsy studies have revealed that histologic prostate cancer occurs in an even larger proportion of men compared to the screening incidence: up to 55% of men in their fifties, and 64% of men in their seventies have prostate cancer diagnosed at autopsy, while only 5–10% are detectable in a screening setting during life [8]. A number of influences might have contributed to the increase of cancer diagnosis over the last decades, apart from the structured screening studies that have been initiated at various places. First, the PSA thresholds for biopsy appears to have been reduced gradually in some areas of the world due to the detection of significant cancers in the low PSA range [9]. Most guidelines, however, still mention the traditional cut-off of 4 ng/ml as an indication for biopsy. Secondly, an increase in the number of core samples per biopsy have been advocated, based on the observation that more cancers are diagnosed when more biopsies are taken. Thirdly, awareness of prostate cancer within the general population has increased stimulated by the information obtained predominantly from the urologic profession [10]. If these current trends continue, the number of living men diagnosed with prostate cancer will increase even further [11]. Mortality What happens to prostate cancer mortality by screening and detecting indolent tumors? Despite this rising incidence, the age-standardized prostate cancer mortality rate has decreased in many countries around the world with or without early detection programs. In The Netherlands, for example, rates fell by 11% between 1989 and 2003, to 28.4 deaths per 100,000 men or 2,349 in total [http://www.cancer.gov]. It is however unclear whether the stage and grade migration observed in screening studies results in a reduction in the mortality, although case-control studies with conflicting results are available [12–14]. A decrease of mortality shown in randomized studies would form a strong argument in favor of population screening. Screening results in the more frequent detection of small volume, low grade and organ confined prostate cancers, which are diagnosed earlier in their course [6, 15]. Many of these tumors have the histological characteristic of autopsy tumors, that is, tumors that have not become symptomatic during life [16]. They have been called indolent or clinically insignificant cancers. Various definitions of clinically insignificant tumors have been designed based on the characteristics of the autopsy studies, of which the Epstein definition is widely known [17]. Detecting such tumors will increase the detection frequency of cancer, but it is unlikely that they will influence the prostate cancer specific mortality, as they do not alter the course of life. Early diagnosis Screen detected tumors are diagnosed more early Screen detected tumors are not only diagnosed more often, but can be expected more early during their natural course. Lead-time is defined as the time period from detection by a screening procedure to the time of diagnosis in absence of screening due to symptoms. If the patient dies during the lead-time period of the tumor, the lead-time is indefinite and therefore equal to overdiagnosis. Early detection by PSA advances prostate cancer diagnosis in time (i.e. lead time) [18]. For men aged 55–75 years lead time amounts to 12.3 years in a screening setting [19]. The lead-time is likely to be shorter for aggressive cancers and longer for indolent ones. Early detection also causes a significant stage shift towards more locally confined and less aggressive cancers [20]. The long lead-time, stage reduction and natural history data cited above indicate that for properly selected cases there is a long ‘time-window’ during which observation is safe without losing the opportunity for cure. This is supported by evidence from nomograms, prognostic tables, and by the only available randomized study of observation against radical prostatectomy [21]. The impact of overdiagnosis and unnecessary treatment and of its side effects on patient health is also unclear; however, application of a mathematical model (the Miscan model) on data from the ERSPC has shown that, in an annual screening program for men aged 55–67 years, 56% of diagnosed cases would constitute instances of overdiagnosis [22]. If this estimate proves to be realistic (as it appears to be the case), nationwide screening programs may not be acceptable using the present screening regimens, even if benefits in terms of mortality reduction were shown. Research aimed at the development of more selective screening tools is therefore very important. The natural course of screen-detected cancers The current knowledge of the natural history of prostate cancer is mainly based on clinically detected cases, diagnosed by disease-related symptoms, or coincidentally at the time of unrelated symptoms of the urinary tract. Natural history data on screen-detected cases of prostate cancer will be created as a result of the large ongoing screening studies worldwide in which tumors might have been treated without invasive therapy [1]. Most studies show histological and cytological grade to be the most important prognostic variable [23, 24]. There is a steep decline in the 5-, 10- and 15-year cancer-specific survival with increasing grade. Most prostate cancer cases diagnosed with present diagnostic techniques fall into the moderately differentiated group (grade 2, Gleason 6), of which the cancer-specific 10–15-year mortality is 18–30%. This is even lower in the group of prostate cancers diagnosed with grade 1 and 2 or Gleason 4–6, and there are subgroups of patients who are not at risk of dying from prostate cancer even within 15 years. Overall mortality is then determined by comorbidity. The majority of these men are currently treated for their prostatic disease with invasive procedures, which might not be needed. Adequate prediction of the outcome of comorbid diseases would be of great support in predicting the outcome of the prostate cancer patient. Overdiagnosis Overdiagnosis and overtreatment, what does it mean? During recent years, increased interest has risen to the possibility that increased detection of prostate cancer may lead to the diagnosis of cancers that rather should not have been diagnosed, and certainly should not have been treated, as their detection and subsequent treatment is unlikely to benefit patients, or even might harm them. Related to this, the terms ‘overdiagnosis’ and ‘overtreatment’ are being used. So, when is prostate cancer overdiagnosed? By using the clinical definition of overdiagnosis, that is diagnosing tumors that would otherwise remain clinically unrecognized until the individual died from other causes, it is clear that this definition can only be applied in retrospect in the evaluation of studies. There are currently no clinical or biological parameters that can identify such tumors 100% adequately at the time of diagnosis. By studying the natural course of prostate cancer, and comparing autopsy results with findings from screened populations, clinical and histological parameters can be identified that predict indolent tumors best. Those indolent tumors are likely to be only a subset of the tumors that are overdiagnosed in retrospect. Overdiagnosis is predominantly being associated with early detection or screening programs. Overdiagnosis appears to be especially harmful when it results in invasive treatment of the tumors that would unlikely to be harmful. This is called overtreatment. Overdiagnosis occurs when screening detects small tumors that would otherwise remain clinically unrecognized until the individual dies from other causes. Such tumors are predominantly found in the low PSA ranges. Unfortunately, an unknown number of biologically more aggressive cancers may hide between the larger number of detectable tumors with favorable stages. Though some of the aggressive tumors can be diagnosed by adverse histological criteria such as high Gleason score in the biopsy, some of these features might be missed due to the heterogeneity of prostate cancers and their representation in the biopsy sampling. This might justify the amount of overtreatment that has been practiced in various areas of the world. Overtreatment is thus defined as unnecessary invasive treatment with respect to the outcome of the natural course of the tumor in combination with its host. One can wonder what number needed-to-treat to prevent one prostate cancer death we are prepared to accept. Based on the Swedish randomized trial of radical prostatectomy versus watchful waiting [22], the Connecticut observation series [15], and the Toronto active surveillance experience [25], a number needed to treat analysis of the benefit of radical treatment of all newly diagnosed favorable-risk prostate cancer patients, compared with a strategy of active surveillance with selective delayed intervention, has been presented by Klotz [25]. This suggested that approximately 73 patients will require radical treatment for each prostate cancer death averted. This translates into a 3- to 4-week survival benefit, unadjusted for quality of life. If any, the number of life-years gained will be small, because of the fact that prostate cancer is a disease of old age. This should be contrasted to the side-effects of all applied treatments. As a minimum, the number of men needed to treat should be higher than the number of men dying from intervention related causes. Informed decision making, reduction of unnecessary prostate biopsies The increasing number of diagnostic procedures and subsequently of prostate cancer diagnosis in various areas of the world is partly fed by an increased awareness of prostate cancer and the anxiety raised of suffering from a devastating disease at the end of life. Men should ask themselves if they are at specific risk for having prostate cancer, and if they want to follow the step wise procedure of PSA testing, urologic investigations, prostate biopsies, and potential treatment. Balanced information regarding this procedure and its consequences should be offered to every man considering prostate testing. Validated information (that is: well understandable text that delivers the information that is required) has been made available in several countries around the world, and it has been shown that such information reduces the number of men who initially wanted to be screened. Based on population data it is possible to provide risk assessments for every step of the screening procedure. Such assessments produce an individual risk calculation based on relevant risk parameters. This might support patients and doctors in their decision to follow or refrain from further steps, dependent on their interpretation of the risk calculated. At the time of cancer diagnosis, it may provide information on the risk of the presence of an indolent tumor, as discussed above. Current risk calculators have incorporated family history, age, micturition complaints, PSA, DRE, TRUS results, and histologic features of the prostate biopsies into their assessment. With an increasing number of relevant parameters, the level of predictive accuracy is enhanced. It is likely that new parameters might be added once tested in population-based biorepositories. Candidate parameters therefore obviously are serum and urine markers for the early diagnostic steps, and histologic markers at the time that biopsies have been taken. Much interest has been given to the increase of specificity of the biopsy procedure in the general population with serum markers like PSA isoforms and kallekreins in order to diminish the number of false negative biopsies [26, 27]. Enhancing specificity always resulted in a reduction of sensitivity of prostate cancers. As we have seen based on autopsy incidence, the number of potentially detectable tumors is manifold the number of currently diagnosed cancers. It is therefore not the absolute number, but the number of clinically relevant cancers that is of interest [28]. The PSA isoforms and kallekreins should therefore ideally be related to the characteristics of the cancers detected. Screening efficiency A small number of studies already have provided evidence that supports strategies to reduce the number of screens in the general population. Such strategies will inevitably lead to the reduction of overdiagnosis of prostate cancer. Information obtained from population based screening studies illustrates that the absolute value of serum PSA is related to the proportion of detectable carcinomas in subsequent screening rounds. Data from the ERSPC as well as from the PLCO [29, 30] showed that men with a PSA less than 1 ng/ml did not develop invasive cancer over the time period of more than 5 years of repeated check-ups. In the Rotterdam site of the ERSPC, 1703 men with an initial PSA of less than 1 ng/ml men were followed during two consecutive 4 year screening rounds. Eighty percent of men attended the second screening round, and 77% the third round. In total, only 8 cancers were found in 47 prostate biopsies on the indication of PSA of >3 ng/ml. In the PLCO screening, every 5 years for baseline PSA less than 1 ng/ml and every 2 years for PSA 1–2 ng/ml could result in a 50% reduction in PSA tests and in less than 1.5% of men missing earlier positive screens. In men who were enrolled onto a cardiovascular study in Sweden, 21,277 men aged <50 years old were assessed over a period of more than 20 years starting between 1974 and 1986. Two decades later, 498 (2.3%) were eventually diagnosed with prostate cancer (outside a structured screening procedure). In retrospect, the level of serum kallikreins (hK2, total PSA, and free PSA) at baseline and thereafter were strongly associated with emerging prostate cancer. This supports the idea of risk stratification for screening on prostate cancer in an early age, that is during the fourth decade of life. Men at low risk may refrain from frequent serum testing for long periods of time based on their individual risk assessment that incorporates the information obtained from currently available and newly validated parameters. Overtreatment Side effects of treatment are substantial Treatment for prostate cancer may involve surgery, external beam radiation therapy, brachytherapy, high intensity focused ultrasound (HIFU), watchful waiting, active surveillance, chemotherapy, cryosurgery, hormonal therapy, or combinations. The most frequently applied treatments for organ confined prostate cancer are radical prostatectomy, external beam radiotherapy and brachytherapy. Although, severe or life-threatening complications with radical prostatectomy are rare, the adverse effects of greatest concern are damage to the urinary sphincter and erectile nerves (nervi erigenti), resulting in urinary incontinence and impotence, respectively. Complete incontinence is uncommon after radical prostatectomy, although a significant number of men experience some degree of stress-urinary incontinence [31–33]. In the Prostate Cancer Outcomes Study, a population-based study of 1,291 men who underwent radical prostatectomy for localized prostate cancer and were followed for 2 years, 1.6% reported no urinary control at 24 months following surgery (compared with 0.7% at baseline prior to surgery), while 7 and 42% reported frequent and occasional leakage, respectively (compared with 2 and 9% at baseline) [32]. Age had an impact on the degree of incontinence; 14% of men aged between 75 and 79 years experienced the highest level of incontinence compared with 0.7–4% of younger men. In the Prostate Cancer Outcomes Study, 42% of men reported that sexual performance was a moderate to large problem at 24 months (compared with 18% at baseline); 60% were not able to have erections firm enough for sexual intercourse (compared with 16% at baseline) [32]. At 24 months postoperatively, men over the age of 60 were more likely to be impotent than younger men (78–85 vs. 61%, respectively). Complications after external beam radiotherapy include bladder irritation (urgency, pain, frequency) in up to 5% of men, and impotence in 40–50% [34]. In contrast to surgery, these complications tend to increase over time. The reported incidence of radiation proctitis ranges from 2 to 39%, depending upon the definition used, and the dose field, and technique of radiotherapy. Prostate inflammation and swelling can occur acutely following brachytherapy, suggesting that men with significant urinary symptoms or a large prostate are not good candidates. Urinary retention can be severe enough to require self-catheterization; transurethral resection to improve micturition is contraindicated until a substantial portion of the radioactivity (usually five half-lives) has dissipated because of the risk of incontinence and radiation risks to the surgeon and pathologist. Later complications include irritative voiding symptoms, urinary retention, rectal urgency, bowel frequency, rectal bleeding or ulceration, and prostatorectal fistulas [35–37]. The incidence of erectile dysfunction ranges from 14 to 52%, depending on whether it is physician- or patient-reported. It is obvious that invasive treatment may influence the quality of life of men with prostate cancer and their families substantially. But so does a potential threat of prostate cancer that is not actively treated or not even diagnosed yet. It is unlikely that quality of life studies will be able to indicate the best balance between these points of view for management decisions on an individual patient level. Active surveillance as alternative to invasive treatment Because not all cancers diagnosed require treatment, one of the major challenges for the future is to determine which diagnosed cancers should be treated, and which can be managed by active surveillance. Active Surveillance manages selected men with prostate cancer expectantly with curative intent. This means men are carefully selected and subsequently actively observed in order to have the possibility to offer them deferred curative treatment once the tumor seems to progress. Active surveillance should be clearly differentiated from watchful waiting. Watchful waiting entails a strategy for all men who are managed expectantly, whereas active surveillance focuses on men for whom therapy is delayed until the tumor becomes progressive and curative treatment can be offered. This offers an attitude of active control over the cancer diagnosed for patients and their doctors. The stage migration that screening provides has resulted in an over-representation of low-risk cancers. Therefore, studies which validate monitoring algorithms in active surveillance regimens are ongoing [38]. Risk stratification for indolent disease Over the last decade, a number of nomograms have been composed to predict the presence of an indolent cancer [39, 40]. The identification of indolent cancers was strongly based on histologic information of prostate biopsies, and power of Gleason score as a predictive parameter for aggressiveness of prostate cancer was unsurpassed. As a single serum parameter at the time of diagnosis, the level of PSA contributed most to prognosis. These nomograms were based on extensive clinical series. A new nomogram recently appeared based on information obtained from a screening series of the general population [41]. Screening series differ from clinical studies, as the incidence of indolent cancer is almost 50% compared to maximal 20% in multicentred clinical series. With the use of the nomogram, at a 70% probability cut-off, at least 69% of all indolent cancers would be diagnosed as such, and be treated with active surveillance. Conclusions It is still too early to say whether population-based prostate cancer screening is a useful tool with regard to cancer mortality. We must wait until the results of ongoing prostate cancer screening trials are available. Until then, opportunistic screening should not be encouraged and those men who do want a PSA test should participate in carefully designed, balanced information program. Even if PSA screening is found to reduce prostate-cancer-specific mortality, levels of overdiagnosis may remain unacceptable for population-based screening. To reduce overdiagnosis in a screening setting, markers are needed that reduce the risk on a positive prostate biopsy, increasing the specificity of this procedure. Men from the age of forty, as well as their advising doctors, need instruments to reduce their doubts and anxiety of the potential presence of a prostate cancer. This, together with balanced information about the benefits and risks of the individual outcome of screening procedures, might induce a more selective and step-wise screening action. Risk assessment, incorporating the main determinants known for the presence of prostate cancer from the age of 50, such as age, family history, and micturition complaints, should form the base of an individual screening approach. Objective values of serum markers might enhance the accuracy of such of risk predictors. Various efforts are performed to find new markers in the proteome and genome of blood and urine. Based on large and longitudinal serum collections of men diagnosed with prostate cancer in screening settings, the EC-sponsored P-MARK consortium evaluates candidate markers as prognostic tools [42]. Until alternative screening tools are found, PSA will continue to be used, and overdiagnosis will remain an unavoidable drawback of prostate cancer screening. The current challenge is to ensure that in the still growing numbers of men diagnosed with prostate cancer world-wide, overdiagnosis does not result in overtreatment. To this end, research efforts presently focus on clarifying which cancers can be managed through active surveillance.

J_Mol_Model-3-1-2039870

π-π stacking tackled with density functional theory

Through comparison with ab initio reference data, we have evaluated the performance of various density functionals for describing π-π interactions as a function of the geometry between two stacked benzenes or benzene analogs, between two stacked DNA bases, and between two stacked Watson–Crick pairs. Our main purpose is to find a robust and computationally efficient density functional to be used specifically and only for describing π-π stacking interactions in DNA and other biological molecules in the framework of our recently developed QM/QM approach "QUILD". In line with previous studies, most standard density functionals recover, at best, only part of the favorable stacking interactions. An exception is the new KT1 functional, which correctly yields bound π-stacked structures. Surprisingly, a similarly good performance is achieved with the computationally very robust and efficient local density approximation (LDA). Furthermore, we show that classical electrostatic interactions determine the shape and depth of the π-π stacking potential energy surface. Introduction Two major factors for the structure and stability of DNA are the hydrogen bonds between two adjacent bases from opposite strands (forming a Watson-Crick pair), and the π-π stacking interactions between two bases within each of the two DNA strands. The hydrogen bonding interactions in DNA are well understood by high-level Density Functional Theory (DFT) calculations [1–3]. They were shown to be provided by nearly equally important contributions from electrostatic attraction and donor-acceptor orbital interactions (e.g., from an N lone pair to an N-H σ* orbital), and to be sensitive to effects from the molecular environment (counter-ions, solvent) [1, 2]. A similar understanding of the π-stacking interactions is currently lacking due to several reasons: (1) π-π stacking interactions have proven to be sensitive to the methodology used (see below); (2) π-π stacking interactions have proven to be very dependent on the size of the basis set used in (ab initio) calculations, resulting in a large basis-set superposition error (BSSE); (3) high-level ab initio benchmark studies (CCSD(T) with at least triple-zeta basis sets) have only recently become available; and (4) the notorious problems of DFT to correctly describe dispersion interactions, [4–16] which constitute one of the components involved in π-stacking interactions (see below). The latter problems of DFT resulted in potential energy surfaces (PES) that, erroneously, lack any equilibrium configuration, i.e., the stacked systems were incorrectly described as being purely repulsive. Because of the technical difficulties involved in obtaining accurate benchmarks (CCSD(T) using large basis sets), Sponer, Hobza and co-workers have studied in a ground-breaking series of papers [17–22] the stacking energies of DNA bases and DNA base analogs using a variety of ab initio methods and basis sets. Earlier studies showed that reasonable interaction energies could be obtained when employing the MP2/6-31G*(0.25) method, which was confirmed by later and more accurate studies [17–22]; in the aforementioned 6-31G*(0.25) basis set, the standard d-polarization functions (with exponent of 0.80) were replaced by more diffuse ones (exponent of 0.25) with the aim to improve description of the dispersion attraction [22]. Thus, for the anti-parallel displaced pyrimidine dimer, the MP2/6-31G*(0.25) energy is only 1.1 kcal mol−1 lower than the CCSD(T)/6-31++G*(0.25,0.15) energy, for the anti-parallel cytosine dimer it is 0.6 kcal mol−1 higher than the CCSD(T)/6-31++G**(0.25,0.15) energy, and for the twisted uracil dimer the MP2/6-31G*(0.25) is equal to the CCSD(T)/6-31++G**(0.25,0.15) energy. Although there seems to be no systematic shift in energy when comparing MP2/6-31G*(0.25) with the more elaborate CCSD(T) stacking energies, the MP2 stacking energies are always larger in magnitude than the corresponding CCSD(T) ones when using the same basis set for both methods. Interestingly, in connection with the quest for reliable ab initio benchmarks, preliminary results [23–27] indicate that the recently developed spin-component scaled MP2 (SCS-MP2) yields systematically lower stacking energies than MP2. This suggests that SCS-MP2 is in good agreement with the CCSD(T) results at only a fraction of the computational cost. Several approaches have been proposed to remedy the “dispersion problem” of DFT, ranging from Time-Dependent DFT frameworks [28] to more ad-hoc procedures such as including (C6) van der Waals terms in the interaction energy (DFT-D) [29] or fitting (reparameterizing) the form of the density functional to some reference energies and/or distances. A more elegant solution was recently proposed by Tozer and co-workers [30], who analyzed the DFT exchange-correlation (xc) potential in comparison with the near-exact multiplicative Zhang-Morrison-Parr (ZMP) potential from coupled cluster Brueckner doubles (BD) densities. Their resulting KT1, and the related KT2, functional [30] produces xc potentials that closely resemble those from ZMP. As a result, KT1 and KT2 produce NMR chemical shifts [4, 31] that in general show smaller deviations from experimental values than those from standard density functionals. Furthermore, the ZMP potential from Brueckner (BD(T)) coupled cluster densities gives dispersion forces in good agreement with near-exact dispersion forces [32]. It is therefore to be expected that the KT1 and KT2 functionals behave properly for dispersion forces as well. However, the application of these new functionals to π-stacking interactions has so far not appeared in the literature. Here, we report the results of a systematic investigation of π-stacking interaction energies, potential energy surfaces (PES) and stacking distances for dimers of, among others, the five DNA/RNA bases. A long list of standard as well as recent density functionals (including the KT1 and KT2 functionals) has been considered in this study, ranging from the Local Density Approximation (LDA), via the Generalized Gradient Approximation (GGA), and meta-GGA, to hybrid functionals. We have also examined BHandH, which was recently [12] shown to give reasonable agreement with ab initio data. The DFT results are compared to benchmark data that were taken from the literature, which in some cases contained energy profiles as a function of the torsion associated with the rotation of one base with respect to the other. The present investigation is part of a larger undertaking. We aim at developing an extended QM/QM scheme, designated QUILD [33], which allows a (complex) molecular system to be decomposed into arbitrary, interpenetrating domains such that each type of interaction can be described with a different method, i.e., the density functional that performs best for that type of interaction or process, for example, electron-pair as well as hydrogen bonding (BP86: see [2, 3]), π-π stacking (LDA, KT1, KT2: this work) or reaction barriers (OLYP, OPBE: see [34, 35]). This is a pragmatic (of course not a fundamental) approach to cope with the present-day shortcomings of DFT, which features density functionals that perform satisfactorily for a number but not all types of interactions and phenomena. The present study focuses on identifying which DFT approaches serve best to describe the π-π stacking interactions in the above-mentioned QM/QM scheme. Furthermore, we have analyzed the π-stacking interactions in more detail by decomposing the interaction energy into three physically meaningful components, namely, electrostatic energy, Pauli repulsion (i.e., the destabilizing interactions between occupied orbitals), and bonding orbital interactions [36]. Our analyses reveal, among others, that the electrostatic attraction between the stacked molecules is the most important component of the π-π stacking interaction (see also [37]) which determines the shape and depth of the PES. We have also analyzed the π-π interactions between two stacked Watson–Crick base pairs with the purpose to differentiate between inter- and intra-strand stacking interactions. Methods Computational details All calculations were performed with the Amsterdam Density Functional (ADF) program. [38–49] Molecular orbitals (MOs) were expanded using a large, uncontracted set of Slater-type orbitals: TZ2P [40]. The TZ2P basis is an all-electron basis of triple-ζ quality, augmented by two sets of polarization functions (2p and 3d on H; d and f on heavy atoms). An auxiliary set of s, p, d, f, and g STOs was used to fit the molecular density and to represent the Coulomb and exchange potentials accurately in each SCF cycle. The equilibrium structure of individual benzene analogs, DNA bases and Watson-Crick base pairs were optimized at the BP86 level of DFT, i.e., using the local density approximation (LDA; Slater exchange and VWN [42] correlation) with non-local corrections due to Becke [43] (exchange) and Perdew [44] (correlation) added self-consistently, which was previously shown to be one of the best DFT methods for the accuracy of geometries [41, 45] and hydrogen-bonding interactions [1–3]. The π-π stacking interaction energies for all density functionals were evaluated post-SCF using LDA as SCF functional. This procedure was recently shown to introduce an error of only a few tenths of a kcal mol−1 [46]. Stacking distances (vertical separation) and orientations (twist angle) were explored with the various density functionals through scans of the potential energy surface (PES) in which the BP86 geometries of the monomers (e.g., DNA bases or Watson–Crick base pairs) were kept frozen. PES scans as a function of the twist angle (see below) were done using steps of 30° in case of homo-base stacks, 60° in the case of hetero-base stacks and 36° for stacks of Watson–Crick base pairs. PES scans as a function of the vertical separation (see below) were done in steps of 0.1 Å. The interaction energy (see below) was studied as a function of the vertical separation between the two monomers in the π-stacked dimer, and as a function of the twist angle between these monomers. The latter is defined as the right-handed rotation of the upper base around the axis that runs through the center of mass of both the upper and lower base, and perpendicular to the plane of the base (see Scheme 1). Scheme 1Definition of the twist angle (TW). The black dot is the center of mass In the stacked DNA systems, the twist angle of 0° is defined as that twist angle at which the respective “glycosidic” N-H bonds (more precisely, the N-H bonds that replace the glycosidic N-C bonds to the sugar in the backbone) are oriented in parallel. Bond-energy decomposition The overall π-stacking energy ΔE is made up of two major components [36] (Equation 1): In this formula, the preparation energy ΔEprep is the energy needed to deform the separate molecular fragments from their equilibrium structure to the geometry that they attain in the overall molecular system. The interaction energy ΔEint is the energy released when the prepared fragments are brought together into the position they have in the overall molecule. It is analyzed for our model systems in the framework of the KS-MO model using a Morokuma-type decomposition [47] into electrostatic interaction, Pauli repulsion (or exchange repulsion), and (attractive) orbital interactions (Equation 2). The term ΔVelstat corresponds to the classical electrostatic interaction between the unperturbed charge distributions of the prepared (i.e. deformed) fragments and is usually attractive. The Pauli-repulsion, ΔEPauli, comprises the destabilizing interactions between occupied orbitals and is responsible for the steric repulsion. The orbital interaction ΔEoi in any MO model, and therefore also in Kohn-Sham theory, accounts for electron-pair bonding, charge transfer (i.e., donor-acceptor interactions between occupied orbitals on one fragment with unoccupied orbitals of the other, including the HOMO-LUMO interactions), and polarization (empty-occupied orbital mixing on one fragment due to the presence of another fragment). The orbital interaction energy can be further decomposed into the contributions from each irreducible representation ℘ of the interacting system (Equation 3) using the extended transition state (ETS) scheme developed by Ziegler and Rauk [48, 49]. Results and discussion We have analyzed π-π stacking interactions using different systems that were taken mainly from reference data present in the literature. We start with the prototypical system for studying π-π stacking, namely, the benzene dimer. The second set of reference data was taken from the supporting information of a paper by Mignon and co-workers [50], who studied hydrogen bonding and stacking in complexes of substituted benzene C6H5X with pyridine at MP2/6-31G*(0.25) with substituents X = H, F, NH2, Cl, CH3, OH, CN, COOH, CHO and NO2. All substitutions of H by another X in benzene were done at the para-position with respect to the nitrogen of pyridine (see Scheme 2). The third set of reference data was taken from a paper by Jurecka and co-workers [21], who investigated hydrogen bonding and stacking interactions in the cytosine dimer with MP2 and CCSD(T) methods, using several orientations (see Scheme 3) to investigate the potential energy surfaces. The fourth set of reference data was taken from a paper by Wu and Yang [14] (based on previous work by Hobza, Sponer and co-workers [17, 19, 22]) who studied the dependence of the stacking energy of DNA bases on the twist angle (see Scheme 4) and vertical separation using MP2/6-31G*(0.25) calculations. Scheme 2Stack of substituted benzene and pyridineScheme 3Orientations of the stacked cytosine dimer from Jurecka and co-workers ([21]). The upper base is drawn in bold, the center of mass is indicated by a black dot, and the carbonylic oxygen and the hydrogen replacing the glycosidic bond are indicated for clarityScheme 4Geometries of stacked base dimers with a twist angle of 0 and 120 degrees. The upper base is drawn in bold, the center of mass is indicated by a black dot, and the hydrogens replacing the glycosidic bond are indicated for clarity. Note that for the homo dimers at zero twist angle the lower base is hidden behind the upper base As mentioned already, the prototypical system for studying π-π stacking interactions is posed by the (parallel sandwich) benzene dimer. Because of its high symmetry (D6h), CCSD(T) energies using large basis sets (aug-cc-pVQZ) are available [51] to compare with (see Table 1). The minimum is found at 3.9 Å for this system with a stacking energy of −1.7 kcal mol−1. The standard density functionals are unable to give a proper description of this system, i.e., they either predict a purely repulsive energy surface (e.g., BP86; see Table 1), or give a shallow well (e.g., OLYP). Interestingly, the KT1 functional predicts a minimum that is very close (3.8–3.9 Å) to that of the CCSD(T) method, and has a well depth that is equally close (−1.6 kcal mol−1; see Table 1). The related KT2 and, surprisingly, the LDA functionals have a minimum at the same distance as CCSD(T) and KT1, but with a somewhat smaller well depth (−1.3 kcal mol−1). Also, the BHandH functional performs reasonably well with a similar equilibrium distance, but a reduced well depth (−0.9 kcal mol−1; see Table 1). Table 1Stacking energy (in kcal mol−1) of the benzene-benzene complex as a function of the vertical separation Rvert (in Å), computed with various density functionalsa and CCSD(T)/aug-cc-pVQZ*bRvertCCSD(T)bLDAKT1KT2BHandHPW91BLYPBP86OLYPB3LYPX3LYP2.913.5314.5914.0817.0824.4131.6327.4638.6730.1328.923.08.609.299.0411.2717.9824.2020.8430.9322.7821.653.15.115.525.477.1313.1818.5315.8724.8517.2316.173.23.712.702.902.984.239.6014.2212.1420.0513.0412.063.31.681.061.101.292.246.9310.949.3416.229.898.983.40.30−0.01−0.090.170.924.958.457.2513.147.526.683.5−0.62−0.68−0.83−0.540.063.486.555.6810.655.734.963.6−1.19−1.07−1.27−0.96−0.452.405.104.508.634.403.693.7−1.51−1.27−1.50−1.17−0.741.593.993.626.983.392.743.8−1.66−1.33−1.58−1.25−0.861.003.152.945.622.632.043.9−1.70−1.31−1.56−1.25−0.890.572.502.434.512.061.524.0−1.67−1.24−1.48−1.18−0.850.252.002.033.601.631.144.1−1.58−1.13−1.37−1.09−0.780.021.611.722.851.300.854.2−1.46−1.02−1.24−0.98−0.68−0.151.311.472.241.050.644.3−0.90−1.11−0.86−0.58−0.271.071.271.750.860.494.4−0.78−0.97−0.75−0.48−0.360.881.111.350.710.374.5−1.08−0.67−0.85−0.64−0.39−0.420.730.971.030.590.285.0−0.58−0.28−0.39−0.26−0.06−0.450.300.520.200.260.095.5−0.27−0.09−0.15−0.070.08−0.310.130.280.000.140.046.0−0.11−0.01−0.050.000.12−0.160.050.15−0.030.080.036.5−0.040.020.000.030.12−0.060.020.08−0.030.050.037.00.030.020.040.11−0.010.000.05−0.010.040.037.50.030.020.040.090.020.000.03−0.010.030.038.00.030.020.090.080.01−0.010.020.000.020.02aPost-SCF using LDA/TZ2P orbitals and densities, see Computational detailsbFrom supporting information of [51] Reference data from Mignon and co-workers Scheme 2 shows the structure of the π-stacked complexes of pyridine with (substituted) benzene, i.e. with benzene, fluorobenzene, aminobenzene, chlorobenzene, toluene, phenol, cyanobenzene, benzoic acid, benzaldehyde and nitrobenzene. For all of these complexes the coordinates were taken from the supporting information of the original paper by Mignon and co-workers [50]. The geometry of each dimer had been fully optimized and therefore the vertical separation is different for each complex; it is found in the range of 3.2–3.4 Å. The interaction energies for several typical density functionals are compared to the original MP2 data in Table 2. Consistent with previous reports, the mean absolute deviations (MAD) with respect to the reference MP2 data are much larger for density functionals containing the Becke88 [43] exchange functional (e.g., BP86, MAD value 7.68 kcal mol−1) than for functionals based on PW91 [52] or PBE [53] exchange (e.g., PW91, MAD value 5.33 kcal mol−1). However, these standard density functionals all show repulsive interactions, in contrast to the MP2 results that indicate bound systems (see Table 2). Much better agreement with the MP2 data is observed for the KT1, KT2 and, surprisingly, LDA functionals, with MAD values of 0.25 kcal mol−1 (see Table 2). The BHandH functional, recently [12] shown to give good agreement with MP2/CCSD(T) energies, shows a larger MAD value of 0.4 kcal mol−1 (see Table 2). Because of the uncertainty of ca. ± 0.5 kcal mol−1 in the reference MP2 data with respect to the high-level CCSD(T) data (see above), it is uncertain which of the four density functionals (LDA, KT1 , KT2 or BHandH) performs best. However, the improvement over standard density functionals is obvious and promising. Table 2Stacking energy (in kcal mol−1) of substituted benzene-pyridine complexes (see Scheme 2), computed with various density functionalsa and MP2/6-31G*(0.25)bX=MP2bLDAKT1KT2BHandHPW91BLYPBP86OLYPB3LYPX3LYPH−2.8−3.4−3.4−3.2−2.72.36.14.611.15.04.1F−2.9−3.4−3.3−3.2−2.82.56.44.911.55.14.2NH2−3.2−3.6−3.6−3.3−3.02.26.14.711.24.93.9Cl−3.4−3.4−3.4−3.2−2.82.66.75.111.95.44.4CH3−3.3−3.3−3.4−3.2−2.81.24.53.58.73.52.7OH−2.7−3.2−3.1−3.0−2.52.86.85.211.95.54.6CN−4.1−3.9−3.9−3.7−3.50.94.23.18.43.12.3COOH−3.5−3.6−3.6−3.4−3.02.46.44.811.55.14.2CHO−3.9−3.8−3.8−3.6−3.30.94.23.28.43.22.4NO2−3.8−3.8−3.7−3.6−3.32.05.84.310.84.63.7MADc0.260.250.240.395.339.077.6813.907.896.99aPost-SCF on LDA/TZ2P orbitals and densities, see Computational detailsbFrom [50]cMean Absolute Deviation (in kcal mol−1) of DFT energies from reference MP2 data Reference data from Jurecka and co-workers Scheme 3 shows the fourteen orientations of the stacked cytosine dimer that have been considered in the paper by Jurecka and co-workers [21] (with a vertical separation of 3.3 Å for the first orientation, and 3.4 Å for all others). The corresponding reference energies and our DFT interaction energies are collected in Table 3. The latter also contains the MAD values with respect to two sets of reference energies, i.e. the usual MP2/6-31G*(0.25) results (MAD1) as well as the MP2 energies obtained after complete-basis-set (CBS) extrapolation corrected for the difference between MP2 and CCSD(T) with the 6-31G*(0.25) basis set (MAD2). Similar to what was found for the benzene-pyridine systems (see above), the MAD values for standard density functionals are found between 5 and 14 kcal mol−1, resulting in many cases in erroneous repulsive stacking interactions between the cytosine fragments (see Table 3). Improved results are again obtained with LDA, KT1, KT2, and to a lesser extent BHandH, which have MAD1 values (i.e. deviations with respect to MP2/6-31G*(0.25)) of 0.9, 0.8, 0.6 and 1.5 kcal mol−1 respectively, and MAD2 values of 0.4, 0.5, 0.7 and 0.5 kcal mol−1 respectively with the CCSD(T) data (see Table 3). Table 3Stacking energies (in kcal mol−1) for several orientations of the cytosine dimer (see Scheme 3), computed with various density functionals and MP2a,borientationMP2b,cMP2ccb,dLDAKT1KT2BHandHPW91BLYPBP86OLYPB3LYPX3LYP12.22.52.72.32.73.98.011.710.916.510.99.92−3.1−3.8−3.7−3.9−3.5−3.32.66.55.412.35.24.13−7.2−8.9−8.8−8.7−8.3−9.0−2.11.70.78.00.2−1.04−8.3−9.9−9.4−9.2−8.9−10.1−2.90.8−0.16.9−0.9−2.150.20.30.60.10.61.56.410.39.315.79.38.360.50.60.80.40.81.86.810.89.716.39.88.87−0.5−1.0−0.7−0.9−0.60.22.85.55.28.75.04.28−7.3−9.1−8.4−8.3−7.9−8.8−3.00.2−0.25.6−1.2−2.29−7.6−9.1−8.7−8.5−8.2−9.4−2.31.50.57.3−0.2−1.410−6.6−8.3−7.9−7.7−7.5−8.3−1.81.80.97.60.3−0.811−7.6−9.4−8.8−8.5−8.3−9.5−2.80.6−0.16.2−1.0−2.112−5.5−7.4−6.7−6.6−6.2−7.0−3.6−1.6−1.41.5−2.4−3.113−7.4−8.8−8.3−8.1−7.9−8.6−3.00.4−0.45.2−1.0−2.014−7.0−9.1−8.8−8.7−8.4−9.4−2.11.60.78.0−0.1−1.3MAD1e0.940.810.601.494.878.357.5813.637.066.03MAD2f0.380.470.720.526.049.528.7514.808.247.21aPost-SCF on LDA/TZ2P orbitals and densities, see Computational detailsbFrom [21]cMP2/6-31G*(0.25) resultsdMP2 with complete-basis-set (CBS) extrapolation, i.e. MP2/CBS, corrected for Δ[CCSD(T)/6-31G*(0.25) - MP2/6-31G*(0.25)]eMean Absolute Deviation (in kcal mol−1) of DFT energies from MP2/6-31G*(0.25) datafMean Absolute Deviation (in kcal mol−1) of DFT energies from MP2/CBS corrected for Δ[CCSD(T)/6-31G*(0.25) - MP2/6-31G*(0.25)] Reference data from Wu and Yang In the paper by Wu and Yang, [14] the potential energy surface (PES) was calculated as a function of the twist angle between two stacked bases (see Scheme 1 and methodological section for the definition of the twist angle) using MP2/6-31G*(0.25), which was chosen as a reliable reference method based on the paper by Hobza and co-workers [22]. Although thymine was not included by Wu and Yang, we report here the energy profiles for all combinations of the five RNA/DNA bases, i.e., thymine, adenine, guanine, cytosine, uracil (see Scheme 4 for the geometries). The vertical separation is 3.4 Å for the C-C, G-C and G-G stacked base dimers, and 3.3 Å for all other systems. This difference in vertical separations was chosen based on earlier papers by Sponer and co-workers [19]. From the tests with the benzene dimer (Table 1), the Mignon (Table 2) and Jurecka reference data (Table 3), it is already evident that standard density functionals do not give reliable interaction energies, which is now shown to be true also for the energy profiles as function of the twist angle. Figure 1 shows the PES for two stacked bases, namely C-C and G-U, which are representative for all possible base pair combinations (the complete figure for all combinations of stacked bases can be found in the Supporting Information). Interestingly, although the standard density functionals typically underestimate the interaction energy, the shape of the PES is in all cases highly similar to that based on the more accurate MP2 data. This suggests that, at least trends and qualitative features in the rotational energy profile can be correctly reproduced with DFT which otherwise shows a (functional and basis-set dependent) constant shift with respect to the more accurate MP2 rotational profile. Fig. 1Stacking interaction (in kcal mol−1) as function of the twist angle (in degree) for π-π stacks of two bases, C-C and G-U, computed with various density functionals. The thick lineswithfilled circles, triangles, squaresand diamonds show the results obtained with LDA, PW91, BLYP, and BP86, respectively. The dashed lines with open squares, trianglesand diamonds show the results obtained with OLYP, B3LYP and X3LYP, respectively. The bold linewithcrosses represents the MP2/6-31G*(0.25) reference data It is, however, more instructive to look at functionals that by themselves already give accurate interaction energies, such as LDA, KT1, KT2 and BHandH. Figure 2 shows the corresponding rotational-energy profiles, again for the stacked bases C-C and G-U. From Fig. 2 it is evident that the LDA, KT1, KT2, and to a lesser extent BHandH are visually (and virtually) indistinguishable from the reference MP2 data. Fig. 2Stacking interaction (in kcal mol−1) as function of the twist angle (in degree) for π-π stacks of two bases, C-C and G-U, computed with various density functionals. The red, blue and green lines show the results obtained with BHandH, KT1 and KT2, respectively. The bold line with crosses represents the MP2/6-31G*(0.25) reference data Figure 3 shows the interaction energy as a function of the vertical separation for both the standard (upper diagrams) and promising (lower diagrams) density functionals, together with MP2 reference curve in each of the four diagrams. Not surprisingly, the density functionals that gave larger errors for the rotational energy profile, also give larger errors for the vertical separation profiles: almost all standard density functionals show potential energy surfaces that are repulsive, or at best weakly attractive with a very shallow minimum at elongated distances. In contrast, the promising functionals (i.e., KT1, KT2, LDA and BHandH) do provide well-defined minima with equilibrium distances (3.2–3.3 Å) close to the reference MP2 data. Similar to the uncertainty in the reference energy data of some 0.5–1.0 kcal mol−1 (see above), we may expect a similar uncertainty for the vertical separation, which we very roughly estimate at 0.1–0.2 Å. The differences between the equilibrium distance of the promising density functionals and that of the reference MP2 falls well within this estimated uncertainty. Thus, KT1, KT2, LDA and BHandH perform well not only for rotational energy profiles but also for the PES as a function of the vertical separation. Fig. 3Stacking interaction (in kcal mol−1) as function of the vertical separation (in Å) for π-π stacks of two bases, A-A and G-G, computed with various density functionals. The thick lines with filled triangles, squares and diamonds in the upper diagrams show the results obtained with PW91, BLYP, and BP86, respectively. The dashed lines with open squares, triangles and diamonds in the upper diagrams show the results obtained with OLYP, B3LYP and X3LYP, respectively. The red, blue, green and orange lines in the lower diagrams that truly show well-defined minima are obtained with BHandH, KT1, KT2 and LDA respectively. The bold black line with crosses represents the MP2/6-31G*(0.25) reference data Decomposition of interaction energy Now that we have established the reliability of the KT1, KT2, LDA and BHandH functionals for the interaction energy of stacked DNA bases, it is interesting to study the actual interaction into greater detail. This is done through a quantitative decomposition of the interaction energy ΔEint into electrostatic attraction ΔVelstat, Pauli repulsion ΔEPauli and orbital interactions ΔEoi (see Methods). Table 4 provides the results of the energy decomposition at KT1/TZ2P for the five π-stacked homo dimers, for each one at a twist angle of 0° and 180° (which is the lowest-energy conformation) and at two different vertical separations. Note that the lowest-energy conformation is always achieved at a twist angle of 180° but that the optimal stacking distance (vertical separation) varies by 0.1 – 0.2 Å between the different π-stacked homo dimers. The energy changes by −6.8 (A-A), −11.8 (C-C), −10.2 (G–G), −11.2 (T-T) and −7.9 (U-U) kcal mol−1, respectively, when going from a twist angle of 0° to 180° (the lowest-energy conformation). This change in energy is almost completely resulting from a change in electrostatics that change by −5.4 (A-A), −13.1 (C-C), −10.3 (G-G), −6.6 (T-T) and −8.3 (U-U) kcal mol−1 respectively. The sum of Pauli repulsion and orbital interactions changes much less, by ca. 1.4 kcal mol−1 or less, except for T-T where the sum of Pauli repulsion and orbital interactions changes by 4.6 kcal mol−1 due to the influence of the peripheral methyl groups. But also the changes in each term individually, i.e., either ΔEPauli or ΔEoi, are much smaller (typically 2 to 5 times, but in some cases even more) than the changes in ΔVelstat (again, only in the case of T-T, the change in ΔEPauli is nearly as large as that in ΔVelstat). Here we note that the same energy decomposition analyses for the five π-stacked homo dimers at LDA/TZ2P instead of KT1/TZ2P yield values that differ by only 1 kcal mol−1 or less (not shown in Table 4). Table 4Decomposition of interaction energiesa (in kcal mol−1) between stacked DNA bases in different geometries, computed at KT1/TZ2P A-AC-CG-GT-TU-UTwist angle 0°(TW, Rvert)(0°, 3.3 Å)(0°, 3.4 Å)(0°, 3.4 Å)(0°, 3.3 Å)(0°, 3.3 Å)ΔEPauli5.352.432.1311.003.72ΔVelstat−3.311.801.97−2.48−0.07ΔEoi−2.30−1.88−2.49−5.66−2.11ΔEint−0.262.351.612.861.54Lowest energy conformation(TW, Rvert)(180°, 3.2 Å)(180°, 3.1 Å)(180°, 3.2 Å)(180°, 3.3 Å)(180°, 3.1 Å)ΔEPauli5.867.014.524.286.13ΔVelstat−8.72−11.32−8.33−9.04−8.38ΔEoi−4.17−5.12−4.80−3.58−4.12ΔEint−7.02−9.44−8.61−8.34−6.36aSee Computational details Additivity of interaction energies of stacked base pair dimers With the good performance of KT1 for stacking interactions kept in mind, we went one step further, and looked also at the interactions within stacked dimersof hydrogen-bonded base pairs (see Scheme 5) [54]. The geometry was obtained by taking the geometry of one hydrogen-bonded base pair and putting another one at 3.4 Å on top of the former. The base pair dimer is stabilized by hydrogen bonding between the bases within one layer, and stacking interactions between the bases in different layers of the stack. In this case, because there are two bases per layer, there are also interactions present between bases that are, in a sense diagonally, on opposite sides and in different layers [55], the so-called cross terms. For example for the AT-AT system as shown in Scheme 5, this refers to the interaction between the thymine of the top layer with the adenine of the bottom layer. Scheme 5Structure of stacked DNA base pair dimer AT-AT with twist angle of 36°. The dot represents the B-DNA helical axis of rotation.[54] In order to investigate in more detail the importance of the various energy terms, in particular, the cross-terms, we approximate the total stacking interaction between the base pairs in the stacked base pair dimer by the sum of four pairwise interactions between two bases, i.e., we approximate the total (stacking) interaction energy ΔEint between the layers AB and CD in Scheme 6 by the sum ΔEadd of interactions A-C, B-D, A-D and B-C as shown in Equations 4a,b: Scheme 6Additivity approximation for the π-π interaction between two stacked Watson-Crick base pairs in terms of pairwise interactions between individual bases The remaining term ΔEcoop that, added to the approximate ΔEadd yields again the real stacking interaction ΔEint, consists of cooperativity effects between the different interactions. In Table 5, we report the energy values resulting from the additivity scheme for the stacked AT-AT, GC-GC and AU-AU base pair dimers at three different values of the twist angle (0, 36 and 180°). Importantly, the additive approximation ΔEadd of the real stacking interaction ΔEint largely accounts for the latter. ΔEcoop has in most cases a positive sign and is small compared to either ΔEadd or ΔEint. Furthermore, the dependence of the ΔEcoop term on the twist angle is much smaller than that of the interaction energy ΔEint. For example, ΔEcoop varies from 1.0 to 1.6 kcal mol−1 whereas ΔEint varies from −1.4 to −10.7 kcal mol−1 for the stacked AT-AT base pair dimer (see Table 5). Larger cooperativity effects are found in the case of the stacked GC-GC base pair dimer, where ΔEcoop values are found between 2.0 and 4.4 kcal mol−1. Note however that the changes in ΔEcoop are again small compared to the changes in ΔEint. Table 5Interaction energy (kcal mol−1) between stacked DNA base pairs in terms of the additivity scheme, computed at KT1/TZ2Pa,bSystemTWΔEACΔEBDΔEADΔEBCΔEaddΔEcoopΔEintAT-AT0−1.23+1.26−1.24−1.24−2.45+1.04−1.41AT-AT36−5.27−3.93−1.11−0.93−11.24+1.11−10.13AT-AT180−1.84+0.39−5.44−5.44−12.33+1.62−10.71AU-AU0−1.23+0.94−1.37−1.37−3.03+1.17−1.86AU-AU36−5.27−3.00−1.20−1.08−10.55+1.20−9.35AU-AU180−1.84+0.28−4.67−4.67−10.90+1.73−9.17GC-GC0+2.15+2.83−4.90−4.90−4.82+4.42−0.40GC-GC36−2.08−1.32−3.44−4.71−11.55+3.92−7.63GC-GC180−7.59−2.60−1.52−1.52−13.23+1.98−11.25aSee Scheme 6: bPost-SCF on LDA/TZ2P orbitals / densities. The cross-terms in the additivity scheme are as important as the non-cross terms, especially when rotating the base pair dimer from a twist angle of 0 degrees to finally a twist angle of 180 degrees. At the end point of 180°, the A and B base have switched, and the cross terms are no longer AD and BC, but AC and BD; this switch occurs after a twist angle of 90°. For instance for the AT-AT base pair dimer at 0°, the cross terms are stabilizing (−2.5 kcal mol−1) and dominate the ΔEadd of −2.2 kcal mol−1. At 180°, the non-cross terms dominate (−10.9 kcal mol−1) with still a substantial contribution from the cross terms (−1.4 kcal mol−1). Note also the dominance of the cross terms (−10.2 kcal mol−1) over the non-cross terms (−3.0 kcal mol−1) for the GC-GC base pair dimer at 180°. This is mainly due to favorable hydrogen-bonding interactions between the cross G-G pair. When rotating base pair dimers from a twist angle of 0° to 36°, the cross terms in the additivity scheme reduce slightly. However, this is dominated by the increase of favorable interactions between the stacked bases. For instance for AT-AT, the cross terms drop from −2.5 to −2.0 kcal mol−1, but the non-cross terms increase from +0.0 to −9.2 kcal mol−1. The same trend is observed for the AU-AU and GC-GC base pair dimers. Again we note that the same energy decomposition analyses for stacked DNA base pairs at LDA/TZ2P instead of KT1/TZ2P yield values that differ by only 1 kcal mol−1 or less (not shown in Table 5). Significance of the observed trends for the structure of DNA The stabilizing interactions for the structure of DNA mainly originate from hydrogen-bonding interactions between bases in one plane and the π-π stacking interactions between stacked bases. However, we have shown here that in addition to these terms there is also an important contribution of the cross terms that arise between bases that are neither hydrogen-bonded within one layer, nor stacked above each other. All these interaction energies can now be understood and quantified with density functional theory using appropriate functionals. For the hydrogen-bonding interactions, the BP86 and PW91 functionals seem to perform best, [3] while for π-stacking interactions KT1, KT2 and (surprisingly) LDA work well. The experimentally observed value for the twist angle (36°) is retrieved in our study on base pair dimers as the orientation with optimal stacking interactions. We have shown that this is predominantly determined by the classical electrostatic component of the stacking energy. The rotation to a twist angle of 36° destroys part of the favorable cross terms (see above), which is however completely overcome by the increase in favorable stacking interactions between the stacked bases. The twist angle and vertical separation that are experimentally observed are of course influenced by both the sugar-phosphate backbone and the presence of solvent and/or counter ions. These factors will be tackled in forthcoming work. Conclusions We have analyzed π-π stacking interactions between two benzenes or benzene analogs, between two DNA bases, and between two Watson-Crick base pairs using Density Functional Theory (DFT) in combination with large basis sets. The interaction energies for a large number of density functionals have been compared with ab initio reference data. In line with previous studies, most standard density functionals recover, at best, only part of the favorable stacking interactions. An exception is the new KT1 functional, which has been constructed to closely resemble the near-exact Zhao–Morrison–Parr (ZMP) exchange-correlation potential. Thus, not unexpectedly, KT1 gives a good description for π-π stacking energies and potential energy surfaces (PES) resulting in bound systems. Surprisingly, however, a similarly good performance is achieved with the Local Density Approximation (LDA). In view of the accurate prediction of PESes and the low computational cost, we recommend the use of either KT1 or LDA for treating the π-π stacking interactions in larger systems, using a QM/QM approach (e.g., our QUILD approach which is under development [33]) that allows this type of interaction to be separated from other types of interaction, such as hydrogen bonding but also regular covalent bonds. To gain insight into the origin of π-π stacking interactions, we have decomposed the interaction energies into the classical electrostatic attraction, Pauli repulsion and orbital interactions. Interestingly, the electrostatic interactions appear to be the most important factor that determines the shape and depth of the PES. In the case of two stacked Watson-Crick base pairs, this classical electrostatic attraction causes a minimum to occur along the energy profile at a twist angle of 36°. Furthermore, the stabilizing contributions to the stacking interaction between two Watson-Crick base pairs is shown to originate from the inter-strand stacking terms, that is, from the interaction between two bases that are in different Watson-Crick pairs and also not directly stacked on top of each other. The above-mentioned insight that electrostatic attraction plays an essential role in π-π stacking interactions nicely consolidates and extends the recent finding by Krapp, Bickelhaupt and Frenking [56] that classical electrostatic attraction is essential for understanding trends in bond strength of diatomic molecules. It is interesting to note that qualitative models of both “regular” chemical bonds and those of π-π stacking interactions often completely ignore classical electrostatic attraction. These qualitative models attempt to rationalize trends in bonding entirely in terms of orbital interactions (in the case of regular bonds) and dispersion interactions (in the case of π-π stacking). Electronic supplementary material Below is the link to the electronic supplementary material. Supporting Information Energy profiles for various π-π stacked systems as a function of the twist angle and the vertical separation computed with MP2 and various density functionals (PDF 1.90 mb)

J_Mol_Med-3-1-1820751

An unexpected new role of mutant Ras: perturbation of human embryonic development

The Ras signaling pathway controls important cellular responses to growth factors, and somatic mutations in RAS genes and other components of the Ras pathway, such as PTPN11 (encoding the protein-tyrosine phosphatase SHP-2) and BRAF, are found in human malignancies. Ras proteins are guanosine nucleotide-binding proteins that cycle between active guanosine triphosphate (GTP)-bound and inactive guanosine diphosphate (GDP)-bound conformations. Neoplasia-associated Ras mutations frequently affect amino acids G12, G13, or Q61 and decrease the intrinsic guanosine triphosphatase (GTPase) activity by ten- to twentyfold. The GTPase activity is crucial for Ras inactivation by hydrolysis and release of a phosphate group from Ras·GTP to produce Ras·GDP. We and others have recently discovered germline mutations in the KRAS gene in individuals diagnosed with Noonan and cardio–facio–cutaneous (CFC) syndrome, two clinically overlapping disorders characterized by short stature, distinct facial anomalies, heart defects, and other abnormalities. Noonan syndrome-associated mutations V14I and T58I K-Ras activate Ras but have milder biochemical effects than somatic mutations encountered in cancers, offering an explanation why these K-Ras lesions are tolerated during embryonic development. Together with recent findings of BRAF, MEK1, and MEK2 mutations in CFC syndrome and HRAS mutations in Costello syndrome, another clinically related disorder, it has now become clear that Noonan-like features (short stature, relative macrocephaly, facial anomalies, learning difficulties) that are found in these three related disorders are a result of constitutive activation of the Ras–Raf–extracellular signal-regulated and mitogen-activated protein kinase pathway. RAS genes The three human RAS genes, KRAS (isoforms A and B), NRAS, and HRAS (isoforms 1 and 2), encode small, highly conserved guanosine triphosphatases (GTPases) that relay growth signals to a number of effector proteins to control fundamental cellular pathways (reviewed in [1]). Ras proteins act as molecular switches by cycling between an active guanosine triphosphate (GTP)-bound and an inactive guanosine diphosphate (GDP)-bound state. Stimulated growth factor receptors recruit a number of adaptor proteins that activate guanosine nucleotide exchange factors (GEFs) to remove guanine nucleotides from Ras. Ras is then activated by binding to GTP, which is present at a tenfold higher concentration than GDP. In the GTP-bound state, the two switch regions of Ras (switch I and II) change their conformation. This conformational switch allows Ras to bind and activate Ras effector proteins such as Raf-1. The “on” position is turned “off” by an intrinsic GTPase activity, which hydrolyses and releases a phosphate group from Ras·GTP to produce Ras·GDP. The conformational transition of the switch I and II regions that is associated with this reaction disrupts the interaction between Ras and its effectors. The intrinsic GTPase activity of Ras is slow and accelerated about 105-fold by GTPase activating proteins (GAPs), such as neurofibromin or p120 GAP. These GAPs mediate Ras·GTP hydrolysis by inserting an arginine residue (arginine finger) into the phosphate-binding pocket of Ras (reviewed in [2, 3]; Fig. 1). Fig. 1Ras cycles between an active GTP-bound and an inactive GDP-bound conformation. In the active state, the two switch regions, switch I and II, change their conformation allowing Ras to activate effector proteins. The intrinsic GTPase hydrolyzes a phosphate group to produce Ras·GDP. This reaction is accelerated by GTPase activating proteins (GAPs). A similar version of this figure has been previously published [61], republication with permission For several decades, it has been well recognized that RAS genes are frequently mutated in human cancers (reviewed in [4]). These mutations predominantly lead to amino acid substitutions at residues G12, G13, or Q61 and lock Ras in the active GTP-bound state by diminishing the intrinsic Ras GTPase activity and/or by causing resistance to GAPs. These mutations were long believed to occur only as somatic events, and in a mouse model system, widespread expression of endogenous K-RasG12D leads to embryonic death [5]. The highest incidence of RAS mutations are found in adenocarcinomas of the pancreas (90%), the colon (50%), and the lung (30%), in thyroid tumors (50%), and in myeloid leukemia (30%) [4]. Noonan syndrome and related disorders Noonan syndrome (NS; MIM 163950) is an autosomal dominant disorder characterized by short stature, distinct facial anomalies (Fig. 2), a typical spectrum of congenital heart defects, including pulmonic stenosis, hypertrophic cardiomyopathy, and septal defects, and developmental delays (reviewed [6]). The disorder occurs in approximately 1 out of 2,000 individuals and shares many features with the much less common disorders of Costello syndrome (CS), cardio–facio–cutaneous (CFC) syndrome, and lentigines, electrocardiographic conduction defects, ocular hypertelorism, pulmonary stenosis, abnormalities of the genitals; retarded growth resulting in short stature, and deafness (LEOPARD) syndrome (LS) [6]. Patients with these disorders have in common Noonan-like facial features, a similar spectrum of cardiac anomalies, delayed growth, and—to a variable degree—developmental retardation, which is stronger in patients with CS or CFC. In addition, each of these disorders is characterized by unique phenotypic patterns: (1) CS patients have nasal papillomata, loose skin, and a strong predisposition to tumors (mainly rhabdomyosarcoma) [7]; (2) CFC patients have ectodermal abnormalities with sparse curly hair, sparse, or absent eyelashes [8]; and (3) LS patients have multiple lentigines typically emerging during adolescence [6]. NS and LS may be familial with an autosomal dominant inheritance pattern (except for rare instances of NS where rare recessive inheritance has been suspected [9]), but most cases of these disorders and virtually all of the more severe conditions, CFC and CS, occur sporadically, suggesting dominant new mutations. Until recently, the genetic basis for these disorders was unknown. It was not clear whether they represented genetically distinct entities or if they were different (allelic) variants of a common disorder. By linkage analysis in families with NS, the disorder was mapped to chromosome 12q24 [10], and subsequently, it has been shown that approximately 50% of patients with NS carry germline mutations in PTPN11 [11]. The PTPN11 gene encodes for tyrosine-protein phosphatase (SHP-2), a phosphatase that relays growth signals from activated growth factor receptors to other signaling molecules, including Ras (reviewed in [12]). Most PTPN11 mutations are predicted to disrupt the auto-inhibition of the catalytic phosphatase domain (PTPase) by the N-terminal src-homology 2 (N-SH2) domain thereby promoting the active conformation of the protein [12]. PTPN11 mutations were not found in patients with CS or CFC syndromes (reviewed in [6]). However, specific mutations in the same gene were uncovered in patients with LS [13]. Surprisingly, in contrast to NS, LS mutants are catalytically defective and may act as dominant negative mutations [14, 15]. Molecular modeling and biochemical studies suggest that LS mutations disrupt the SHP-2 catalytic domain to result in open, inactive forms of SHP-2. Thus, the pathogenesis of LS and NS is distinct. It is unknown why the clinical phenotypes of LS and NS are similar although the underlying mutations have opposite biochemical effects. There are several possible explanations for this paradox. These are discussed in detail by Kontaridis et al. [14]: (1) The NS and LS phenotypes potentially result from differential effects of mutant SHP-2 on different receptor tyrosine kinase pathways at distinct developmental times. For example, recent work by others suggests that the main effect of NS mutants is to enhance epithelial–mesenchymal transformation/mesenchymal cell proliferation by increasing ErbB2/3 (and/or ErbB3/4) signaling. By contrast, LS mutants antagonize HB-EGF/ErbB1 signaling at later times [14]; (2) Other phenotypes common to NS and LS such as facial abnormalities and short stature might involve defective migration and/or differentiation, which might result from increased or decreased signaling involving the same pathway; (3) Yet, undetermined or poorly understood functions of SHP-2 might underlie pathogenesis of one or both of these disorders [14]. Fig. 2a Craniofacial phenotype of young children with NS, CFC, and CS aged between 10 and 18 months. Their genotypes are PTPN11 G503R, BRAF K499E, and HRAS G13C, respectively. Note the similarities of facial features, including hypertelorism, broad forehead, and low-set ears. Coarse facial features are particularly typical of CS. b The same disorders in older children and adolescents with the mutations PTPN11 N308D, MEK2 F57I, and HRAS G12S, respectively. Facial features become more distinct with age, although the similarities of the three syndromes are still evident. Courtesy of Prof. Rainer König, Frankfurt (images of CFC patients) and Prof. Kerstin Kutsche, Hamburg (images of CS patients) The Noonan gene PTPN11 also acts as oncogene One of the myeloid malignancies that has been found to be particularly related to perturbed Ras signaling is juvenile myelomonocytic leukemia (JMML), a myeloproliferative disorder (MPD) of early childhood [16], in which mutations occur in NRAS or KRAS (∼25%) or in NF1 (clinical diagnosis of neurofibromatosis type 1 in ∼11%) [17–19]. In a murine model, somatic activation of K-Ras in hematopoietic cells initiates a rapidly fatal MPD modeling JMML [20, 21]. Despite a low annual incidence estimated at 1–2 per million, JMML has attracted many clinical and basic researchers because of the severe and often lethal clinical course and the association with NS [22] and neurofibromatosis type 1 (NF1; MIM 162200; reviewed in [19]). Shortly after the discovery of PTPN11 mutations in individuals with NS, specific germline PTPN11 alterations were identified in young children with NS who developed a JMML-like disorder within the first few weeks of birth (NS/JMML) [23, 24], and somatic PTPN11 mutations were subsequently uncovered in JMML cells from 35% of children with non-syndromic JMML [23–25]. Somatic PTPN11 mutations also occur in B-cell precursor acute lymphoblastic leukemia [26] and rarely other malignancies [27]. Somatic mutations observed in patients with JMML differ from those mutations found in patients with NS/JMML and from those detected in patients with NS alone. In sporadic JMML, the most common mutation predicts an E76K substitution. This mutation has never been found in patients with NS (N308D most common) or NS/JMML (T73I most common) [6, 25]. In elegant functional experiments, several groups have shown that somatic PTPN11 mutations associated with sporadic JMML exhibit stronger biochemical and biological effects than germline PTPN11 mutations, leading to the concept that only milder SHP-2 activation may be tolerated during embryonic development [28–30]. The situation is too complex however to be explained by a simple model of strong activating somatic vs less activating germline mutations alone. Enzymatic, structural, and mathematical modeling analyses show that these mutants can affect basal activation, SH2 domain-phosphopeptide affinity, and/or substrate specificity to varying degrees, and there is no absolute correlation between the mutants’ extents of basal activation and the diseases they induce [31]. A murine knock-in Ptpn11D61G/+ model of NS has been constructed, revealing that endocardial cushions of these mice have increased activation of extracellular signal-regulated kinase [32]. This finding suggests that the phenotype exhibited by PTPN11 mutations is mediated through hyperactive Ras signaling. Neurofibromatosis type 1 JMML is also associated with NF1 (MIM 162200), an autosomal dominant disorder that occurs in 1 of 4,000 births and is characterized by pigmentary anomalies (multiple café-au-lait spots) and a predisposition to benign and malignant tumors of mainly neurogenic origin. The disorder is caused by mutations in the NF1 tumor suppressor gene, which encodes the RasGAP neurofibromin. The incidence of JMML is increased approximately 200-fold in children with NF1 (reviewed in [33]). Loss of the normal NF1 allele (LOH, loss of heterozygosity) is common in JMML cells from children with NF1 [34, 35], and this results in severely deregulated Ras signaling and causes aberrant growth of hematopoietic progenitor colonies in vitro [36]. In addition, adoptive transfer of homozygous Nf1 mutant fetal liver cells or somatic inactivation of a conditional mutant Nf1 allele in hematopoietic cells induces a JMML-like MPD in mice [37, 38]. Remarkably, many NF1 patients have mild features reminiscent of NS [39], and some may even develop a mixed phenotype, which has prompted the definition of neurofibromatosis–NS (NFNS; MIM 601321) as a separate entity. Most patients who are diagnosed with this condition harbor NF1 mutations without an obvious genotype–phenotype correlation [40]. Additional genes mutated in Noonan syndrome To date, the identification of additional NS genes was hampered by the fact that the vast majority of PTPN11 mutation-negative NS-patients represent sporadic cases [41], rendering genetic linkage studies impossible. However, recent findings by our group and others identified germline mutations in components of the Ras signaling pathway in individuals with NS or related syndromes [42–46]. We systematically screened patients with NS and CFC for RAS mutations after having discovered a novel de novo germline KRAS mutation in a patient with NS/JMML [42]. The mutation c.173C>T (p.T58I) detected in the index patient affects a highly conserved amino acid residue of K-Ras flanking the switch II region (amino acids 59–67) of the protein. De novo KRAS mutations were identified in 4 of 174 sporadic cases with NS previously excluded for PTPN11 mutations. All germline KRAS mutations were novel and not known to occur in human cancer. NS-associated KRAS alleles included recurrent mutations, V14I and D153V. The latter was confirmed in patients with more severe phenotypes [44, 46]. Analogous to the dual role of PTPN11 as both oncogene and developmental gene, this discovery led to the hypothesis that activating KRAS mutations do not only act as oncogenes; aberrant K-Ras can also cause developmental disorders when mutations—probably conferring relatively mild effects—emerge in the germline. To prove our hypothesis, we studied the functional properties of V14I and T58I K-Ras [42] and found both NS-associated mutants V14I and T58I to have intermediate biochemical (e.g., ability to hydrolyze Ras·GTP) and biological properties (e.g., growth behavior in response to growth factors after retroviral transfection) when compared with wild-type K-Ras and the oncogenic mutant K-Ras G12D [42]. The two aforementioned mutations, V14I and T58I, are located adjacent to amino acid residues that are typically altered in cancer (G12, G13, or Q61). They locate to regions of Ras that are known to be involved in GTP binding. By contrast, in a subset of NS patients, KRAS mutations lead to substitutions in protein regions not obviously involved in GTP binding (e.g., D153V K-Ras), indicating the existence of previously unappreciated mechanisms of Ras activation. Carta et al. [46] performed a structural analysis on the two K-Ras mutants, V152G and D153V, that these investigators identified in two patients with severe NS. Their computer-based analysis indicated that both mutations disturb the conformation of the guanine ring-binding pocket favoring the active GTP-bound conformation by increasing the guanine nucleotide dissociation rate. Additionally, these residues are predicted to be important for binding the RasGEF son of sevenless (SOS) [47]. In the meantime, we have detected a number of additional KRAS mutations associated with NS (Q22R, P34L, P34Q, I36M), all of which affect highly conserved amino acid residues and are assumed to confer (mild) gain of function [48]. In this study on additional 236 PTPN11-negative NS patients, we detected KRAS mutations in seven individuals [48], thus confirming that KRAS accounts for less than 5% of NS cases. Genes mutated in cardio–facio–cutaneous syndrome In our initial study, we also discovered a novel K-Ras mutation, K-Ras P34R, in 1 of 12 patients with CFC syndrome [42]. This mutation is now known to be located in another mutational hotspot associated with NS [48]. A P34R H-Ras lesion has been previously characterized by Stone et al. [49]. These investigators employed a mutagenesis strategy during which they found that H-Ras P34R binds to GTP in vivo. In vitro, H-Ras P34R is not stimulated by GAPs [49]. Considering the highly conserved G-domain structure of Ras proteins, the P34R K-Ras mutation presumably has very similar (if not identical) biochemical properties. The presence of K-Ras mutations in CFC syndrome was confirmed by Niihori et al. [44] who analyzed DNA specimens from 43 individuals with CFC syndrome and found two KRAS germline mutations (G60R and D153V) in three patients. Notably, the D153V K-Ras mutation also has been described in patients with NS [42, 46, 48]. In a more recent study, we identified additional K-Ras mutations in individuals with CFC or NS/CFC including Q22E and F156I. The observations that mutations, such as K-Ras F156I, may be associated with an overlap of NS and CFC suggest that there is not a very strict genotype–phenotype correlation [48]. Simultaneous to the finding of mutated K-Ras in CFC, Niihori et al. identified BRAF germline mutations in 16 of 43 CFC patients [36]. At the same time, another group found BRAF germline mutations in 18 of 23 CFC patients and MEK1 or MEK2 germline mutations in 3 of the remaining 5 CFC patients studied [45]. Like PTPN11 and RAS, BRAF is a known oncogene, and somatic mutations are frequently found in cancer [50]. The molecular basis of Costello syndrome Shortly before germline KRAS mutations were described in patients with NS and CFC, HRAS germline mutations were reported to cause CS [43]. Later, other investigators also detected these mutations in the majority (∼90%) of CS patients studied [51–53]. Surprisingly, these germline lesions affect the same amino acid residues of H-Ras that are also mutated in cancer. The observation that activating oncogenic H-Ras mutations affecting codons G12 or G13 are tolerated in the germline, whereas oncogenic K-Ras mutations affecting these residues are not, underscores the notion that although H-Ras and K-Ras are structurally highly similar, they play different roles during embryonic development. Knockout studies have shown that only K-Ras is essential for embryonic development, whereas N-Ras and H-Ras are not [54]. Different phenotypes exhibited by mutations in various Ras isoforms may be due to heterogenous expression patterns of these proteins. Additionally, various Ras isoforms undergo different processing, e.g., de/repalmitoylation kinetics that regulate subcellular localization and activity of Ras isoforms [55, 56]. Although the vast majority of patients with CS harbor HRAS mutations, very recent studies have indicated that a CS phenotype may occasionally be associated with specific mutations of BRAF [57] or KRAS [48]. It remains to be determined if these patients also have the increased risk of neoplasia, which is typical of CS. Therefore, we doubt it is useful to classify these patients with mutations in Ras pathway genes other that HRAS as CS. Neuro–cardio–facial–cutaneous syndrome The molecular explanation of how different germline K-Ras or H-Ras mutations cause a wide spectrum of different phenotypes is still largely unknown. Activating germline K-Ras mutations may be associated with the broadest spectrum of clinical manifestations [42, 44, 46]. However, these syndromes are not just a less or more severe expression of essentially the same disorder. Therefore, it is conceivable that the different RAS mutations associated with divergent phenotypes do not only vary quantitatively in their degree of constitutive activation. Unique lesions may also have different qualitative effects on downstream signaling pathways. Additionally, modifier loci may play a role. Recently, the term ‘neuro–cardio–facial–cutaneous’ (NCFC) syndrome [58] was coined to illustrate that clinically overlapping disorders of the NS spectrum, including NF1, NS, CS, LS, and CFC syndrome, are caused by mutations in components of the Ras signaling pathway (Fig. 3). We speculate that the phenotypic variability between these disorder results from (1) different expression patterns of affected genes/isoforms and (2) variable mechanisms by which certain mutants interact with downstream effectors or regulatory proteins; these mutants therefore perturb Ras signaling in varying degrees. Further research is required to determine the basis of genotype–phenotype correlations in NS and related disorders. Fig. 3The Ras signaling pathway relays growth signals from activated growth factor receptors to the nucleus. Somatic mutations in several molecules of the pathway have been implicated in cancer. It is now recognized that germline mutations of identical molecules may cause disorders of the Noonan spectrum. A similar version of this figure has been previously published [61], republication with permission Neurofibromatosis Noonan syndrome and NS-like features in neurofibromatosis The rapidly increasing knowledge of the Ras signaling pathway and its relation to developmental disorders of the NS spectrum suggests that Noonan-like features are the clinical correlate of any genetic abnormality occurring in the germline and leading to a generalized mild deregulation of the Ras signaling cascade(s) during embryonic development. The new insights gained by recent research in this field may explain the phenotypic similarities between individuals with NF1 and NS. In addition to the well-known symptoms defining NF1, namely multiple café-au-lait spots and cutaneous neurofibromas, patients with NF1 are known to exhibit NS features, including relative short stature, relative macrocephaly, mild facial anomalies, thorax deformities, and learning difficulties. NFNS probably just represents the extreme end of a highly variable expression of these features [59]. Although the association of NF1 and NS was described in one family with independently segregating mutations in NF1 and PTPN11 [60], this double chance event is not the causative mechanism in many other patients with NF1 and NS-like features. The majority of patients with the NFNS phenotype have been found to harbor NF1 mutations, some of which also occur in patients with “pure” NF1 [40, 59]. We propose that the NS-like features that are often present in patients with NF1 are potentially due to NF1 haploinsufficiency leading to decreased inactivation of Ras·GTP. This might cause mild constitutional deregulation of Ras signaling, which may occasionally reach a level that leads to a NF1-NS phenotype. Frequently, patients with NF1 have mild features of NS. These may be explained by a mild activation of Ras, which is due to loss-of-function of one NF1 allele. Unknown genetic modifiers may play a role. Malignant cells arising in patients with NF1 somatically loose the wild-type NF1 allele. This second somatic hit ablates the GAP function of neurofibromin resulting in strongly enhanced Ras signaling (Fig. 4). Fig. 4Model illustrating the molecular basis of how Ras signaling may be increased in patients with neurofibromatosis type 1 leading to Noonan-like feature in these patients. Although only one NF1 allele is inactive in the germline, tumors of patients with neurofibromatosis type 1 somatically lose the second, wild-type NF1 allele Work in progress Although the vast majority of cases with CFC and CS are explained by mutations in KRAS, BRAF, MEK1, MEK2, and HRAS, the underlying genetic defect in 50% of NS and 10–30% of CFC cases remains unknown. Based on the assumption that the NS phenotype results from hyperactive Ras signaling, we and others are currently screening DNA specimens from patients for mutations in genes encoding for other components, including negative regulators, of the Ras pathway. Analogous to PTPN11 and KRAS, currently unknown genes of the Ras pathway mutated in NS are presumably playing double roles in both development and oncogenesis. Mutations in BRAF and MEK1/2 have been excluded as major NS genes (M.Z., unpublished data). It will be of interest to study how the different expression patterns of various RAS genes and isoforms influence clinical phenotypes when these genes/isoforms are mutated in the germline. It will be crucial to elucidate the structural mechanisms to understand how these new lesions perturb signaling on a molecular level. Characterization of the biological consequences of these mutations will be largely improved by the construction of murine knock-in models. Note added in proof After this paper was accepted we uncovered mutations in SOS1 encoding for the homonymous RasGEF in patients with NS. Meanwhile, however, two other groups have published elegant reports on activating SOS1 mutations in approximately 10% of cases with NS: Roberts AE, Araki T, Swanson KD, Montgomery KT, Schiripo TA, Joshi VA, Li L, Yassin Y, Tamburino AM, Neel BG, Kucherlapati RS. Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nat Genet. 2006 Dec 3; [Epub ahead of print] Tartaglia M, Pennacchio LA, Zhao C, Yadav KK, Fodale V, Sarkozy A, Pandit B, Oishi K, Martinelli S, Schackwitz W, Ustaszewska A, Martin J, Bristow J, Carta C, Lepri F, Neri C, Vasta I, Gibson K, Curry CJ, Siguero JP, Digilio MC, Zampino G, Dallapiccola B, Bar-Sagi D, Gelb BD. Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome. Nat Genet. 2006 Dec 3; [Epub ahead of print]”.

Dev_Biol-1-5-2082130

Differential activities of the core planar polarity proteins during Drosophila wing patterning

During planar polarity patterning of the Drosophila wing, a “core” group of planar polarity genes has been identified which acts downstream of global polarity cues to locally coordinate cell polarity and specify trichome production at distal cell edges. These genes encode protein products that assemble into asymmetric apicolateral complexes that straddle the proximodistal junctional region between adjacent cells. We have carried out detailed genetic analysis experiments, analysing the requirements of each complex component for planar polarity patterning. We find that the three transmembrane proteins at the core of the complex, Frizzled, Strabismus and Flamingo, are required earliest in development and are the only components needed for intercellular polarity signalling. Notably, cells that lack both Frizzled and Strabismus are unable to signal, revealing an absolute requirement for both proteins in cell–cell communication. In contrast the cytoplasmic components Dishevelled, Prickle and Diego are not needed for intercellular communication. These factors contribute to the cell–cell propagation of polarity, most likely by promotion of intracellular asymmetry. Interestingly, both local polarity propagation and trichome placement occur normally in mutant backgrounds where asymmetry of polarity protein distribution is undetectable, suggesting such asymmetry is not an absolute requirement for any of the functions of the core complex. Introduction The term planar polarity was first used to describe the polarisation of structures within the plane of the insect cuticle (Nübler-Jung et al., 1987); however, the phenomenon is widespread in nature (reviewed in Klein and Mlodzik, 2005). Genetic analysis, particularly in Drosophila, has identified a planar polarity or PCP (planar cell polarity) pathway, dependent on the function of Frizzled (Fz) family receptors. Interestingly, not only are elements of this pathway conserved throughout the animal kingdom, but it is also required for developmental patterning processes that are distinct from planar polarity, such as polarised cell rearrangements during vertebrate gastrulation (Wallingford et al., 2002). To date, planar polarity patterning has been best studied in the Drosophila wing, which provides a simple model in which each cell becomes coordinately polarised and produces a single distally pointing trichome (Fig. 1A). It is widely considered that this pattern is produced by three tiers of gene activity (Tree et al., 2002a; Klein and Mlodzik, 2005; Strutt and Strutt, 2005). At the top of the hierarchy the type II transmembrane protein Four-jointed (Fj) and the atypical cadherins Dachsous (Ds) and Fat (Ft) act (probably with other unidentified factors) to provide a long-range (or “global”) patterning cue across the axis of the tissue (Adler et al., 1998; Zeidler et al., 2000; Strutt and Strutt, 2002; Ma et al., 2003). In a manner which is not understood, but is possibly dependent on widerborst gene function (Hannus et al., 2002), this long-range cue is thought to be interpreted by the middle tier of genes which include fz and a number of other factors known as the “core” polarity genes (Shulman et al., 1998). The final tier consists of tissue-specific effectors, which modulate cellular behaviours such as polarisation of the cytoskeleton and transcription, in response to activity of components of the core. The definition of the “core” polarity proteins is somewhat fluid, but was originally used to refer to factors that act together with Fz in all tissues examined in Drosophila. A notable property of Fz during planar polarity patterning is that it adopts an asymmetric subcellular localisation in polarising cells, for instance in the wing becoming localised to the junctional zone at the distal cell edge (Strutt, 2001). Five other proteins that act with Fz also adopt asymmetric localisations, either at the proximal or distal edges of wing cells, and loss of any one of these proteins prevents the distal localisation of Fz. As these proteins colocalise to junctions with Fz and are required for Fz localisation, it seems reasonable to regard them as the “core”. They consist of the multidomain cytoplasmic protein Dishevelled (Dsh) and the ankyrin repeat protein Diego (Dgo) that localise distally with Fz (Axelrod, 2001; Das et al., 2004), the fourpass transmembrane protein Strabismus (Stbm, also known as Van Gogh [Vang]) and the LIM-domain protein Prickle that localise proximally (Bastock et al., 2003; Tree et al., 2002b), and the sevenpass transmembrane cadherin Flamingo (Fmi, also known as Starry Night [Stan]) that localises both proximally and distally (Chae et al., 1999; Usui et al., 1999) (Fig. 1B). We note, that by this definition, the Gαo subunit encoded by the brokenheart gene may also be regarded as a component of the “core” (Katanaev et al., 2005), but this requires further investigation. Fz is thought to perform at least three functions in planar polarity patterning. The first is to receive long-range pattering information from upstream cues, for instance provided by the activities of Fj/Ds/Ft. Experiments analysing the temporal requirements of fz and ds suggest that such coupling may occur around 6 to 24 h of pupal life (Strutt and Strutt, 2002; Matakatsu and Blair, 2004). Recent models have suggested that this information could be provided either by generation of a gradient of Fz activity across the whole axis of the wing or alternatively via generation of a gradient of Fz activity across the axis of individual cells (Lawrence et al., 2004; Amonlirdviman et al., 2005). Notably, there is currently no evidence that other components of the core are involved in this coupling. Second, Fz is involved in a process of cell–cell communication that locally coordinates cell polarity (Adler et al., 2000; Ma et al., 2003; Lawrence et al., 2004) and also occurs after 6 h of pupal life (Strutt and Strutt, 2002). Historically, models to explain this coordination have invoked the production of a diffusible ligand for Fz (Park et al., 1994; Zheng et al., 1995; Adler et al., 1997). However, more recent models based on the observation of core polarity protein localisation to cell junctions have suggested that cell–cell signalling is contact-dependent (Tree et al., 2002b; Lawrence et al., 2004; Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). Generally, it has been assumed that all components of the core act with Fz in local coordination of polarity, but the exact roles of each protein have not been defined. The third function of Fz is to provide a subcellular cue for trichome growth, apparently via its localisation to the distal cell edge (Wong and Adler, 1993; Strutt, 2001). In the absence of Fz, or several other core components, trichomes form in the cell centre. Provision of Fz activity after 24 h of pupal life is sufficient to permit asymmetric localisation and polarised trichome growth (Strutt and Strutt, 2002); however, distal polarity is lost, presumably due to disruption of earlier fz functions. As all core components asymmetrically localise together with Fz prior to trichome formation, it is tempting to conclude that all are required for trichome placement, but this has not been definitively demonstrated. Asymmetric localisation of the core components only becomes clearly visible during pupal life by about 24 h of pupal life (but has also been observed earlier in development, see Classen et al., 2005), and hence it has been suggested that this probably follows the cell–cell communication phase (Strutt and Strutt, 2002; Lawrence et al., 2004). However, other workers have argued that asymmetric complex formation may occur progressively over a longer period of pupal life, and be intrinsically required for cell–cell communication and local coordination of polarity (Tree et al., 2002b; Amonlirdviman et al., 2005). In this context, it is important to consider that the spatial relationships observed during asymmetric complex formation (Fz, Dsh, Dgo and Fmi/Stan colocalising at distal cell edges; Stbm/Vang, Pk and Fmi/Stan at proximal edges) may not necessarily reflect earlier functional relationships. Notably, associations have also been reported between Dsh and Pk (Tree et al., 2002b; Jenny et al., 2005), Dsh and Stbm/Vang (Bastock et al., 2003; Jenny et al., 2005), Dgo and Pk (Das et al., 2004) and Dgo and Stbm/Vang (Das et al., 2004). In this manuscript, we address three key issues: First, which components of the core act together with Fz during the different planar polarity patterning processes? Second, are the spatial relationships seen during the later phase of asymmetric localisation also relevant during the phase of cell–cell communication and local coordination of polarity? Third, is asymmetric core protein localisation absolutely required for planar polarity patterning? Materials and methods Fly strains and genetics Alleles and transgenes used are described in FlyBase, except where noted. Temporal rescue of polarity phenotypes in the wing and eye was carried out and analysed as described (Strutt and Strutt, 2002). Actin ≫ fz-EYFP and Actin ≫ stbm-EYFP have been described (Strutt, 2001; Strutt et al., 2002), Actin ≫ dsh-ECFP, Actin ≫ fmi-FLAG, Actin ≫ pkpk, sev-stbm and sev-pksple were constructed as previously (Strutt and Strutt, 2002). Note that the pk locus produces two protein isoforms, of which the Pk variant is sufficient for wing patterning and the Sple variant is sufficient for eye patterning (Gubb et al., 1999). For double mutant clones, rescue of fz activity on the X and 2R was provided by Arm-fz-EGFP transgenes (Strutt, 2001) and rescue of stbm/Vang activity on the X was provided by an Actin-stbm-EYFP transgene. fz;stbm twinclones were generated by inducing clones of FRT42 stbm6 Arm-fz-EGFP in a fz background, resulting in cells homozygous for stbm6 Arm-fz-EGFP juxtaposed to twinspot cells lacking the transgene. Clones in the wing were generally induced using Ubx-FLP, kindly provided by Jürgen Knoblich. Exact genotypes used are as follows: Figure 1 Temporal rescue of stbm/Vang in wing: w hsFLP1; stbm6/stbmVang-A3; Act-FRT-polyA-FRT-stbm-EYFP/+ Temporal rescue of pkpk-sple in wing: w hsFLP1; pkpk-sple-13/pkpk-sple-13; Act-FRT-polyA-FRT-pk/+ stbm/Vang phenotype in eye: w; FRT42 stbm6/FRT42 P[w+] stbmVang-A3 Rescue of stbm/Vang phenotype in eye by sev-stbm: w; FRT42 stbm6/FRT42 P[w+]stbmVang-A3; sevE-sevP-stbm7.1/+ pkpk-sple-13 phenotype in eye: FRT42 pkpk-sple-13 cn sp/FRT42 pkpk-sple-13 cn Rescue of pkpk-sple phenotype in eye by sev-pksple: w; FRT42 pkpk-sple-13 cn/FRT42 pkpk-sple-13 cn; sevE-sevP-sple2.2/+ Genotypes shown in graph in (S): w; stbm6/FRT42 P[w+] stbmVang-A3 w; FRT42 stbm6/FRT42 P[w+] stbmVang-A3; sevE-sevP-stbm2.2/+, w; FRT42 stbm6/FRT42 P[w+] stbmVang-A3; sevE-sevP-stbm7.1/+ Genotypes shown in graph in (T): FRT42 pkpk-sple-13 cn/FRT42 pkpk-sple-13 cn w; FRT42 pkpk-sple-13 cn/FRT42 pkpk-sple-13 cn; sevE-sevP-pksple[2.2]/+ w; FRT42 pkpk-sple-13 cn/FRT42 pkpk-sple-13 cn; sevE-sevP-pksple[14.2]/+ Figure 2 fz clones using rescuing transgene on 2R: w; FRT42D/FRT42D Arm-fz-EGFP, Arm-lacZ; fz15/fz21, Ubx-FLP stbm/Vang clones: y w Ubx-FLP/+; FRT42D stbm6/FRT42D Arm-lacZ stbm/Vang; fz double clones: w; FRT42D stbm6/FRT42D Arm-fz-EGFP; fz21/fz21, Ubx-FLP stbm/Vang and fz twin clones: w; FRT42D stbm6, Arm-fz-EGFP/FRT42D Arm-lacZ; fz21/fz21, Ubx-FLP fmi/stan clones: y w Ubx-FLP/+; FRT42D fmiE59/FRT42D Arm-lacZ fmi/stan; fz double clones: w; FRT42D fmiE59/FRT42D Arm-fz-EGFP, Arm-lacZ; fz21/fz21, Ubx-FLP stbm/Vang fmi/stan double clones: y w Ubx-FLP/+; FRT42D stbm6 fmiE59/FRT42D Arm-lacZ Figure 3 dsh3 clones: y w dsh3 FRT18A/w Arm-lacZ FRT18A; FLP38/+dsh; fz double clones: y w dsh3 f36a FRT19A/w Arm-fz-EGFP FRT19A; fz21/fz21, Ubx-FLP dsh; stbm/Vang double clones: y w dsh3 f36a FRT19A/w Act-FRT-polyA-FRT-stbm-EYFP FRT19A; stbm6/stbm6, Ubx-FLP pkpk-sple clones: y w Ubx-FLP/+; FRT42D pkpk-sple-13/FRT42D Arm-lacZ pkpk-sple; fz double clones: w; FRT42D pkpk-sple-13/FRT42D Arm-fz-EGFP; fz21/fz21, Ubx-FLP pkpk-splestbm/Vang double clones: y w Ubx-FLP/+; FRT42D pkpk-sple-13 stbm6/FRT42D Arm-lacZ Figure 4 stbm/Vang dgo double clones: y w Ubx-FLP/+; FRT42D stbm6 dgo380/FRT42D Arm-lacZ pkpk-spledgo double clones: y w Ubx-FLP/+; FRT42D pkpk-sple-13 dgo380/FRT42D Arm-lacZ pkpk-spledgo; fz triple clones: w; FRT42D pkpk-sple-13 dgo380/FRT42D Arm-fz-EGFP; fz21/fz21, Ubx-FLP Figure 5 stbm/Vang overexpression in fz background: y w hsFLP1/+; Act-FRT-y+-FRT-GAL4, UAS-lacZ/+; fz15, UAS-stbm/Df(3L)fzD21 fz-EGFP overexpression in stbm/Vang background: w hsFLP1/+; Act-FRT-y+-FRT-GAL4, stbm6/stbm6, UAS-fz-EGFP fz-EGFP overexpression in pkpk-sple background: w hsFLP1/+; Act-FRT-y+-FRT-GAL4, pkpk-sple-13/pkpk-sple-13, UAS-fz-EGFP fz-EGFP overexpression in pkpk-sple dgo380 background: w hsFLP1/+; FRT42D pkpk-sple-13 dgo380/FRT42D pkpk-sple-13 dgo380; Act-FRT-CD2-FRT-GAL4, UAS-fz-EGFP/+ fz-EGFP overexpression in dsh1: w dsh1/Y; FLP38/Act-FRT-y+-FRT-GAL4, UAS-lacZ; UAS-fz/+ fz-EGFP overexpression under ptc-GAL4 control in wings containing dsh3 clones: y w dsh3 f36a FRT19A/y w w+FRT19A; ptc-GAL4/+; UAS-fz-EGFP, Ubx-FLP/+ Figure 6 Act-fz-EYFP expression in pkpk-sple background: w hsFLP1/+; pkpk-sple-13, Act-FRT-polyA-FRT-fz-EYFP/pkpk-sple-13 Act-fz-EYFP expression in dsh1 background: w dsh1/Y; Act-FRT-polyA-FRT-fz-EYFP/FLP38 dgo clones: y w Ubx-FLP/+; FRT42D dgo380/FRT42D Arm-lacZ pkpk-sple clones: y w Ubx-FLP/+; FRT42D pkpk-sple-13/FRT42D Arm-lacZ pkpk-spledgo double clones: y w Ubx-FLP/+; FRT42D pkpk-sple-13 dgo380/FRT42D Arm-lacZ dsh3 clones: y w dsh3 FRT18A/w Arm-lacZ FRT18A; FLP38/+ Supplementary Figure 1 Temporal rescue of fz: y w hsFLP1; Act-FRT-polyA-FRT-fz-EYFP/+; fz21 Temporal rescue of fmi/stan: y w hsFLP1; fmiE45, GAL4-1407/fmiE59; Act-FRT-polyA-FRT-fmi-FLAG/UAS-fmi GAL4-1407 and UAS-fmi provide rescue of fmi activity in the embryonic nervous system (Usui et al., 1999) Temporal rescue of dsh: w dsh1/Y; FLP38/+; Act>FRT-poly-FRT-dsh-ECFP/+ Supplementary Figure 2 w hsFLP1; stbm 6 /stbm Vang-A3 ; Act-FRT-polyA-FRT-stbm-EYFP/+ y w hsFLP1; fmiE45, GAL4-1407/fmiE59; Act-FRT-polyA-FRT-fmi-FLAG/UAS-fmi w dsh 1 /Y; FLP38/+; Act>FRT-poly-FRT-dsh-ECFP/+ w hsFLP1; pk pk-sple-13 /pk pk-sple-13 ; Act-FRT-polyA-FRT-pk/+ Supplementary Figure 3 stbm/Vang clones: w hsFLP1; FRT42 stbm6/FRT42 P[w+] stbm/Vang clones rescue by sev-stbm: w hsFLP1; FRT42 stbm6/FRT42 P[w+]; sevE-sevP-stbm7.1/+ Note that fz21, stbm6, dsh3, fmiE59, pkpk-sple-13 and dgo380 have been molecularly characterised and are thought to be null alleles on the basis of being unable to give rise to functional proteins (Jones et al., 1996; Wolff and Rubin, 1998; Wehrli and Tomlinson, 1998; Usui et al., 1999; Gubb et al., 1999; Feiguin et al., 2001). fmiE45 contains a missense mutation that generates an amorphic mutation in the wing by genetic criteria (Usui et al., 1999). fz15 contains a nonsense mutation that gives rise to a truncated protein that has been characterised as amorphic in the wing (Jones et al., 1996). stbmVang-A3 has not been molecularly characterised, but has been defined by genetic criteria to be amorphic in the wing (Taylor et al., 1998). dsh1 contains a missense mutation in the DEP domain which has been reported to be a strong mutation for planar polarity functions of the gene (Perrimon and Mahowald, 1987; Axelrod et al., 1998; Boutros et al., 1998). Histology Pupal wings were processed for immunofluorescence and imaged as previously (Strutt, 2001). Primary antibodies used for experiments or confirmation of genotypes were mouse monoclonal anti-βgal (Promega), rabbit anti-βgal (Cappel), rabbit anti-GFP (Abcam), mouse monoclonal anti-Fmi#74 (DSHB, Usui et al., 1999), rabbit anti-Pk (Tree et al., 2002b), rabbit anti-Stbm (Rawls and Wolff, 2003), rat anti-Dsh (Shimada et al., 2001) and rabbit anti-Dgo (Feiguin et al., 2001). Actin was visualised using Texas-Red-conjugated phalloidin (Molecular Probes). Adult wings were mounted in GMM and eye sections were prepared as described (Tomlinson and Ready, 1987). Results Differing temporal requirements of the core polarity proteins during wing development We previously analysed the temporal requirements of fz for planar polarity patterning in the wing, by rescuing the phenotype of fz mutant flies using an inducible fz-expressing transgene (Strutt and Strutt, 2002). Expression of the transgene is activated at different times during pupal development, by administration of a heat-shock, allowing determination of the latest timepoint that gene expression is sufficient to permit normal patterning. These studies found no requirement for fz function prior to 6 h after prepupa formation (APF). Progressively later heat-shocks up to 24 h APF produced stronger phenotypes that were qualitatively and quantitatively different from the reported fz loss-of-function phenotype. We classified this stronger phenotype as ds-like, as Fz protein was still localising at cell edges and specifying the site of trichome formation, but due to a loss of non-autonomous coordination of polarity Fz localisation was seen in a swirling pattern rather than uniformly at distal cell edges (Strutt and Strutt, 2002). Heat-shocks after 28 h APF resulted in the reported fz loss-of-function phenotype, consistent with this being produced by loss of the later autonomous function that places trichomes at the cell edge. As the core polarity gene stbm/Vang shows similar phenotypes to fz, exhibiting both strong domineering non-autonomous effects on trichome polarity and being required for trichome placement at the cell edge (Taylor et al., 1998), we considered it a good candidate for sharing common functions with fz. Using the same methodology, we analysed its timecourse of requirement in wing patterning (Figs. 1C–I). In common with fz, stbm/Vang is not required prior to 6 h APF, but then shows progressively stronger phenotypes when induced between 12 and 24 h APF, with induction at 30 h APF mimicking the normal loss-of-function phenotype (seen when no heat-shock is administered, Fig. 1I). For comparison, we repeated our analysis of the timecourse of fz-requirement (Supplementary Fig. 1A), but this time using the molecularly characterised fz21 null allele (Jones et al., 1996). This gave the same timecourse as observed for stbm/Vang, although generally with slightly stronger phenotypes being observed. Next we analysed the temporal requirement of the core polarity gene pk, which produces a protein that colocalises with Stbm/Vang at the proximal cell edge and which has been implicated in cell–cell coordination of planar polarity (Tree et al., 2002b). Interestingly, induction of pk expression as late as 20 h APF resulted in only negligible polarity defects in the adult wing, with induction at 24 h and 28 h still providing partial rescue of pk function (Figs. 1J–N). These results indicate that whereas stbm/Vang shares an early requirement with fz in the wing, pk has only a relatively late function. We further extended these results by investigating the requirements of the other two core components fmi/stan and dsh (Supplementary Figs. 1B, C). To circumvent the embryonic lethality of dsh null alleles, we analysed rescue of the strong planar polarity phenotype of the viable dsh1 allele (Perrimon and Mahowald, 1987) (the core component dgo was not examined, as the adult wing phenotype is too subtle for this approach to be feasible, Feiguin et al., 2001). Induction of fmi/stan expression between 12 and 24 h APF resulted in progressively stronger phenotypes that differ from the loss-of-function phenotype (Supplementary Fig. 1B), as observed for fz and stbm/Vang. Conversely, induction of dsh at 16 to 20 h APF resulted in relatively minor defects, although later induction revealed a strong requirement for dsh function after 20 h APF (Supplementary Fig. 1C). Hence, fmi/stan appears to share early requirements with fz and stbm/Vang, whereas dsh exhibits later temporal requirements. However, we cannot rule out the possibility that the dsh1 allele exhibits residual activity in planar polarity, which might contribute to the apparently later requirement. For all genotypes, early transgene induction can rescue, indicating that the transgenes provide appropriate levels of expression throughout the wing. Consistent with this, almost all cells express detectable protein after transgene induction (Supplementary Fig. 2). Furthermore, without induction, we observe the expected loss-of-function phenotype seen in the absence of the transgene, indicating that our results are unlikely to be due to “leaky” expression from the transgenes. We tested whether the differences might be due to transmembrane proteins taking longer to be synthesised and targeted to the appropriate subcellular sites; however, we found that after induction both Fz-EYFP and Dsh-ECFP show the appearance of junctional staining within 2–3 h (data not shown). Differing temporal requirements of the core polarity proteins during eye development We also find a common early requirement for fz and stbm/Vang in the eye. We previously distinguished between early and late activities of fz in the eye, by expressing fz under control of the sevenless promoter which is not active until the time of photoreceptor differentiation (Strutt and Strutt, 2002). Providing fz activity only at the time of photoreceptor differentiation resulted in defects in the dorsoventral polarity of ommatidia, indicating that fz activity is specifically required prior to photoreceptor differentation for correct specification of dorsoventral polarity. However, lack of fz activity after photoreceptor differentiation results in randomisation of all aspects of ommatidial polarity including both dorsoventral and anteroposterior polarity and rotation. Hence, fz shows two phases of requirement during eye development, an early phase needed just for dorsoventral patterning and a later phase required for dorsoventral and anteroposterior polarity and rotation of ommatidia. In contrast, we showed that ommatidial polarity and rotation defects within null mutant dsh3 tissue can be rescued completely by expression of sev-dsh, indicating that dsh does not share the early dorsoventral patterning function with fz, but nevertheless is required for the later phase of activity. We have now extended this work to stbm/Vang and pk. Rescuing the phenotype of stbm/Vang in the eye by expression of sev-stbm reduces general polarity and rotation defects but reveals an underlying randomisation of dorsoventral polarity (Figs. 1O, P, S) indicating that stbm/Vang shares with fz an early dorsoventral patterning function. Conversely, the pk phenotype is almost completely rescued by a sev-pksple transgene (Figs. 1Q, R, T) which expresses the Sple isoform of the Pk protein which is specifically required for eye patterning (Gubb et al., 1999). Thus pk, like dsh, does not exhibit an early patterning function in eye development. Interestingly, while investigating the functions of stbm/Vang in the eye, we found that stbm/Vang clones also show equatorial non-autonomy of the polarity phenotype. In a number of cases we observed dorsoventral polarity inversions in ommatidia on the equatorial sides of clones, in which all 8 photoreceptors of the ommatidium retain stbm/Vang activity (Supplementary Fig. 3). This is consistent with the observed non-autonomy of clones in the wing (Taylor et al., 1998). However, a previous analysis of over 169 misoriented ommatidia on the edges of clones found no significant evidence of non-autonomy of the polarity phenotype of stbm/Vang (Wolff and Rubin, 1998). Re-examination of this original data set in the light of our results again failed to find evidence of non-autonomy (T. Wolff, personal communication). The reasons for this discrepancy are currently unclear (but see legend to Supplementary Fig. 3 for further discussion of this issue). Mutual dependence of fz, stbm/Vang and fmi/stan for intercellular communication The common early function of fz, stbm/Vang and fmi/stan is likely to be either receiving long-range patterning cues and/or local coordination of polarity. Little is understood about how the long-range signal might be received, rendering this activity difficult to study. However, the effects of fz and stbm/Vang on local coordination of polarity can be assayed, as groups of cells lacking the activity of either gene cause neighbouring cells to mispolarise: fz clones cause neighbouring cells to point their trichomes towards the clone (arrow Fig. 2A, Gubb and García-Bellido, 1982; Vinson and Adler, 1987), whereas trichomes point away from stbm/Vang clones (arrow Fig. 2B, Taylor et al., 1998). As during later pupal life Fz and Stbm/Vang are seen localised to adjacent cell boundaries (Strutt, 2001; Bastock et al., 2003), it has been proposed that polarity coordination requires signals to pass between Fz and Stbm/Vang expressing cells. Some evidence for this has been presented in the abdomen (Lawrence et al., 2004), and recent models for planar polarity coordination in the wing are based on this hypothesis (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). However, there is no rigorous experimental evidence for signals passing between Fz and Stbm/Vang expressing cells in the wing. Furthermore, if such signalling does occur, it is not known whether signals might pass monodirectionally from Fz to Stbm/Vang, monodirectionally from Stbm/Vang to Fz or bidirectionally. To address this issue, we generated clones of cells simultaneously mutant for both fz and stbm/Vang. We reasoned that if signals pass strictly monodirectionally from Stbm/Vang to Fz, then wild-type cells outside of a clone would receive the same aberrant polarity cue from a stbm/Vang; fz double mutant clone as from a stbm/Vang single mutant clone. Thus, stbm/Vang; fz double mutant clones should show the same polarity phenotype as stbm/Vang single mutant clones. Conversely, if signals pass strictly from Fz to Stbm/Vang, cells outside should polarise as if neighbouring a fz clone and not a stbm/Vang clone. In this case, cells require Fz to send polarity cues and cells mutant for both fz and stbm/Vang provide the same aberrant polarity cue as cells mutant for only fz. However, if there is a bidirectional interaction, such that cells expressing Fz need to contact cells expressing Stbm/Vang and vice versa, then the result is harder to predict. In this case, signal receiving cells would require both Fz and Stbm/Vang and similarly signal sending cells would require both Fz and Stbm/Vang. Hence, one possibility is that clones of cells doubly mutant for fz and stbm/Vang would send or receive no polarity signals, and thus might have no effect on the polarity of their neighbours. A precedent for this prediction has been provided by work in the abdomen, where experimental results suggest that cells that lack Fmi/Stan are unable to send or receive polarity cues, and in this case the neighbours to exhibit normal polarity (Lawrence et al., 2004). However, it is also possible that a failure to send or receive cues might result in neighbouring cells adopting a randomised polarity. Control clones lacking only fz activity (marked by lack of lacZ expression) show trichomes pointing towards the clone (arrow Fig. 2A); whereas stbm/Vang clones (marked by lack of lacZ) show trichomes pointing away (arrow Fig. 2B). We then generated double mutant stbm/Vang; fz clones using null alleles of both fz and stbm/Vang and an Arm-fz-EGFP transgene (which rescues fz activity, see Strutt, 2001) located on the same chromosome arm as stbm/Vang. This resulted in genetically mosaic wings containing clones of cells of the genotype stbm6/stbm6; fz21/fz21 juxtaposed to twinspot tissue of the genotype Arm-fz-EGFP/Arm-fz-EGFP; fz21/fz21 or heterozygous tissue of the genotype Arm-fz-EGFP/stbm6; fz21/fz21 (see Materials and methods). Such clones of stbm/Vang; fz cells (marked by lack of Fz-EGFP, green) show negligible effects on the polarity of trichomes in neighbouring cells (Fig. 2C, trichomes visualised by labelling for Actin, magenta). This result fits the hypothesis that bidirectional interactions occur between Fz and Stbm/Vang expressing cells, and that lack of communication with cells within a clone leads to neighbouring cells adopting a wild-type polarity. Interestingly, within the double mutant clones, trichome polarity is also relatively unperturbed (Fig. 2C). This is in contrast to single mutant fz and stbm/Vang clones, where trichomes of a sufficient age adopt polarities consistent with those shown by trichomes outside the clone (e.g. Fig. 2B). We do not fully understand this phenomenon; however, it is well-established that trichomes within fz and stbm/Vang tissue largely emerge in the cell centre without obvious polarity (Wong and Adler, 1993; Taylor et al., 1998). We surmise that such “apolar” trichomes subsequently align themselves with the strongly polarised trichomes in the wild-type tissue surrounding the clone- possibly as a result of cytoskeletal interactions between adjacent cells. We also examined the distribution of the core polarity protein Fmi/Stan on the boundaries of clones of cells singly or doubly mutant for fz and stbm/Vang. It has previously been shown that Fmi/Stan strongly localises to the boundaries between fz+ and fz− tissue and stbm/Vang+ and stbm/Vang− tissue (arrowheads Figs. 2A′, B′; Usui et al., 1999; Bastock et al., 2003). Although not formally proven, it is widely thought that such localised protein localisation might mediate cell–cell communication (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). Consistent with this view, there is no strong localisation of Fmi/Stan on the boundaries of stbm/Vang; fz double clones (Fig. 2C′). So far our results suggest that Fz in one cell and Stbm/Vang in the adjacent cell is necessary for cell–cell communication and polarisation of trichomes. We next investigated whether Fz and Stbm/Vang in adjacent cells were sufficient for this process. To do this, we examined the effect of juxtaposing cells that lack fz activity to cells that lack stbm/Vang activity. This was achieved by generating clones homozygous for the genotype FRT42D stbm6, Arm-Fz-EGFP; fz21 juxtaposed to twinspots of the genotype FRT42D Arm-lacZ; fz21 (see Materials and methods), such that cells lacking stbm/Vang activity also lacked lacZ expression, whereas cells lacking both fz activity and the rescuing Arm-fz-EGFP transgene exhibited high levels of lacZ expression. On the boundaries where stbm/Vang tissue is juxtaposed to fz tissue, we observe strong Fmi/Stan localisation (Fig. 2D, arrowheads in Fig. 2D′) resembling that seen on the edges of fz or stbm/Vang clones (Figs. 2A′,B′). Notably, at the edges of fz and stbm/Vang clones, localised Fmi/Stan is associated with production of polarised trichomes (Figs. 2A, B), apparently as a result of assembly of a polarised asymmetric core polarity protein complex with Fz on one side of the cell–cell boundary and Stbm/Vang on the other side (Fig. 1B). We also observe polarised trichomes produced at the site of Fmi/Stan localisation on boundaries between stbm/Vang and fz tissue, which point towards the fz tissue (Figs. 2Dʺ,D‴). Taken together, the localisation of Fmi/Stan and the production of polarised trichomes suggest that a functional core polarity protein complex assembles on the boundaries between stbm/Vang and fz tissue and that this complex is sufficient to specify polarised trichome formation. We note that within the stbm/Vang and fz mutant tissue, there is no assembly of asymmetric complexes and trichome placement is unpredictable (Figs. 2D, D′), as expected from previous work (Wong and Adler, 1993; Taylor et al., 1998). We next analysed the role of Fmi/Stan in cell–cell communication of polarity cues in the wing. Unlike fz or stbm/Vang clones, clones of cells lacking fmi/stan activity do not strongly affect the polarity of neighbouring cells (Fig. 2E, Chae et al., 1999; Usui et al., 1999). This could be interpreted to suggest that fmi/stan is not required for cell–cell communication and coordination of cell polarity. However, the Fmi/Stan protein is thought to act as a homophilic cell adhesion molecule (Usui et al., 1999) and so alternatively Fmi/Stan may be required in both sending and receiving cells for coordination of cell polarity and loss of fmi/stan blocks cell–cell communication. Support for this second view comes from experiments in the abdomen, where cells overexpressing Fz or Stbm/Vang are unable to repolarise their neighbours if they also lack fmi/stan activity (Lawrence et al., 2004). We generated clones of cells double mutant for either fz and fmi/stan or for stbm/Vang and fmi/stan. Both showed a phenotype typical of single mutant fmi/stan clones (Figs. 2F, G), indicating that Fmi/Stan is required in both Fz and Stbm/Vang expressing cells for cell–cell communication, and thus by extension is required on both sides of the cell–cell boundary. Thus, for the fz-dependent process of cell–cell communication that is thought to locally coordinate cell polarity, we have demonstrated that Fz and Stbm/Vang are required in opposite cells and that Fmi/Stan is required in both cells. This spatial arrangement is as seen in the asymmetric complex that assembles at the site of trichome initiation (Fig. 1B), and supports models in which a subset of this complex is also involved in intercellular communication. dsh and pk are not required for intercellular communication In the late asymmetric complex, Dsh associates with Fz at distal cell edges whereas Pk localises with Stbm/Vang at proximal cell edges. Hence, it is possible that the association of Dsh with Fz is essential for signalling to Stbm/Vang in the adjacent cell, and similarly that Pk association with Stbm/Vang is required for signalling to Fz in the adjacent cell. However, interactions have also been reported between Dsh and Pk and between Dsh and Stbm/Vang (Tree et al., 2002b; Bastock et al., 2003; Jenny et al., 2005), and during an early phase of cell–cell communication these interactions might also be important. Clones of cell mutant for dsh alone do not strongly affect the polarity of neighbouring cells (Fig. 3A, Theisen et al., 1994), in this respect resembling fmi/stan clones. Thus, like fmi/stan, it is possible that dsh might be required on both sides of cell–cell boundaries for communication to occur, consistent with its known physical associations with both Fz and Stbm/Vang. However, we found that both dsh; fz and dsh; stbm/Vang double mutant clones showed non-autonomous effects on the polarity of neighbouring cells, typical of either single mutant fz or stbm/Vang clones respectively (arrows Figs. 3B, C), with normal accumulation of Fmi/Stan at clone boundaries (arrowheads Fig. 3B′). These results demonstrate that dsh activity is not required for Fz-Stbm/Vang-dependent intercellular communication to occur, in either Fz-expressing or Stbm/Vang-expressing cells. We carried out similar experiments using a null allele of pkpk-sple. Single mutant clones of pk also do not significantly affect the polarity of neighbouring cells (Fig. 3D, Gubb et al., 1999). However, pk-sple; fz and pk-sple stbm/Vang double mutant clones still show the typical non-autonomous effects of fz or stbm/Vang clones respectively (arrows Figs. 3E, F) and accumulation of Fmi/Stan at clone boundaries (arrowheads Fig. 3E′). We conclude that pk activity is also not essentially required for Fz-Stbm/Vang-dependent intercellular communication. The range of non-autonomous alterations in cell polarity around fz and stbm/Vang clones is generally up to 10 cell diameters; however, it varies with clone size, shape and position (Vinson and Adler, 1987; Taylor et al., 1998; Adler et al., 2000). We observed similar ranges of non-autonomy for the double mutant clones generated with null dsh and pk alleles, suggesting that the strength of intercellular signalling remained in the normal range. Analysis of dgo function It has been recently reported that during planar polarity patterning of the Drosophila eye disc, the core polarity gene dgo acts redundantly with stbm/Vang and pk (Das et al., 2004). If such a situation also pertained in the wing, we reasoned that this might mask specific roles of either pk or dgo in intercellular signalling. However, we find that stbm/Vang dgo clones still exhibit the proximal non-autonomy typical of stbm/Vang single clones (arrow Fig. 4A) and pk dgo clones behave like pk single clones, showing no non-autonomous effect on trichome polarity (Fig. 4B). In an attempt to test the hypothesis of redundant functions of core polarity proteins as rigorously as possible, we generated clones of cells triply mutant for pk, dgo and fz. These also behaved like single mutant fz clones, showing typical non-autonomous effects on trichome polarity (arrow Fig. 4C). In the eye, dgo has been particularly implicated in cooperating with pk and stbm/Vang to maintain the junctional localisation of Fmi/Stan (Das et al., 2004). Interestingly, in the pupal wing Fmi/Stan remains junctional in stbm/Vang dgo clones (Fig. 4D), although proximodistal asymmetric localisation is lost as previously reported for stbm/Vang clones (Bastock et al., 2003). The same is true of Fmi/Stan localisation in pk dgo clones (Fig. 4E). We also find that contrary to the reported situation in the eye, Stbm/Vang apicolateral localisation is maintained in pk dgo clones (Fig. 4F). Polarity defects propagate through tissue mutant for pk, pk dgo and dsh to different degrees Mosaic experiments so far described have analysed the ability of clones of cells lacking the function of one or more core polarity genes to influence the polarity of neighbouring cells via intercellular signalling. However, for polarity defects to propagate away from such clones, intracellular signalling is required across the axes of individual cells, in addition to intercellular signalling between cells. The nature of intracellular signalling is poorly understood. One recent model proposes that it depends upon detection of levels of intracellular fz activity by transmembrane receptors on different sides of the cell (Lawrence et al., 2004), whereas others suggest that it relies on asymmetric assembly of protein complexes on one cell edge in response to the presence of an asymmetric complex at the opposite cell edge (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). Although dsh, pk and dgo do not have essential functions in intercellular signalling, they might nevertheless be required for intracellular communication. However, in this context, it is interesting to note that polarity defects can still propagate around fz clones in abdomens wholly mutant for a null allele of pk (Lawrence et al., 2004) and in wings mutant for the pk1 mutation which mutates one of the pk isoforms (Adler et al., 2000). Taken at face value, these data suggest that pk may not be essentially required for either intercellular or intracellular signalling. To investigate this further, we generated clones of cells with altered fz or stbm/Vang activity in wings wholly mutant for the function of other core polarity genes. As it simplified the generation of the appropriate fly strains, in these experiments we generated clones of cells with increased Fz activity (which cause neighbouring trichomes to point away from the clone, see e.g. Strutt, 2001) or Stbm/Vang activity (which cause trichomes to point towards the clone, Amonlirdviman et al., 2005). For control experiments, we analysed wings entirely mutant for fz or stbm/Vang activity. These factors are required both for intercellular communication and trichome placement at the cell edge, so overexpression clones in these backgrounds are not expected to alter the polarity of neighbouring cells (e.g. Taylor et al., 1998). Consistent with this, clones of cells that overexpress Stbm/Vang cannot alter trichome polarity in fz wings (Fig. 5A), and clones of cells overexpressing Fz cannot alter trichome polarity in stbm/Vang wings (Fig. 5B). However, if Fz is overexpressed in clones of cells in wings wholly mutant for a null allele of pk, we observed a strong effect on trichome polarity in neighbouring cells (Fig. 5C), which extends 8–9 cells (average 8.6, n = 11) from the clone boundary, similar to the effect seen in wild-type wings and in agreement with previous observations (Adler et al., 2000; Lawrence et al., 2004). Interestingly, in wings double mutant for pk and dgo, clones of cells overexpressing Fz also affect the polarity of neighbouring cells (Fig. 5D), but the effect only extends for 5–6 cells (average 5.6, n = 8). Hence, although patterning is essentially normal in dgo wings, and polarity defects propagate a normal distance though pk tissue, when both factors are absent the propagation of polarity defects is substantially reduced, revealing an unexpected redundancy between these factors for this process. We also investigated the effect of overexpressing Fz in clones of cells in wings mutant for dsh. To circumvent the lethality of dsh null mutations, we used the dsh1 point mutation which affects only planar polarity functions. We again observed an effect of the clones on the polarity of neighbouring cells (Fig. 5E); however, in this case, polarity defects only propagated at most 3–4 cells from the clone (average 3.7, n = 7). Finally, we tested propagation through cells mutant for a null allele of dsh. Non-autonomous polarity defects were induced by overexpressing Fz-GFP in the patched expression domain at the anteroposterior compartment boundary (Adler et al., 1997), in wings containing dsh3 clones (Fig. 5F). Within the clones, we observed trichomes emerging largely in the cell centre as previously reported (Wong and Adler, 1993), and were unable to detect propagation of polarity defects. Asymmetry of polarity protein activity exists in the absence of asymmetry in distribution Recent models have suggested that asymmetric localisation might be essential for polarity propagation (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). This is at variance with observations of propagation of polarity defects in pk or dsh1 backgrounds (Figs. 5C, E and Adler et al., 2000; Lawrence et al., 2004), in which asymmetry is not observed (Figs. 6A, B and e.g. Axelrod, 2001; Strutt, 2001; Usui et al., 1999). However, it is possible that weak asymmetry exists in these backgrounds, which is difficult to detect. We investigated this more closely by generating clones of cells expressing Fz-EYFP. In general, a subtle increase or decrease in Fz-EYFP at the edge of one cell might be masked by Fz-EYFP localisation on the adjacent boundary of a neighbouring cell (as it is not possible to distinguish between localisation at adjacent cell boundaries by visible light microscopy). However, by looking at Fz-EYFP distribution on the edges of clones, it should be easier to discern subtle asymmetry of proximodistal localisation (see Strutt, 2001). Hence we examined the distribution of Fz-EYFP expressed in clones in null pk and dsh1 backgrounds, but still were unable to observe any evidence of asymmetric localisation (Figs. 6A′,B′). Notwithstanding our failure to find asymmetry of distribution of polarity proteins in pk and dsh1 wings, we nevertheless suppose that there must be asymmetry of activity in order for propagation of polarity and asymmetric trichome placement to occur. Interestingly, in clones of cells lacking dgo or pk activity, trichome formation occurs both at the same time as in adjacent non-mutant tissue and at the distal cell edge (Figs. 6C, D). Hence in these backgrounds there is clearly an asymmetric signal for trichome formation. In contrast, in clones of cells doubly mutant for dgo and pk, trichome formation in often seen to be delayed and when it does occur is often seen in the cell centre (Fig. 6E), indicative of loss of the asymmetric trichome placement cue. The phenotype in pk dgo clones is similar to that seen in tissue mutant for dsh, fz or stbm/Vang (Fig. 6F, Wong and Adler, 1993; Taylor et al., 1998). Hence, consistent with the differing effects of dsh, pk and dgo on propagation of polarity defects, we see a similar progression of effects on the asymmetric placement of trichomes at the cell edge, suggesting that these two processes are linked. Discussion As described in the Introduction, it is possible to define a core group of polarity proteins, but this does not imply that all components of the core make equal contributions to planar polarity patterning. In this work, we have attempted to systematically analyse the contributions of the core proteins to the processes of coupling to the global cue, local coordination of polarity and asymmetric trichome placement. Our key findings are as follows:(i)The transmembrane proteins Fz, Stbm/Vang and Fmi/Stan have a common early function during planar polarity patterning in the wing and eye, with the cytoplasmic factors Dsh and Pk playing only later roles.(ii)The transmembrane core of Fz, Fmi/Stan and Stbm/Vang is absolutely required for intercellular communication. We demonstrate that the asymmetric relationship of these proteins seen at the time of trichome placement, with Fmi/Stan in both communicating cells and Fz in one cell juxtaposed to Stbm/Vang in the adjacent cell, is also necessary and sufficient for such intercellular communication. In addition we provide evidence that information passes both from Fz to Stbm/Vang expressing cells and vice versa.(iii)Intercellular communication does not require Dsh or Pk in either Fz or Stbm/Vang expressing cells and Dgo also does not play redundant roles with Pk in intercellular signalling.(iv)Pk and Dgo act redundantly in propagation of polarity from cell to cell, most likely by promoting intracellular communication. Dsh plays a prominent role in such propagation, greater than that of both Pk and Dgo. We speculate that the intracellular communication required for such polarity propagation is dependent on establishing intracellular asymmetries of protein activity.(v)Although even subtle asymmetry of Fz localisation is not apparent in pk tissue, not only does polarity propagate between cells, but trichome placement also occurs at the normal time and place. Hence, asymmetry of polarity protein activity exists in the absence of detectable asymmetry of localisation.(vi)Neither Pk nor Dgo are directly required for determining the site of trichome placement. Coupling to the global polarity cue As noted in the Introduction, one of the putative functions of the core polarity proteins is to couple to long-range polarity patterning cues. It has been suggested that these cues are provided by fj/ds/ft (Ma et al., 2003; Yang et al., 2002), but little is understood regarding the molecular mechanism of any such coupling. We suppose that fz, stbm/Vang and fmi/stan are involved as they show the earliest requirement. Comparison with the temporal requirements of ds (Matakatsu and Blair, 2004), argues against a role for pk, and probably dsh (with the caveat that we were unable to analyse a null allele). Interestingly, it has been argued that during abdomen patterning, pk may play a particular role in “rectifying” the global signal in different compartments (Lawrence et al., 2004). In the wing, such a function is not necessary, possibly explaining why we do not find a corresponding early role for pk. Local coordination of polarity A better understood and major function of Fz and the core polarity proteins is the local coordination of cell polarity. All recent models for this coordination have proposed a role for cell–cell contact mediated signalling, as opposed to schemes requiring the secretion of a diffusible ligand. A key feature of such models is that they require both intercellular communication to pass polarity cues between adjacent cells and intracellular communication to pass information across the axes of individual cells. Experimental support for cell–cell contact mediated signalling has been provided by experiments in the abdomen, showing that the atypical cadherin Fmi/Stan is required in both signal sending and receiving cells, suggesting that signals pass between Fmi/Stan homodimers (Lawrence et al., 2004). Theoretical evidence has also been provided by a number of mathematical models that have confirmed the feasibility of locally coordinating polarity via assembly of asymmetric junctional complexes containing Fz in one cell and Stbm/Vang in the adjacent cell (Amonlirdviman et al., 2005; Le Garrec et al., 2006). Our results rigorously demonstrate that, in the wing, intercellular signalling events that locally coordinate polarity require Fmi/Stan in both communicating cells and Fz in one cell and Stbm/Vang in the other. This supports models in which the asymmetric junctional distributions that are observed by immunofluorescence are required for intercellular signalling (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). However, it should be noted that although signalling may require the assembly of complexes with Fz in one cell adjacent to Stbm/Vang in the next, this does not necessarily imply that detectable asymmetric subcellular distribution of proteins within cells is necessary. Indeed the persistence of signalling in pk and dsh1 backgrounds where subcellullar asymmetry of the core polarity proteins is not observed argues against this being essential. In addition, our data raise the possibility of bidirectional cell–cell communication via Fz-Stbm/Vang, and are inconsistent with a monodirectional signal as proposed to occur in the abdomen (Lawrence et al., 2004). It is also evident from our results that intercellular signalling does not require association of Dsh, Pk or Dgo with Fz or Stbm/Vang. Indeed, fz or stbm/Vang clones that also lack any of these factors are not obviously impaired in their ability to alter the polarity of neighbouring cells. What then are the roles of Dsh, Pk and Dgo? We show that propagation of polarity defects away from a clone is reduced in dsh and pk dgo tissue, indicating that they are required for local relay of polarity cues. Hence, a likely role would be in the intracellular signalling required to pass polarity cues across the axes of cells. Previous experiments in which polarity was seen to propagate normally in a pk background (Adler et al., 2000; Lawrence et al., 2004) argued against such a function for pk, which is only revealed when dgo function is also absent. One proposed mechanism for intracellular signalling is that each cell acquires a particular level of Fz activity, which is communicated by intercellular signalling to all surrounding cells (Lawrence et al., 2004). In this case, roles for Dsh, Pk and Dgo in modulating intracellular levels of Fz activity could be envisaged. However, the majority of models suggest roles for Dsh and Pk in intracellular feedback loops that amplify differences in the asymmetric localisation of the core polarity protein complexes within cells (Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). For instance, it has been proposed that association of Dsh with Fz might be antagonised by high local concentration of Stbm/Vang-Pk (Amonlirdviman et al., 2005), or alternatively that Fz-Dsh antagonise Stbm/Vang-Pk interactions (Le Garrec et al., 2006). In either case, if asymmetric complexes containing Fz, Fmi/Stan and Stbm/Vang were somehow stabilised by addition of Dsh, Pk and/or Dgo to the complex, then such antagonistic interactions would provide feedback that would amplify asymmetries of protein localisation across the axes of individual cells. Notably, our experimental results suggest that if such feedback is occurring, then the relative importance of the cytoplasmic factors in stabilising complex formation follows the hierarchy Dsh > Pk > Dgo. Such a scheme would also explain the redundant functions of pk and dgo, even though these factors act at opposite cell edges, as Pk on one side of a cell–cell boundary could bind to and stabilise a complex that was also being stabilised by Dgo binding on the opposite side of the cell–cell boundary. Simultaneous loss of both Pk and Dgo would have a greater destabilising effect than loss of either factor alone. However, models that depend on amplification of differences in asymmetric subcellular protein distribution have to be reconciled with the failure to observe protein asymmetries in pk or dsh1 tissue, through which polarity can still propagate. Possibly in these backgrounds there are subtle asymmetries which cannot be observed—notably at least one recent model predicts such subtle asymmetry in pk clones (Amonlirdviman et al., 2005). But another explanation is that receptor proteins such as Fz may be uniformly distributed, but nevertheless exhibit differential signalling activity across the axes of cells. Despite the apparently non-essential role of protein asymmetry either in polarity propagation over short distances or in trichome placement, it nevertheless seems likely that it is an active mechanism in ensuring robust coordination of polarity and correct trichome placement over the whole wing (Ma et al., 2003; Amonlirdviman et al., 2005), as otherwise the failure of long-range coordination of polarity in pk wings cannot be explained. Interestingly, it has recently been reported that asymmetry is present from as early as the third instar stage of development, but is subsequently lost during junctional remodelling in pupal stages (Classen et al., 2005), suggesting that such asymmetry could be playing a role from much earlier in development than previously suspected. Specification of the site of polarised trichome production The precise mechanism by which asymmetric trichomes are generated remains unknown, although there appears to be a role for asymmetric subcellular activities of polarity effector proteins such as Inturned (Adler et al., 2004). As asymmetric trichomes can be generated in the absence of Pk or Dgo, it seems unlikely that either of these proteins interacts directly with the trichome placement machinery; however, all of the other core polarity proteins are candidates for such a role. Concluding remarks In conclusion, we note that there has recently been great interest in attempting to mathematically model the processes underlying propagation of planar polarity between cells (e.g. Lawrence et al., 2004; Amonlirdviman et al., 2005; Klein and Mlodzik, 2005; Le Garrec et al., 2006). Although the presented models have been very successful at reproducing known phenomena, they have nevertheless been based on limited experimental data. This work both provides support for some of the assumptions of such models, for instance by directly testing the central role of core transmembrane proteins such as Fz, Fmi/Stan and Stbm/Vang in intercellullar signalling, but also provides challenges, for instance by demonstrating the propagation of polarity in the absence of visible protein asymmetry.

Eur_Spine_J-2-2-1602194

Textiloma: a case of foreign body mimicking a spinal mass

Items such as cotton or gauze pads can be mistakenly left behind during operations. Such foreign materials (called textilomas or gossypibomas) cause foreign body reaction in the surrounding tissue. The complications caused by these foreign bodies are well known, but cases are rarely published because of medico-legal implications. Some textilomas cause infection or abscess formation in the early stage, whereas others remain clinically silent for many years. Here, we describe a case of textiloma in which the patient presented with low-back pain 4 years after lumbar discectomy. Imaging revealed an abcess-like mass in the lumbar epidural space. Introduction Textiloma and gossypiboma are non-medical terms used to describe a mass of cotton matrix that is left behind in a body cavity during an operation [9, 18, 20]. Such foreign bodies can often mimic tumors or abscesses clinically or radiologically. Although these masses and their associated complications may occur, they are rarely reported due to medico-legal implications [7]. Most cases of textiloma in the literature have been connected with abdominal or thoracic surgery, very few have been linked with spinal surgery. From 1965 to 2006, only 32 cases of spinal or paraspinal textiloma have been documented in the literature [1, 3–7, 9, 10, 12, 14, 16, 19–21]. Here, we describe a case in which cotton, a foreign body, was left behind during an operation for lumbar disc herniation. The patient presented 4 years later with a symptomatic mass in the lumbar epidural space, and imaging indicated a possible abscess. Case A 62 year old woman presented a complaint of low-back pain. She had had this problem for 1 year, and the pain had been radiating to her left calf for 1 month. The patient had undergone surgery for lumbar disc herniation 4 years prior to presentation. Physical examination indicated good health status. There was no tenderness, swelling or erythema at the incision site. The straight leg-raising test was positive at 60° on the left. The patient’s left hamstring muscle strength was 4/5, and she had diminished patellar deep tendon reflex on that side. Routine laboratory testing (complete blood count, erythrocyte sedimentation rate, blood biochemistry panel) revealed nothing abnormal. Magnetic resonance imaging (MRI) of the lumbosacral spine showed the laminectomy defect at L3 and a mass lesion in the posterior paravertebral region at this level. The mass appeared hypointense to spinal cord tissue on T1-weighted images and hyperintense on T2-weighted images. Injection of contrast medium revealed an enhanced hyperintense rim around a hypointense center (Figs. 1, 2). The lesion was interpreted as a possible epidural abscess. At surgery, a foreign body composed of cotton was found and was completely removed (Fig. 3). No infection or abscess was detected. Histological examination of the tissues around the material revealed only chronic inflammatory infiltration and granuloma formation. One month after the operation, there were no abnormal findings either on physical examination or on MRI of the lumbosacral spine. Fig. 1MRI of the lumbosacral spine showing the mass lesion in the posterior paravertebral regionFig. 2The sagittal section of MRI showing the left laminectomy at L3 vertebra and the mass lesionFig. 3Cotton materials extracted from the patient during the operation Discussion Cotton pads, towels and sponges are used to achieve hemostasis during surgical procedures, including dissection for intervertebral disc herniation and other spinal problems. Although precautions are taken to avoid leaving such materials behind, mistakes do happen and the resultant foreign bodies can cause various clinical and radiological manifestations [1, 5, 8, 12]. In the early period after surgery, these forgotten materials can lead to infections and abscess formation. However, some remain clinically asymptomatic for many years, and then cause a foreign body reaction in the surrounding tissue, with new clinical signs indicating significant mass effect [3, 5, 6, 15, 22]. Cotton is not the only material that can lead to such problems. The literature contains reports of other hemostatic materials (such as gelfoam and Surgicel) causing foreign body reactions that could not be distinguished from recurrent tumors on MRI [9, 13]. The introduction of MRI has made it possible to diagnose most foreign bodies accurately. Findings differ according to the radiological modality that is used to investigate the patient. Cotton sponges and cotton fibers exhibit characteristic features on plain radiographs, whereas the findings on computerized tomography and ultrasonography are less diagnostic [3, 6, 11, 12, 14, 16, 20]. The MRI appearance of foreign materials that are left behind during surgery can differ greatly depending on the time since the operation and the type of foreign body reaction that occurs. There are two types of foreign body reactions: aseptic fibrous tissue reaction, which involves adhesion formation, encapsulation and granuloma formation, or the exudative-type tissue reaction, which leads to abscess formation [3, 6, 8, 14–16, 18, 22]. Karcnik et al. [7] reported a case of foreign body reaction that manifested in a later period after an anterior cervical fusion operation. MRI showed a granuloma that was hypointense on T1-weighted images and hyperintense on T2-weighted images, and that mimicked a solid tumor. In general, most lesions caused by foreign bodies are hypointense on T1-weighted images and hyperintense on T2-weighted images [3, 5, 8, 15, 16]. In our case, the MRI findings were consistent with an epidural abscess, but direct observations at surgery, microbiologic testing and pathologic examination revealed no infection. Foreign bodies that are left behind during operations may organize and increase in size but such changes are not correlated with time. To date, the case reported by Taylor et al. [17] features the longest period from surgery to manifestation of symptoms. They detected an intrapulmonary foreign body 43 years after thoracotomy. The longest reported interval in the neurosurgery literature is 40 years. In that case, a cotton pad was left posterior to the lumbosacral vertebrae during a laminectomy operation, and the material eventually caused a cavitary lesion [16]. Our patient developed a textiloma 4 years after lumbar disc surgery. Civil lawsuits brought against surgeons for surgical complications are becoming more frequent, and this is prompting surgical teams to be even more careful. It is possible to to overlook cotton and gauze pads in the surgical field. Such materials should always have a tag that allows them to be easily located and removed, and all materials that are placed in the wound temporarily, must be counted many times with meticulous care. Cotton pads are not safe because they can break into fragments during manipulation; other materials are preferred for securing hemostasis. Once hemostasis is achieved, the operative site should be flushed with saline and carefully examined for foreign materials.

Osteoporos_Int-4-1-2267485

FRAX™ and the assessment of fracture probability in men and women from the UK

Summary A fracture risk assessment tool (FRAX™) is developed based on the use of clinical risk factors with or without bone mineral density tests applied to the UK. Introduction Several multi-factorial diseases (e.g., diabetes, hypertension) are defined on the basis of important risk factors for the clinical outcome. Hypertension, for example, is defined from the measurement of blood pressure that provides information on the likelihood of stroke. Hypercholesterolaemia and osteoporosis are also examples. In the case of osteoporosis, bone mineral density (BMD) is measured both to provide a diagnosis, and to yield information on fracture risk [1]. Many well-controlled prospective studies with dual energy X-ray absorptiometry (DXA), particularly in elderly women, indicate that the risk of fracture about doubles for each SD reduction in BMD [2, 3]. The measurement of a risk factor for diagnostic use, however, can only capture one aspect of the likelihood of the outcome when the disease is multifactorial, and in osteoporosis, assessment with BMD captures a minority of the fracture risk. For example, the annual incidence of hip fracture increases approximately 30-fold between the ages of 50 and 90 years; but, from the known relationship between BMD and fracture risk and the loss of bone with age, it is expected that hip fracture risk would rise only fourfold [4–6]. Thus, the increase in risk with age is approximately sevenfold greater than can be explained on the basis of BMD alone. The imperfect capture of risk with BMD alone poses several problems for the clinical assessment of fracture risk. In the context of population screening with BMD alone, the performance characteristics of the test are less than optimal in terms of the trade-off between sensitivity and specificity [1, 7, 8]. Thus, osteoporotic fractures affect a substantial minority of the population, but intervention thresholds based on BMD alone lack sensitivity over most reasonable assumption, i.e., the detection rate is low. For example, at the age of 50 years the proportion of women with osteoporosis is approximately 5% [4]. The proportion of these who will fracture in the next 10 years (i.e., positive predictive value) is about 20%. The detection rate for these fractures (sensitivity) is, however, low, and 96% of fragility fractures would arise in women without osteoporosis given a test like BMD where the fracture risk doubled for each SD decrease [9]. Low sensitivity is one of the reasons why wide-spread population screening is not widely recommended in women at the menopause [1]. Moreover, a normal BMD measurement is no guarantee that a fracture will not occur. The use of risk factors that add information on fracture risk independently of BMD improves the sensitivity of the assessment for any specificity [8, 9]. Over the past several years, we have undertaken a series of meta-analyses to identify clinical risk factors for fracture that provide independent information on fracture risk [3, 10–16]. The analyses were based on the primary data from prospective population based studies. This permits the inter-dependence of each of the candidate risk factors to be examined so that they can be accurately combined for clinical use. In the case of the clinical risk factors, BMI is used as a continuous variable, so that its distribution is preserved by the addition of dichotomous variables [8]. For hip fracture prediction, the gradient of risk (increase in fracture risk per standard deviation increase in risk score) for the CRFs is comparable to the use of DXA alone, and the gradient of risk is further enhanced by the addition of BMD to the CRFs [17]. These inter-relationships, assessed from multiple populations and validated in independent cohorts, permit the more accurate identification of individuals who will fracture, so that the average risk in any given proportion of the population identified for treatment will be higher [8]. These considerations indicate that assessment can be improved by the integration of clinical risk factors with or without BMD. In other words, treatment should be directed not only on the basis of T-score for BMD, but also on the independent contribution of other validated risk factors. However, the use of different metrics (the T-score, gradients of risk, risk ratios, etc) is confusing for clinicians and patients alike. For this reason, there has been interest in the development of algorithms that express absolute risk, or the probability of fracture within a given time period [18–20], as has been done for cardiovascular and other diseases [21–25]. The aim of the present study was to develop a model for the clinical assessment of fracture probability in men and women based on the epidemiology of the UK. Methods Cohorts In order to identify the relevant risk factors, we used baseline and follow-up data from nine prospective population-based cohorts comprising the Rotterdam Study, The European Vertebral Osteoporosis Study (later the European Prospective Osteoporosis Study (EVOS/EPOS), The Canadian Multicentre Osteoporosis Study (CaMos), Rochester, Sheffield, Dubbo, a cohort from Hiroshima and two cohorts from Gothenburg. Details of each of the cohorts have been recently published elsewhere [10–16]. Baseline and outcome variables Height and weight were measured using standard techniques in all cohorts. Body mass index (BMI) was calculated as weight divided by height squared (kg/m2) and used as a continuous variable. BMD was assessed at the femoral neck by DXA with the exception of the two Gothenburg cohorts in which BMD was measured elsewhere. Femoral neck BMD was used as a continuous variable (cohort-specific Z-scores excluding the two cohorts from Gothenburg). The clinical risk factors utilised were those identified from the previous meta-analyses [3, 10–16]. These comprised a parental history of hip fracture, exposure to systemic glucocorticoids, a prior history of fragility fracture, current smoking, high intake of alcohol (3 or more units daily on average) and the presence of rheumatoid arthritis as an indicator for secondary osteoporosis. Fracture ascertainment in the primary cohorts was undertaken by self-report (Sheffield, EVOS/EPOS, Hiroshima) and/or verified from hospital or central data-bases (Gothenburg, CaMos, DOES, Sheffield, EVOS/EPOS, Rochester, Rotterdam). Models used Four models were constructed from the risk factor analysis to compute fracture probabilities. These comprised the probability of hip fracture, with and without BMD, and the probability of other major osteoporotic fractures (clinical spine, forearm and proximal humerus), with and without BMD. For each model, fracture and death as continuous hazard functions were computed using a Poisson regression [26, 27] and detailed in the Appendix. In brief, for each risk factor, all significant interactions terms that were identified by meta-analysis were entered (with age, time, sex and the risk factor) with and without BMD [17]. Interactions that were significant for hip fracture risk were also entered into the model for other osteoporotic fractures, and also included in the model for death. Where interactions noted in the “mega-analyses” were no longer significant for both hip fracture and other osteoporotic fractures, these were omitted in a step-wise manner by dropping the interaction with the largest p value. For the death hazard, all significant interactions for fracture risk were included and thereafter omitted if appropriate in a step-wise manner, as described for the fracture hazard. Epidemiology The incidence of hip, forearm and proximal humerus fractures in the UK was taken from Singer et al. [28]. Vertebral fractures can be classified as clinically overt fracture, that is, a symptomatic fracture that comes to clinical attention, or a morphometric fracture, which includes both symptomatic and asymptomatic fractures. For the purposes of this study the clinical definition of a vertebral fracture was used. Because data on vertebral fracture risk in the UK are scarce, with a great deal of variation in reported rates [29], the clinical vertebral fracture incidence was calculated by assuming that the ratio of clinical vertebral fracture to hip fracture would be similar in the UK compared to Sweden [30]. The validity of the assumption cannot be directly tested, but in the case of long bone fractures, there is a close concordance of relative incidence at different fracture sites despite marked differences in absolute risk [31]. Swedish fracture risk data were taken from Kanis et al. [32]. The models were then calibrated so that the mean hazard functions of fracture (and death) equalled that of the UK, as detailed in the Appendix. Input and output variables Individual patient details comprise age (50 to 90 years), sex, weight (in kg) and height (in cm). BMI is automatically computed from height and weight. Dichotomised risk variables are then entered: A prior fragility fracture (yes/no)Parental history of hip fracture (yes/no)Current tobacco smoking (yes/no)Ever long-term use of oral glucocorticoids (yes/no)Rheumatoid arthritis (yes/no)Other causes of secondary osteoporosis (yes/no)Daily alcohol consumption of three or more units daily (yes/no) A distinction is made between rheumatoid arthritis and other secondary causes of osteoporosis. Rheumatoid arthritis carries a fracture risk over and above that provided by BMD [11]. Whereas this may hold true for other secondary causes of osteoporosis, the evidence base is weak. Of the many secondary causes of osteoporosis, the following have been consistently documented to be associated with a significant increase in fracture risk: Untreated hypogonadism in men and women, e.g., bilateral oophorectomy or orchidectomy, anorexia nervosa, chemotherapy for breast cancer, hypopituitarism [33–40]Inflammatory bowel disease, e.g., Crohn’s disease and ulcerative colitis [41–43]. It should be noted that the risk is in part dependent on the use of glucocorticoids, but an independent risk remains after adjustment for glucocorticoid exposure [44].Prolonged immobility, e.g., spinal cord injury, Parkinson’s disease, stroke, muscular dystrophy, ankylosing spondylitis [45–50]Organ transplantation [51–54]Type I diabetes [55–58]Thyroid disorders, e.g., untreated hyperthyroidism, over-treated hypothyroidism [59–63] Whereas there is strong evidence for the association of these disorders and fracture risk, the independence of these risk factors from BMD is uncertain. It was conservatively assumed, therefore, that the fracture risk was mediated via low BMD, but with a risk ratio similar to that noted in rheumatoid arthritis. From an operational view, where the field for rheumatoid arthritis is entered as ‘yes’, a risk is computed with and without BMD. If the field for other secondary osteoporosis is also filled as ‘yes’ this does not contribute to the calculation of fracture probability. Conversely, where the field for rheumatoid arthritis entered as ‘no’, and the field for secondary osteoporosis is ‘yes’, the same β coefficients as used for rheumatoid arthritis contribute to the computation of probability where BMD is not entered. In the presence of BMD, however, no additional risk is assumed in the presence of secondary osteoporosis, since its independence of BMD is uncertain. If any of the fields for dichotomous variables is not completed, a negative response is assumed. Fractures probability can then be calculated. The output (without BMD) comprises the 10-year probability of hip, clinical spine, shoulder or wrist fracture and the 10-year probability of hip fracture (Fig. 1). Fig. 1Input and output for the FRAX™ model Femoral neck BMD can additionally be entered either as a Z-score or a T-score. The transformation of Z- to T-score and vice versa is derived for the NHANES III database for female Caucasians aged 20–29 years [64]. When entered, calculations give the 10-year probabilities as defined above with or without the inclusion of BMD. Results Clinical risk factors The contribution of single clinical risk factors is shown in Table 1 for men and women aged 65 years. In the example, the BMI is set at 25 kg/m2. In the absence of BMD, hip fracture probabilities were higher in women than in men. Each clinical risk factor had a different significance for hip fracture probability, with a family history having the least and a prior fracture the greatest weight in the absence of BMD. The rank order of weighting differed for the major osteoporotic fractures. For example, a parental history of hip fracture was a strong risk factor, close to that provided by a prior fragility fracture. The contribution of age to the assessment of probability is shown in Table 2. In both men and women, there was greater than 100-fold difference in hip fracture probability between the age of 50 years (no risk factors) and 80 years (parental history of hip fracture). Table 1Ten-year probability (%) of a major osteoporotic fracture or hip fracture in men and women aged 65 years according to the presence of a single clinical risk factor Without BMDT-score −2.5 SDMenWomenMenWomenOsteoporoticaHipOsteoporoticaHipOsteoporoticaHipOsteoporoticaHipNo clinical risk factors4.90.88.61.39.83.612.43.0Parental history of hip fracture9.31.016.01.716.53.722.13.2Current cigarette smoking5.11.19.21.911.05.613.75.1Alcohol intake >2 units daily6.01.210.42.012.55.415.44.6Rheumatoid arthritis6.81.411.72.312.85.016.14.3Oral glucocorticoids7.51.513.72.715.06.119.75.5Previous fragility fracture9.61.916.43.216.05.920.25.0BMI is set at 25 kg/m2. The right-hand panels show probabilities at a T-score of −2.5 SD at the femoral neckaHip, clinical spine, humeral or forearm fractureTable 2Ten-year probability of fracture (%) at the sites shown for men and women with a BMI of 25 kg/m2 according to age and the presence or absence of a single risk factor in the absence of BMD Osteoporotic fractureaHip fracture5060708050607080(a) MenNo clinical risk factors2.83.95.77.20.10.41.33.4Parental history of hip fracture5.57.69.1150.20.62.811Current cigarette smoking2.84.15.97.50.20.71.84.2Alcohol intake >2 units daily3.34.77.19.50.20.72.05.1Rheumatoid arthritis3.75.38.0110.20.82.35.8Oral glucocorticoids4.46.18.5100.30.92.45.5Previous fragility fracture5.87.911120.51.32.75.2(b) WomenNo clinical risk factors3.56.011170.20.72.37.0Parental history of hip fracture6.91217310.30.95.022Current cigarette smoking3.66.512190.31.13.49.5Alcohol intake >2 units daily4.17.314220.31.13.610Rheumatoid arthritis4.78.215250.41.34.112Oral glucocorticoids5.69.818260.51.54.813Previous fragility fracture7.41220280.82.14.911aClinical spine, hip, humeral or forearm fracture The presence of more than one risk factor increased fracture probability in an incremental manner. For example, in women aged 65 years with a BMI of 25 kg/m2, the 10-year hip fracture probability was 1.3%. With one clinical risk factor, the probability ranged from 1.7% to 3.2%, depending on the risk factor. With two risk factors, the range was 2.5–6.6%, and with 3, 4 and 5 risk factors the ranges were 3.8–11%, 6.7–17% and 13–24%, respectively. In the presence of all six clinical risk factors, the 10-year probability of hip fracture was 30%. The effect of several clinical risk factors on the 10-year probability of a major osteoporotic fracture is shown in Fig. 2 for a woman aged 65 years and a BMI of 25 kg/m2. In women with rheumatoid arthritis, there was a 33% increase in fracture probability compared with those without rheumatoid (from 9% to 12%). When women additionally took oral glucocorticoids and had a prior fracture there was a fourfold increase in fracture probability. Fig. 2Effect of combinations of clinical risk factors on the 10-year probability of a major osteoporotic fracture in women aged 65 years and a BMI of 25 kg/m2. [05Ca201] A higher or lower BMI had a marked effect on fracture probability (Fig. 3). For example, in women at the age of 65 years the 10-year hip fracture probability in the absence of risk factors was 2.3% with a BMI of 20 kg/m2, but was fourfold lower (0.6%) at a BMI of 40 kg/m2. At each level of BMI, fracture probability increased with an increasing number of clinical risk factors. Fig. 3Ten-year probability of hip fracture (%) in women aged 65 years according to the number of clinical risk factors with a BMI of 20 kg/m2 (left-hand panels) and a BMI of 40 kg/m2 (right-hand panels). The intervals reflect the different weights afforded by different risk factors and provide a range of probabilities. [05Ca070] Bone mineral density The relationship between fracture probability and BMD is shown in Table 3. In general, fracture risk increased with increasing age and decreasing T-score. At low T-scores, however, fracture probabilities decreased with age, a phenomenon more marked in men than in women. For example, at the age of 50 years, the 10-year probability of hip fracture was 16.0% in men with a T-score of −4 SD and this fell progressively with age, so that at the age of 70 years, the probability was 12.9% and at the age of 90 years was 8.3%. Table 3Ten-year fracture probability (%) in men and women from the UK with a BMI of 25 kg/m2 and no clinical risk factors according to age and T-score at the femoral neckAgeT-score (SD)(years)+10−1−2−3−4(a) Hip fracture men50<0.10.10.41.34.716.0600.10.20.61.95.615.6700.20.51.12.65.912.9800.61.11.93.46.110.7901.21.82.53.85.68.3(b) Hip fracture women50<0.10.10.20.83.211.7600.10.20.41.44.413.5700.10.40.92.46.316.2800.51.02.14.49.519.7901.22.03.55.89.816.9(c) Osteoporotic fracturea - men502.52.73.55.19.220.9603.03.54.56.811.522.2703.64.35.58.212.820.9803.24.05.27.611.316.9903.13.95.16.99.713.5(d) Osteoporotic fracturea - women503.03.33.85.28.517.7604.24.85.67.912.623.0705.66.88.411.518.230.4805.87.510.113.821.734.3905.17.19.913.820.030.3aHip, clinical spine, humerus or forearm fracture The differences in probabilities between men and women were much less marked for any given age and T-score than the differences seen with the use of clinical risk factors alone. At a fixed BMD (−2.5 SD in Table 1), the difference in probability between men and women was less evident. For example, the hip fracture probability ratio (women/men) with glucocorticoid use was 1.8 in the absence of BMD but 0.9 at a T-score of −2.5 SD. In the case of all major osteoporotic fractures, probabilities remained higher in women than in men at a T-score of −2.5 SD but the difference in probabilities between men and women was less marked. Figure 4 compares the probability of hip fracture in men and women with a prior fracture according to age. In the absence of BMD there was approximately a twofold difference in probability between men and women. When the BMD was set at an average for women (Z-score = 0), there was little difference in probabilities up to the age of 75 years. Thereafter, men had a lower probability than women due to the higher death hazard. Fig. 4Ten-year hip fracture probability (%) in men and women with a prior fracture according to age. The left-hand panel gives probabilities in the absence of BMD. In the right-hand panel probabilities are shown at an average BMD for women at each specific age (i.e., a Z-score = 0). A BMI is set at 24 kg/m2. [05Ca065] The clinical risk factors added to the information provided by BMD. Isopleths for 10-year fracture probability are shown in Fig. 5 for the combination of several of the clinical risk factors. The clinical risk factors were somewhat less predictive in the presence of BMD in the models. For example, in women aged 65 years and a BMI of 20 kg/m2, the 10-year hip fracture probability in the absence of BMD ranged from 2.3% in the absence of clinical risk factors to 27.9% with four risk factors. When BMD was set constant at a T-score of −2.5 SD, the range was from 2.8% with no clinical risk factor to 19.7% with four risk factors (Fig. 6). The effect of variations in BMI was even more markedly affected by BMD. For example, in women aged 65 years the 10-year hip fracture probability was 2.3% at a BMI of 20 kg/m2 and decreased progressively at higher levels of BMI to 0.6% with a BMI of 40 kg/m2. When BMD was fixed, hip fracture probability remained constant irrespective of BMI (Fig. 7). Fig. 510-year probability of a major osteoporotic fracture in men and women aged 65 years according to T-score and clinical risk factors. Body mass index is set at 25 kg/m2. [05Ca136]Fig. 6Ten-year probability of hip fracture (%) in women aged 65 years, according to the number of clinical risk factors. The left-hand panel shows the probabilities without BMD at a BMI fixed at 20 kg/m2. The right-hand panel illustrates the effects with BMD fixed at the threshold for osteoporosis. [05Ca073]Fig. 7Effect of variations in BMI on 10-year hip fracture probability (%) in women aged 65 years. Probabilities with BMD are computed at a T-score of −2.8 SD. [05Ca074] Discussion The present study provides a model for the assessment of fracture probability in men and women. The model (FRAX™) uses data derived from nine cohorts from around the world, including centres from North America, Europe, Asia and Australia and has been validated in 11 independent cohorts with a similar geographic distribution [17]. The use of primary (but anonymized) data for the model construct permits the interaction of each of the risk factors to be determined to improve the accuracy whereby fracture probability can be computed. The large sample permitted the examination of the general relationship of each risk factor by age, sex, duration of follow up and, for continuous variables (BMD and BMI), the relationship of risk with the variable itself in a manner hitherto not possible. The use of primary data also eliminates the risk of publication bias. The validity of the clinical risk factors identified are supported by the expected relationships between BMD and fracture risk [3]. In the present study, the FRAX™ model has been calibrated to the epidemiology of the UK, but could be calibrated to any country where the epidemiology of fracture and death is known (see Appendix). FRAX™ models for the UK and some other countries are available through the web (http://www.shef.ac.uk/FRAX/index.htm). The approach uses easily obtained clinical risk factors to estimate risk. The estimate can be used alone or with BMD to enhance fracture risk prediction. Several previous studies have developed models to predict fracture risk from the combination of clinical risk factors and BMD [18, 65–78]. The risk factors used include activities of daily living, impaired cognition, liability to falls, poor overall health, history of stroke, seizure disorder and several different medications. A limitation of many of these studies is that, with the exception of the SOF study [18], and one of the GPRD studies [76], they have not been tested in other cohorts. The model described in this paper has been validated in 11 independent prospectively studied cohorts with in excess of one million patient years [17]. The use of risk factors for case finding presupposes that the risk so identified is responsive to a therapeutic intervention. To test this hypothesis, it would be necessary to recruit patients selected on the basis of the risk factor(s) to a randomised controlled trial (RCT). The risk factor that is best evaluated in this way is BMD, and indeed the vast majority of therapeutic studies have recruited patients on the basis of low BMD as recommended by regulatory agencies in the US and Europe [79, 80]. In recent years, other trials have recruited patients on the basis of age, gender, a prior vertebral fracture and current exposure to glucocorticoids irrespective of BMD, and have shown therapeutic effects similar to those noted in RCT’s based on BMD selection [14, 81–83]. For other risk factors, comparable data are lacking. In the absence of empirical data, an alternative approach is to demonstrate that the presence (or absence) of a risk factor does not adversely influence therapeutic efficacy against fractures. Several studies have shown no significant interaction between response to treatment and the presence or absence of the risk factors used in the present study including age, height, family history of fracture, low body weight or BMI, smoking, alcohol intake or prior non-vertebral fracture [84–88]. In contrast, some risk factors may be associated with less therapeutic efficacy. For example, patients selected on the basis of risk factors for falling may respond less completely to agents that preserve bone mass than patients selected on the basis of low BMD [89]. This concern is greatest in models that omit BMD, because pharmacological agents may not be equally effective across the entire range of BMD [90]. The present model has several unique features. FRAX™ uses Poisson regression to derive hazard functions of death and fracture. Such hazard functions are continuous as a function of time, unlike Cox’s regression for which the corresponding hazard functions are zero except at the time points of a fracture or death. There are also several advantages of the Poisson model over logistic regression analysis. Logistic regression does not take account of when a fracture occurred, nor whether individuals without a fracture died or when death occurred. Secondly, for the assessment of 10-year probabilities by logistic regression, the observation period should be for 10 years. Moreover, information longer than the 10-year period cannot be used for analysis. The cost of ignoring information when fractures occur and whether and when deaths occur is on the precision of the estimate. In simulation experiments, the Poisson model gives the same precision as logistic regression with fewer numbers of individuals. In our own simulations in the present context (data on file), the Poisson model gave the same precision as logistic regression with half the number of individuals. Finally, the Poisson model allows adjustments to be made for time trends. The ability to use several Poisson models permits the use of data from different sources to integrate fracture and death hazards, and to calibrate to different countries. A further feature of the FRAX™ model is that it takes account of deaths from all causes. In several recent models of disease probability, this has not been accounted for [23–25]. For example, the probability of stroke has been determined as a function of age, race, smoking, body mass index, atrial fibrillation, HbA1c, systolic blood pressure, ratio of total to HDL cholesterol and duration of diabetes [25], but the risk of dying from other reasons was not taken into account. In the context of osteoporosis, fracture probabilities are markedly underestimated when no account is taken of the competing death hazard [27, 35, 36]. For example, in a study of men followed after orchidectomy, the cumulative incidence of fractures was 19% after 15 years, but the figure was 40% when deaths were considered as a competing event [36]. FRAX™ also takes account of the impact of risk factors on the death hazard. For example, smoking and low BMD are risk factors for fracture but also significant risk factors for death. Thus, at very low T-scores for BMD, hip fracture probabilities decrease with age (see Table 3), in part related to the higher mortality associated with the lower values for BMD. There are several limitations that should be mentioned. As with nearly all randomly drawn populations, non-response bias may have occurred. The effect is likely to exclude sicker members of society, and may underestimate the absolute fracture risk for example by age. The analyses also have significant limitations that relate to the outcome variables and the characterisation of risk factors. The definition of what was considered to be an osteoporotic fracture was not the same in all cohorts, but the effect of this inconsistency is likely to weaken rather than strengthen the associations that were found. For the hip fracture outcome, the definition was similar in all cohorts, and may explain in part the higher risk ratios associated for this fracture rather than for osteoporotic fracture. Also, the analyses were confined to clinical fractures, and the results might differ from vertebral fractures diagnosed by morphometry or as an incidental radiographic finding. There are also limitations with the risk factors themselves. In the case of BMI, this was chosen rather than weight as the measure for body composition. This has the advantage that there is less variability across countries and between sexes. A potential drawback is that BMI can be influenced by height loss associated with vertebral deformities. Therefore, in individuals with important loss of height, the risk conferred through BMI could be underestimated [91]. The use of maximal attained height, rather than current height, might be a solution in the future, if it were shown that fracture risk prediction could be improved. Further problems relate to the construct of the questions to elicit the presence or absence of risk factors, which varied between cohorts. These included questions on family history, prior fracture, smoking and glucocorticoid use. The effect of this heterogeneity is likely to weaken rather than strengthen the associations found. Recall is also subject to errors and was not validated in any of these cohorts. This is particularly problematic in the elderly. In addition, the validity of self-reported alcohol intake is notoriously unreliable [92]. Indeed, alcohol consumption was significantly less in both men and women than that assessed in the UK [93]. Given that these studies were prospective, however, much of this error (with the exception of alcohol intake) should be random, giving rise to non-directional misclassification. Thus, the associations may actually be stronger than reported here. Any underestimate may have limited consequences for case-finding, since the populations to be tested are similar to the populations interrogated. Biases that arise have more significance where causality is inferred. A further limitation is that several of the clinical risk factors identified take no account of dose-response, but give risk ratios for an average dose or exposure. By contrast, there is good evidence that the risk associated with excess alcohol consumption and the use of glucocorticoids is dose-responsive [14, 94]. In addition, the risk of fracture increases progressively with the number of prior fractures [95]. These limitations are nearly all conservative. It should be acknowledged that there are many other risk factors that might be considered for incorporation into assessment algorithms. These include BMD at other skeletal sites, ultrasonography, quantitative computed tomography and the biochemical indices of bone turnover. The available information was too sparse to provide a meta-analytic framework, but they should be incorporated into risk assessment algorithms when they are more adequately characterised. Notwithstanding, the present model provides a mechanism to enhance patient assessment by the integration of clinical risk factors alone and/or in combination with BMD. The application of this methodology to clinical practice will demand a consideration of the fracture probability at which to intervene, both for treatment (an intervention threshold) and for BMD testing (assessment thresholds). These are currently being developed for the UK, based on cost-effectiveness analyses [96]. Intervention thresholds developed for the United Kingdom may not be applicable to other countries. The 10-year probability of fracture varies markedly in different countries [97]. For countries with low hip fracture rates, as found in developing countries, the relative risk at which intervention is cost-effective will be higher, though the absolute risk at which intervention is cost-effective would not change assuming comparable costs. Intervention thresholds would, however, change with differences in costs, particularly fracture costs, which vary markedly world wide. There is also the issue of affordability or willingness to pay for a strategy. The gross domestic product (GDP) per capita provides an index of affordability. The GDP varies markedly in different regions of the world. In the UK, the GDP per capita is estimated at US$ 25,300 in 2002, as compared with US$ 7,000 in Turkey. Thus, for the same fracture risk and the same costs, treatment will be less affordable (at least to health services) in Turkey than in the UK. Nevertheless, individuals in Turkey, rather than society as a whole, may be willing to pay “United Kingdom prices” for health care. There is also a marked heterogeneity in the proportion of GDP devoted to health care, and in the proportion of the population at risk from osteoporotic fracture (i.e., elderly people) [98, 99]. For all these reasons, it is important to define intervention and assessment thresholds on a country by country basis that takes into account the setting for service provision and willingness to pay, as well as considerations of absolute costs.

Clin_Auton_Res-3-1-1914255

Leg blood flow measurements using venous occlusion plethysmography during head-up tilt

We tested whether venous occlusion plethysmography (VOP) is an appropriate method to measure calf blood flow (CBF) during head-up tilt (HUT). CBF measured with VOP was compared with superficial femoral artery blood flow as measured by Doppler ultrasound during incremental tilt angles. Measurements of both methods correlated well (r = 0.86). Reproducibility of VOP was fair in supine position and 30° HUT (CV: 11%–15%). This indicates that VOP is an applicable tool to measure leg blood flow during HUT, especially up to 30° HUT. Introduction The main mechanism responsible for maintaining blood pressure during orthostatic stress is arteriolar vasoconstriction [6]. In order to quantify the response in vascular resistance to postural stress, in particular in the leg, it is necessary to measure leg blood flow accurately. Venous occlusion plethysmography (VOP) is a well-established method to measure calf blood flow, which has been used in a variety of conditions, i.e. exercise [13] and reactive hyperaemia [17]. However, its use for measuring leg blood flow in standing or head-up tilt (HUT) position remains controversial, since an empty venous system has been suggested to be requisite for this method [3, 4, 12]. Since in the upright posture veins are already distended, due to an increase in hydrostatic pressure, further collection of blood may be defined by venous compliance rather than arterial inflow. This may question the validity of measuring blood flow using VOP in dependent limbs or in the upright posture. Although VOP has been used during HUT, accuracy or reproducibility of this method has not been reported [22–24]. Therefore, the purpose of this study was to assess the applicability and reproducibility of VOP for blood flow measurements in the calf (CBF) during HUT at different tilt angles (0°, 30°, 45°, and 70°). In a subgroup CBF measurements by VOP were compared with blood flow measurements using Doppler ultrasound. To assess reproducibility of VOP, measurements were performed twice. Materials and methods Subjects In total eighteen healthy, normotensive subjects aged 21–30 years volunteered to participate in this study. In eight subjects blood flow measurements using VOP were compared with blood flow measurements using Doppler ultrasound (DU). In nine other subjects the VOP measurements were repeated within two weeks to assess reproducibility. Baseline subject characteristics are illustrated in Table 1. None of the subjects used cardiovascular medication or suffered from cardiovascular disease. All were non-smokers and had no history of syncope. All volunteers refrained from caffeine and alcohol for at least eighteen hours and from food intake for three hours prior to testing. The local medical ethical committee approved the study. All subjects gave their written informed consent. Table 1Subject characteristicsMean ± SDAge, years25 ± 4Length, cm187 ± 9Body mass, kg80 ± 11Calf circumference, cm38 ± 2Systolic blood pressure, mmHg125 ± 12Diastolic blood pressure, mmHg74 ± 5Heart rate, bpm64 ± 10 Measurements The subjects lay in supine position on a manually driven tilt table and were supported by a saddle. The venous occlusion cuff was placed around the right thigh and connected to a rapid cuff inflator (Hokanson Stopler E-20, Bellevue, WA 98005, USA). The mercury-in-silastic strain gauge was placed around the thickest part of the calf and was connected to the plethysmograph. Red blood cell velocities and systolic and diastolic vessel diameter of the right superficial femoral artery were measured with a pulsed-colour Doppler device, which is described in detail elsewhere [8]. Reproducibility of DU in the superficial femoral artery was 1.5% for diameter, 14% for blood flow [9]. Protocol Supine blood flow measurements using DU and VOP were performed after subjects were 30 minutes quietly in supine position. When the subject was 2 minutes into 30° HUT, DU measurements started until 3.5 minutes where after CBF continued for another 3.5 minutes. The venous occlusion pressure was adjusted to the hydrostatic pressure column, which is derived from the vertical distance heart level–thigh level and was calculated as the sinus of the tilt angle * actual distance heart–thigh, and was 75 mmHg during 30° HUT. The same procedure was repeated for 45°, and 70° HUT using a venous occlusion pressure of 87, and 105 mmHg, respectively. Data analysis CBF in ml·100 ml−1 · minute−1 was calculated as described previously [25] and values from minute 3.5 until 7 were averaged to calculate CBF for each HUT position. Doppler ultrasound measurements were analyzed as described before [8] to obtain superficial femoral artery blood flow. To compare CBF measurements with superficial femoral artery blood flow, CBF measurements in ml·100 ml−1 minutes−1 were multiplied by lower leg volume (ml) as measured by water displacement. Statistics Data are expressed as mean ± standard deviation (SD). The results of each method were correlated and agreement evaluated according to the method described by Bland and Altman [5]. The limits of agreement are defined as the mean of the relative differences between the two methods ± 2 SD. Student’s t-test was used to test for systemic differences between the two methods. Reproducibility of the CBF was assessed by calculating the coefficient of variance (CV) from two measurements [25]. To determine whether hemodynamic responses were dependent on the angle of tilt one-way repeated measures ANOVA’s were applied. If significant effects of tilt were observed post-hoc paired t-tests with Bonferroni correction for multiple testing were used. A P-value of <0.05 was considered to indicate significance. Results In five volunteers, CBF could not be measured during 70° head-up tilt (HUT), due to a poor plethysmography signal or near fainting of the subject. Hemodynamic responses to HUT (Figure 1) CBF and superficial femoral artery blood flow (BF SFA) decreased significantly from supine to 30° with no further decrease with increasing tilt angle (Figure 1). The relative decrease in CBF (46% ± 11%) from supine to 30° was significantly larger than the decrease in BF SFA (40% ± 12%) (P < 0.001). Fig. 1Absolute values of calf blood flow (CBF) measured with venous occlusion plethysmography and superficial femoral artery blood flow (BF SFA), heart rate (HR) and mean arterial pressure (MAP) during supine position, 30°, 45°, and 70° HUT. One-way repeated measures ANOVA showed a significant effect of tilt for all parameters. *P < 0.05 and indicates significantly different from the position before. ■ = supine;  = 30° HUT;  = 45° HUT; □ = 70° HUT Agreement VOP and DU The Pearson correlation between the two methods was 0.86 (P < 0.001) for all data points (Figure 2). The agreement between the two methods was evaluated by plotting the relative difference in each measurement against the mean for all data points and separated for the different tilt angles (Figure 3). The relative mean difference ((VOP–DU)/DU) was −14% ± 22% for all data points in supine position and HUT indicating that overall CBF (VOP) is lower than BF SFA (DU); for supine position the relative mean difference between VOP and DU was 1.5% ± 24%; for 30°: −13% ± 23%; for 45°: −23% ± 19%; for 70°: −23% ± 15%. Limits of agreement for all data points in supine position and during HUT were −58% to 31% and became smaller during HUT. The limits of agreement are reasonable, although CBF during HUT is lower than BF SFA. Fig. 2Blood flow in the superficial femoral artery (BF SFA) measured by Doppler ultraound during different angles of head-up tilt versus calf blood flow (CBF) measured by venous occlusion plethysmography corrected for lower leg volume. Pearson correlation coefficient is 0.86Fig. 3Relative difference between the blood flow measured by venous occlusion plethysmography (VOP) and the superficial femoral artery blood flow measured by Doppler ultrasound (DU) versus the mean of both flow for each individual subject at different tilt angles Reproducibility of VOP during head-up tilt The coefficient of variation (CV) of CBF ranged between 8.7% and 15.0% (Table 2). In 70° HUT, the CV for both parameters was calculated over four subjects only. Table 2Values of calf blood flow (n = 9)SubjectSupine30° HUT45° HUT70° HUT (n = 4)test 1test 2test 1test 2test 1test 2test 1test 2Mean ± SD2.6 ± 0.62.6 ± 0.81.3 ± 0.31.3 ± 0.3 1.1 ± 0.31.1 ± 0.21.1 ± 0.2 n = 41.2 ± 0.2 n = 4%change ± SD−45 ± 11−49 ± 12−54 ± 11−57 ± 13−44 ± 17−44 ± 14CV15.0%(CI 10.1–29.1)11.0%(CI 7.4–21.3)14.9%(CI 10.0–28.9)8.7%(CI 4.9–33.2)Values of calf blood flow in ml·100 ml−1 · minute−1 and mean absolute and relative data ± SD in supine position, 30°, 45°, and 70° head-up tilt (HUT) for the first and second test. Coefficients of Variation (CV). Missing data in 70° HUT are due to near fainting or a poor plethysmography signal. Discussion Calf blood flow (CBF) measured with VOP correlates well with superficial femoral artery blood flow (BF SFA) measured with DU, and can be measured reproducibly during HUT. Since the most profound changes in blood flow with both techniques were already measured in 30° HUT, and the increase in hydrostatic and venous pressure, and concomitant technical difficulties are smallest from supine to 30° HUT we recommend to use VOP for leg blood flow measurements during HUT up to 30°. The decrease in leg blood flow assessed with VOP and DU, is comparable to tilt-induced blood flow changes in other studies using DU (33%–59%) [2, 7, 10, 11]. The strong relationship between VOP and DU blood flow measurements in the present study is in line with previous studies reporting correlation coefficients varying from 0.57 to 0.99 at rest and during exercise [14, 16, 27]. Head-up tilt affects muscle blood flow more than skin blood flow [21, 28]. Since skin blood flow contributes more to superficial femoral blood flow than to calf blood flow, this may explain the observed discrepancy between the decrease in CBF (∼48%) versus the decrease in superficial femoral artery blood flow (∼40%) in response to HUT. Reproducibility of baseline CBF (15.0%) is in range with other studies using similar techniques to measure leg blood flow [1, 18, 25]. The coefficient of variation of CBF during HUT was even better (11.0%–17.9%), which indicates that VOP is a reproducible tool to measure tilt-induced vasoconstriction repetitively. The low coefficients of variation of CBF during 70° (8.7%–8.9%) are not representative since these coefficients of variation were calculated over no more than 4 subjects. Not all subjects were able to abstain from moving their legs in 70° HUT position, and some subjects fainted in this position. Moreover, the quality of the plethysmographic tracing became worse at 70° HUT whereas at the lower tilt angles the plethysmography signal is of good quality indicated by the volume pulsations in the plethysmographic tracing for the period of venous occlusion. Our data and previous studies [15, 19, 20, 26] show that at 30° HUT peripheral vascular responses are accomplished to a large extent. The increase in hydrostatic pressure and concomitant increase in venous pressure is low at 30°. From Figure 4B it can be concluded that at 30° HUT venous compliance is still at the steep portion of the venous compliance curve whereas during 45°, and 70° HUT the venous compliance has shifted to the non-linear part. At 30° HUT increase of the plethysmography signal during venous occlusion is linear while at 45° and 70° HUT, venous distensibility is reduced and consequently results in a non-linear increase in leg volume during inflation of the venous occlusion cuffs, which is illustrated in Figure 5. We therefore recommend using VOP at 30° HUT. For studies focussing on syncope at the endpoint of HUT, which requires larger tilt angles, other techniques to measure leg blood flow should be used. Fig. 4(A) Pressure-Volume curve and (B) Pressure Compliance curve based on data of a similar group of volunteers measured by venous occlusion plethysmography at different cuff occlusion pressures (for method and protocol see de Groot et al., Journal of applied Physiology, 2005). The increases in calf volume in response to 30°, 45°, and 70° HUT are marked by the dotted lines in the pressure volume curve (A). The different tilting angles correspond with different venous pressures (x-axis). Transferring these venous pressures into the pressure-compliance curve (B) clearly demonstrate that during 30° HUT venous compliance is still on the steep linear part of the curve, whereas during 45°, and 70° HUT the venous compliance is compromisedFig. 5Typical plethysmographic tracing of one individual subject during a complete experiment. Blood flow measurements at 30° HUT start when the plethysmography signal does not change anymore, meaning that venous volume reached a steady state situation. Besides, looking at a typical VOP tracing at 30° HUT, the increase in venous volume is linear during the first 5 seconds of cuff inflation, indicating that blood flow measurements using VOP during 30° HUT are not compromised by a decrease in venous compliance Limitation Using VOP, blood flow is defined as limb volume changes over time. During HUT, when the leg is below heart level, volume changes can still be measured using VOP, however, the physiological determinants of these volume changes are complex and it is no longer possible to say with reasonable certainty that a change in volume over time, which most likely reflects flow, is determined by resistance vessel tone. For example, limb blood flow measured using VOP in HUT position can decrease due to an increase in venous pressure, as a result of venous congestion and the associated fall in arterio-venous pressure gradient, without any increase in resistance at the arteriolar level. In conclusion, this study demonstrates that CBF measured by VOP during HUT is suitable and reproducible. The method is easy applicable and recommended in tilt angles equal to 30° to avoid high hydrostatic and leg venous pressures.

Aesthetic_Plast_Surg-4-1-2175019

Cell-Assisted Lipotransfer for Cosmetic Breast Augmentation: Supportive Use of Adipose-Derived Stem/Stromal Cells

Background Lipoinjection is a promising treatment but has some problems, such as unpredictability and a low rate of graft survival due to partial necrosis. Autologous fat transplantation is one promising treatment for facial rejuvenation and soft tissue augmentation because it results in no incisional scar or complications associated with foreign materials. However, certain problems remain, such as unpredictability and a low rate of graft survival due to partial necrosis. Many innovations to overcome these problems have been reported [1, 2, 4–6, 18] and reviewed previously [4, 14]. On the basis of these reports, we tentatively concluded that we could harvest fat with a 2.5-mm cannula or 18-gauge needle at a vacuum lower than 700 mmHg and reinject it using an 18-gauge needle without significant adipocyte damage [14]. Lipoinjection can be used to treat facial changes associated with aging and to correct various types of depressed deformities such as hemifacial microsomia and pectus excavatum. It also has been used in breast augmentation by a limited number of plastic surgeons [3], although the use of autologous fat for breast augmentation has been controversial. Consensus is lacking on whether lipoinjection is safe and appropriate because of microcalcifications that may cause confusion in the evaluation of mammograms. Recently, autologous fat injection has been reevaluated as a potential alternative to artificial implants for breast augmentation [3, 15, 16, 19]. This reevaluation may reflect recent advances in autologous fat transfer and the radiologic detection of breast cancer. To overcome the problems associated with autologous fat transfer, we use a novel strategy known as cell-assisted lipotransfer (CAL) (Fig. 1). Findings have shown that tissue-specific progenitor cells in the adipose tissue have the capacity to differentiate into various cell lineages [21]. Thus, the progenitors, currently known as adipose-derived stem/stromal cells (ASCs), are expected to become a valuable tool in a wide range of cell-based therapies. Fig. 1Scheme of cell-assisted lipotransfer. Relatively adipose-derived stem/stromal cell (ASC)-poor aspirated fat is converted to ASC-rich fat by supplementing ASCs isolated from the other half of the aspirated fat. The ASCs are attached to the aspirated fat, which is used as a scaffold in this strategy The therapeutic concept of CAL was described in our previous report on preclinical studies [9]. We found that aspirated fat has approximately half the number of ASCs found in excised whole fat. There are two main reasons for this relative deficiency. First, a major portion of the ASCs is located around large vessels and left in the donor site after liposuction [9]. Second, a part of the ASCs is released into the fluid portion of liposuction aspirates [20]. The relative deficiency of ASCs may induce postoperative long-term atrophy of injected fat, as partially confirmed in animal studies [8, 9, 11]. With the CAL strategy, autologous ASCs are used to enhance angiogenesis, to improve the survival rate of grafts, and to reduce postoperative atrophy. In CAL, half the volume of the aspirated fat is processed for isolation of the stromal vascular fraction (SVF) containing ASCs. During the isolation process, the other half of the aspirated fat is prepared for grafting. Freshly isolated SVF, which we characterized previously [20], is attached to the aspirated fat, with the fat acting as a living scaffold before transplantation. Finally, the SVF-supplemented fat is injected into the target sites. Thus, ASC-poor fat is converted to ASC-rich fat in the preparation process of the injectable material. In this report, we describe the preliminary results experienced by patients who underwent CAL for cosmetic breast augmentation. This is the first report on the clinical use of ASCs for cosmetic purposes. Materials and Methods Patients From 2003 to 2007, we performed CAL for 70 patients: in the breast for 60 patients (including 8 patients who had breast reconstruction after mastectomy), in the face for 12 patients, and in the hip for 1 patient. For three patients, CAL was performed at two sites. Informed consent was obtained from all the patients. The study protocol conformed to the guidelines of the 1975 Declaration of Helsinki and was approved by individual institutional review boards. In this study, 40 patients with healthy thoraxes and breasts underwent CAL for purely cosmetic breast augmentation. Patients undergoing breast reconstruction for inborn anomaly or after mastectomy were not included. At this writing, 19 of these 40 patients have been followed for more than 6 months, and the maximum follow-up period has been 42 months. All the patients were Japanese women with a mean body mass index (BMI) of 19.1 ± 1.9. Their ages varied from 20 to 62 years (mean, 35.8 ± 9.1). The mean volume of injected fat was 268.1 ± 47.6 ml on the left side and 277.3 ± 39.1 ml on the right side. The demographic and surgical data for these patients are summarized in Table 1. Table 1Patient dataNo. of cases40Sex40 F, 0 MAge (years) 35.8 ± 9.1BMI19.7 ± 1.9 Surgical procedure  Group A6  Group B2  Group C32Site of liposuction  Thighs25  Thighs and abdomen13  Thighs and lower legs2Total volume of suctioned fat (ml) 1111.8 ± 164.0Volume of injection (ml)  Left268.1 ± 47.6  Right277.3 ± 39.1Operation time (min)257.1 ± 39.1 Surgical Techniques Before the procedure began, the liposuction site was infiltrated with combined saline solution and diluted epinephrine (0.001%). With the patient under general anesthesia, adipose tissue was suctioned using a cannula with a 2.5-mm inner diameter and a conventional liposuction machine. Approximately half of the collected liposuction aspirate was used for isolation of the SVF from both the adipose and fluid portions of the liposuction aspirates, as described previously [20]. This cell processing procedure required about 90 min. During the processing period, the other half of the lipoaspirate was harvested as graft material. The adipose portion of the liposuction aspirates was either washed several times and placed in an upright position to obtain clear separation of fluids and oil (groups A and B) or centrifuged at 700 g for 3 min without washing (group C), then put into a metal jar (500 ml), which was placed in water with crushed ice. In groups A and C, the fresh SVF isolated from both the adipose and fluid portions was added to the graft material. After gentle mixing and a wait of 10 to 15 min for cell adherence to the aspirated fat, the cell-supplemented fat was put into an injection syringe. In Group B, the freshly isolated SVF was resuspended in 60 ml of saline, then diffusely injected into the whole breast mounds separately (30 ml for each breast) immediately after conventional lipoinjection. There were 6 patients in group A, 2 patients in group B, and 32 patients in group C. For the injection syringe, a 10-ml LeVeen inflator (Boston Scientific Corp., Boston, MA, USA) or our original 20-ml syringe was used because each is a screw-type syringe (with a threaded plunger), and the threaded connections fit both the connecting tube and the needle to allow precise control during injection. To reduce the time of the procedure, two syringes were used. While the one syringe was being used for an injection, the other was being filled with the graft material in preparation for the next injection. An 18-gauge needle (150 mm long) was used for lipoinjection and inserted subcutaneously at one of four points indicated in Figs. 2A and B. Fig. 2Schematic instruction of the injection method. (A) A small amount of fat tissue is injected as small aliquots or as a thin string with a long needle, using a syringe with a threaded plunger, while the needle is continuously withdrawn. (B) The needle is inserted from either one of two points on the areola margin or one of two points at the inframammary fold in variable directions and planes to achieve a diffuse distribution The operator took care to insert and place the needle horizontally (parallel to the body) to avoid damaging the pleura and causing a pneumothorax. The needle was inserted in several layers and directions. It was continuously and gradually retracted as the plunger was advanced. This technique was used to obtain a diffuse distribution of the graft material (Figs. 2 and 3). The grafts were injected into the fatty layers on, around, and under the mammary glands, and also into the pectoralis muscles. Fig. 3A clinical view of injection. The injection needle is rigidly manipulated by an operator while an assistant rotates the plunger according to the operator’s instruction. A high-pressure injection can be performed using a disposable syringe with a threaded plunger. A 150-mm-long, 18-gauge needle is connected to the syringe with a connecting tube threaded at both ends Results The transplantation of adipose tissue was successfully performed in all cases, and the time of the injection process ranged from 35 to 60 min for both breasts. Subcutaneous bleeding, occasionally seen in some parts of the breasts, resolved in 1 to 2 weeks. Transplanted adipose tissue was gradually absorbed during the first 2 postoperative months (especially during the first month), and the breast volume showed a minimal change thereafter, although skin tension sometimes lessened after 2 months. Three representative surgical sites are shown in Figs. 4 to 9. The difference in breast circumference (chest circumference at the nipple minus the chest circumference at the inframammary fold) had increased in all cases by 4 to 8 cm at 6 months, which corresponds to two to three brassiere cup sizes. This circumference increase appeared to correspond to the 100- to 200-ml increase in the volume of each breast mound, which was partially confirmed by our preliminary evaluation using a three-dimensional quantitative measurement system. Fig. 4Clinical views of a patient in group A (patient 1). Preoperative (left) and postoperative (right) views at 24 months. A 22-year-old woman underwent breast augmentation using cell-assisted lipotransfer (CAL) (290 ml in each breast), with satisfactory results at 24 months. Her breast circumference increased by 5 cm, and her augmented breast mounds remained soft and natural appearing without injection scars or subcutaneous indurationsFig. 5Radiologic views showing the chest of patient 1. (A) A preoperative computed tomography (CT) image in the horizontal plane of the nipples. (B) A horizontal image 12 months after surgery. Note that the adipose tissue is augmented both subcutaneously and under the mammary glands. (C) Mammograms at 12 months show no calcification or other abnormal signsFig. 6Clinical views of a patient in group C (patient 2). Preoperative (top) and postoperative (bottom) views at 12 months. A 32-year-old woman underwent breast augmentation with cell-assisted lipotransfer (CAL) (280 ml in each breast). Her breast circumference difference increased from 9 cm (baseline) to 14.5 cm (at 12 months). The breast mounds are soft and natural appearing with no visible injection scarsFig. 7Radiologic views showing the chest of patient 2. (A) A preoperative computed tomography (CT) image in the horizontal plane at the level of the nipples. (B and C) Horizontal images by magnetic resonance imaging (MRI) 12 months after surgery: (B) T1-image; (C) T2-image. The adipose tissue is augmented around and under the mammary glands. A small cyst (<10 mm) appears in the fatty layer under the right mammary gland. (D) Mammograms at 12 months show no abnormal signs such as calcificationsFig. 8Clinical views of a patient in group C (patient 3). Preoperative (top) and postoperative (bottom) views at 24 months. A 30-year-old woman underwent breast augmentation with cell-assisted lipotransfer (CAL) (310 ml in each breast). Her breasts were dramatically augmented with an increase in breast circumference difference by 8 cm at 24 months. The breast mounds were soft with no subcutaneous indurations. An original inframammary fold on the left breast is slightly visible, but injection scars are not visibleFig. 9Radiologic views of patient 3. (A) A preoperative computed tomography (CT) image in the horizontal plane at the level of the nipples. Only a very thin fatty layer is observed around the mammary glands. (B) A horizontal magnetic resonance image (MRI) (T1 weighted) 24 months after surgery. Transplanted adipose tissues survived and formed thick layers around and under the mammary glands. (C) Mammograms 24 months after surgery show no abnormal signs Compared with breast augmentation using implants of the same size, augmentation with CAL showed a lower height but a more natural contour of the breasts. All cases but one (see later) showed natural softness of the breasts without any palpable nodules at 6 months, and all the patients were satisfied with the resulting texture, softness, contour, and absence of foreign materials despite the limited size increase possible with autologous tissue—Cyst formation (<12 mm) was detected by magnetic resonance imaging (MRI) in two patients, and microcalcification was detected by mammogram in two patients at 24 months. In one of two patients in group B, fibrous breast tissue and fibrosis on the sternum were observed by computed tomography (CT) scan at 6 months, and the breasts were found to be harder than in other cases. Discussion Modifications of lipoinjection techniques to improve the survival rate for injected fat have been attempted. From these, it is well accepted that adipose tissue should be placed as small aliquots [3], preferably within an area 3 mm in diameter [1]. Because it takes a long time to perform ideally diffuse distribution of suctioned fat [3], we have used a disposable syringe with a threaded plunger and connections, a very long needle (150 mm), and an assistant to rotate the plunger. As a result, only 35 to 60 min are required for injection in both breasts. These devices are critical to performing large-volume lipoinjection safely and precisely in a short time. In addition, the harvesting, preserving, and refining of graft materials also are important, as repeatedly indicated in the literature. We used a relatively large suction cannula, centrifuged the aspirated fat in some cases, and kept it cooled until transplantation. In this study, the clinical results (increase in breast circumference) appeared to be better in group C (using centrifuged fat) than in group A (using noncentrifuged fat), although quantitative measurement and statistical comparison were not done. In a previous study, we found that centrifugation of aspirated fat is substantially influential because centrifugation at 1,200 g decreases the fat volume by 30%, damaging 12% of the adipocytes and 0% of the ASCs. This leads to the concentration of cell numbers per volume of adipocytes and ASCs by 25% and 43%, respectively [7]. In addition, centrifugation may be especially beneficial in our treatment because water content in the graft material may disturb the adherence of ASCs to the adipose tissue and interfere with differentiation into expected lineages. Any ASCs floating in a solution, which is a nonphysiologic environment, may migrate over distances, penetrate into the lymphatic flow, and differentiate unexpectedly. We believe that such migration and altered cell differentiation caused the development of fibrotic tissue on the sternum of one patient in group B. Thus, we conclude that centrifuged fat combined with ASCs as cell pellets (group C procedure) was best among the three methods used in this study. Although small cystic formation and microcalcification were detected in some cases, the microcalcification was easily distinguished from that associated with breast cancer, and the overall cosmetic results were generally satisfactory and encouraging. Almost all the patients were satisfied with their enlarged and soft breasts with a natural contour. Both CT scans and MRI showed that transplanted fat tissue survived and formed a significant thickness of the fatty layer not only subcutaneously on and around the mammary glands, but also between the mammary glands and the pectoralis muscles. Breast volume stabilized 2 to 3 months after transplantation. Maximum breast augmentation using the described technique varied among the patients and appeared to be 100 to 200 ml. Although these volumes may be smaller than those achieved with large artificial implants, a definite advantage is that patients need not be concerned about postoperative complications induced by artificial implants such as rupture, infection, capsular contracture, unnatural contour, hardness, neurologic symptoms, and immune response. Compared with our dozens of patients who underwent conventional autologous lipoinjection to the breasts, augmentation effects were apparently higher with CAL. A 2- to 3-cm increase in breast circumference was common with the conventional procedure, compared with the 4- to 8-cm increase seen in this trial of CAL, although the augmentation effect varied among patients. The measurement system we recently devised may help to quantify the difference in augmented volume in the future. Adipose tissue contains not only adipogenic progenitor cells, but also multipotent stem cells, which can differentiate into fat, bone, cartilage, and types of tissue [21, 22]. Suctioned fat appears to lose a significant number of these precursors during liposuction and the preparation processes as compared with nonsuctioned adipose tissue [9]. This relative deficiency of precursors may contribute to the low survival rate and long-term atrophy of transplanted lipoaspirates. In CAL, the deficit of ASCs was compensated by supplementing ASCs. To maximize the biologic function and avoid unexpected behavior of ASCs, it seems important to ensure adherence of supplemented ASCs to adipocytes or connective tissue. With this novel treatment, ASCs have four possible roles, which were partly confirmed in preclinical studies [8, 9, 11]. First, ASCs can differentiate into adipocytes and contribute to the regeneration of adipose tissue. Second, ASCs can differentiate into endothelial cells and also probably into vascular mural cells [8, 10, 12], resulting in the promotion of angiogenesis and graft survival. Third, ASCs are known to release angiogenic growth factors in response to hypoxia and other conditions [13], and these factors influence surrounding host tissue. In their final role, which may be the most influential, ASCs survive as original ASCs [9]. In the adipose tissue, ASCs reside between adipocytes or in the extracellular matrix, especially around vessels, and contribute to the turnover of adipose tissue, which is known to be very slow (2 years or more) [17]. However, adipose grafts probably turn over during the first 2 to 3 months after transplantation because they experience temporary ischemia followed by reperfusion injury. This turnover, the replacement process of the adipose tissue, is conducted by tissue-specific progenitor cells, which are ASCs. The relative deficiency of ASCs in aspirated fat may affect the replacement process and lead to postoperative atrophy of grafted fat, which is known to occur commonly during the first 6 months after lipoinjection. The freshly isolated SVF used in CAL contains not only ASCs but also vascular endothelial cells, pericytes, blood cells (both white and red), and other cells, as previously described [20]. After transplantation, ASCs may interact with other cells such as vascular endothelial cells, and supplementation with the SVF may be superior to supplementation with ASCs alone in this treatment. However, further studies are needed to elucidate the synergistic effects of ASCs with other cells contained in the graft. In this preliminary study, satisfactory clinical results were generally achieved without any major complications. Thus, we can conclude that CAL is sufficiently safe for continuation of the study, though controlled investigations and accumulated long-term results are needed to elucidate the overall safety and efficacy of the treatment. A variety of new innovations, including stem cell technology, may be developed and may contribute to the improvement of autologous tissue transplantation and regeneration. Further improvements of the technique may cause autologous tissue transfer to become the first choice for breast augmentation in the future.

Behav_Genet-3-1-1824713

The Association between Conduct Problems and the Initiation and Progression of Marijuana Use during Adolescence: A Genetic Analysis across Time

The present study used a prospective, longitudinal design to investigate genetic and environmental influences on the association between earlier conduct problems and the initiation and progression of marijuana use during adolescence. Parent- and teacher-reported conduct problems assessed at Time 1 (1996) and self-reported marijuana use assessed at Time 2 (2004) were available for 1088 adolescent twin pairs participating in the Cardiff Study of All Wales and North West of England Twins (CaStANET). Using a novel approach to the modeling of initiation and progression dimensions in substance use, findings suggested that the initiation of marijuana use in adolescence was influenced by genetic, common and unique environmental factors. The progression (or frequency) of marijuana use was influenced by genetic and unique environmental factors. Findings for conduct problems indicated that while the presence or absence of conduct problems was largely heritable, the relative severity of conduct problems appeared to be more strongly environmentally influenced. Multivariate model fitting indicated that conduct problems in childhood and early adolescence made a small but significant contribution to the risk for marijuana use 8 years later. Marijuana use can pose a major risk to adolescent health and well-being, yet it remains the most commonly used illicit drug in both the United States and the United Kingdom. In the USA, both availability and rates of marijuana use among adolescents increased sharply during the 1990s, with only a slight decline in recent years (Johnston et al. 2005). In the UK, there was a gradual increase in adolescents reporting marijuana use in the past 12 months, from 10% in 1998 to 13% in 2003, with a slight decline in 2004 to 11% (National Centre for Social Research 2005). In the United States, 50% of adolescents reported the use of marijuana by the age of 17 (Johnston et al. 2005) while in the United Kingdom, 38% of 15–16 year olds had used marijuana (Hibell et al. 2004). Marijuana use is related to a range of deleterious outcomes including lower academic achievement, criminality and mental health problems such as depression and suicidal behaviour (Fergusson et al. 2002; Penning and Barnes 1982; van den Bree and Pickworth 2005). Recent research using a twin design has identified conduct problems in childhood and adolescence as a major risk factor for substance use, and marijuana use specifically (e.g. Miles et al. 2002; Silberg et al. 2003; Young et al. 2000). A series of studies have documented genetic and environmental influences on adolescent marijuana use (e.g. Maes et al. 1999; McGue et al. 2000; Miles et al. 2001). Given the complex processes thought to underlie the link between initiation and progression of drug use however, it is argued that the question should not be whether or not there is a genetic component to drug use per se, but rather what role genetic influences play in the aetiology of drug use over time (Rutter et al. 1999; e.g. Rhee et al. 2003). Recent research has highlighted that the initiation and progression of substances may not be characterised as a single liability of risk (e.g. Agrawal et al. 2005; Heath et al. 1991, 1997; Koopmans et al. 1999). Rather, the initiation and progression phases of substance use may be more appropriately characterised as partially overlapping (or even, independent; Heath et al. 1991). Indeed, while evidence from adult twin studies indicates that there are additive genetic, common environmental and unique environmental factors that are shared between the initiation and progression of marijuana use, there is also evidence of genetic and unique environmental influences that are specific to heavier use of marijuana (Agrawal et al. 2005). The increased influence of genetic factors for heavy substance use possibly reflects the increased involvement of biological processes in chronic use (Kender 2001; van den Bree 2005). For many individuals, initiation and experimentation with substances occurs during adolescence (Fuller et al. 2005). Adolescents may however, have lower tolerance levels for substances, including marijuana, and become dependent at lower doses than adults (e.g. Chen et al. 1997). Furthermore, adolescent-onset substance misuse is characterized by more rapid development of multiple drug dependencies and more severe psychopathology (e.g. Clark et al. 1998). With this in mind, it is important to consider the relative role of genetic and environmental influences on initiation and progression of substance use in this age group and whether the relationship between these dimensions of use identified with adult samples is also observed in adolescents. Antisocial behaviour during childhood and adolescence has been linked to substance use in adolescence and early adulthood (e.g. Moffitt et al. 2002; Silberg et al. 2003). Moreover, in the context of other disruptive behaviour disorders, conduct disorder has been shown to be the most predictive of substance use and illicit drug use (e.g. Lynskey and Fergusson 1995; Moss and Lynch 2001). A recent follow-back study of a prospective, longitudinal cohort found that adults with a substance use disorder (alcoholism, marijuana and other drug dependence) were significantly more likely than those without such disorder to have had a conduct and/or oppositional defiant disorder before 15-years old (Kim-Cohen et al. 2003). Strong associations have also been found between conduct disorder symptoms and marijuana use in adolescence for both males and females (Miles et al. 2002). This association was moderately influenced by genetic factors and to a lesser extent, by non-shared environmental factors (see also Young et al. 2000). The findings of a recent longitudinal twin study indicated that conduct problems in adolescence temporally preceded substance use and that the covariation between conduct problems and later substance use was influenced by genetic and common environmental factors (Silberg et al. 2003). These findings mark an important shift away from documenting cross-sectional associations between conduct problems and substance use, from which it is impossible to determine the direction of effects, toward the use of genetically sensitive research designs that assess relationships as they unfold over time. Indeed, in the absence of an experimental design, a primary way in which the nature of the relationship between conduct problems and the initiation and progression of marijuana use during adolescence can begin to be disentangled is to use a prospective, longitudinal twin design. The present study is among the first to investigate whether the initiation and progression of marijuana use during adolescence reflects a common underlying liability. Extending previous research on the relationship between conduct problems and substance use, this study also examines whether genetic and environmental influences on conduct problems are related to the later initiation of marijuana use in adolescence and in addition, whether conduct problems are implicated in the continued use of marijuana once initiation has occurred. Bivariate genetic analyses were conducted to establish whether marijuana initiation and progression represented a single liability of risk or alternatively, whether these behaviours were more accurately represented as independent liabilities (see Fig. 1). It was hypothesised that genetic, common and unique environmental influences would contribute to the initiation of marijuana use, consistent with the role of social factors in the initial experimentation phase of drug use (Rutter et al. 1999). In accordance with previous studies (Agrawal et al. 2005; Kendler et al. 1999; van den Bree et al. 1998) we hypothesized heavier use (i.e. the progression of marijuana use, rather than initiation) to be more strongly influenced by genetic factors with a reduced role for common environment. Multivariate analyses assessed the relationship between conduct problems and later marijuana use. It was hypothesised that earlier genetic and environmental influences on conduct problems would be related to the initiation of marijuana use 8 years later. Initiation of marijuana use was expected to remain the strongest predictor of progression of marijuana use after controlling for the influence of conduct problems. Fig. 1A conceptual model of the bivariate causal covariance contingency model. Note: Superscript i refers to factors for initiation. Superscript p relates to factors specific to progression Method Sample The sample used in the present analyses was drawn from the second (1996) and fourth wave (2004) of data collection of the longitudinal Cardiff Study of All Wales and North West of England Twins (CaStANET; Rice et al. 2002; van den Bree et al. in press). The CaStANET register is a population-based twin register, including twins born between 1976 and 1991 in the Cardiff area of South Wales and between 1980 and 1991 for the rest of Wales and the North West of England. This twin register includes families from a systematically ascertained, population-based register of twin births between 1980 and 1991 in Wales and Greater Manchester, UK. Zygosity was assigned using a twin similarity questionnaire completed by parents that is more than 90% accurate in distinguishing monozygotic (MZ) from dizygotic (DZ) twins and, in a subsample, validated by genotyping DNA markers (Cohen et al. 1975; Nichols and Bilbro 1966; Payton et al. 2001). The CaStANET study received ethical approval from the Multi Centre Research Ethics Committee for Wales, UK. At the second wave of data collection (1996), questionnaires were mailed to parents of twins aged 5–16 years old. Of 2846 families contacted, 2082 parents returned questionnaires, representing a response rate of 73% (see Thapar et al. 2000 for a more detailed description of the sample). Parental consent was also obtained to contact the twins’ teacher. Of 2168 teachers contacted, 1913 returned questionnaires, representing a response rate of 88%. At the fourth wave of data collection in 2004, questionnaires assessing various aspects of family functioning, parent health and twin psychological adjustment were mailed to families (parents and twins) on the CaStANET twin register with twins aged 11–19 years. Families were sent a reminder postcard, reminder questionnaires and finally, a reminder letter. Non-responding families who might have moved address were traced through General Practitioners. Previous research has shown that adolescent smokers and marijuana users are less likely to respond at follow-up in longitudinal studies (Siddiqui et al. 1996). Extra efforts were therefore made to contact families via telephone where one twin had reported substance use. Of 1755 families with adolescent twins re-contacted in 2004, at least one family member from 1214 families returned questionnaires (1081 parents; 1125 twin pairs, where either or both twins replied) representing a response rate of 69%. Family members who returned questionnaires received a high street store voucher as a token of appreciation for their participation. Demographic statistics indicated that the sample was representative of British families living in the UK region of England and Wales with regard to family constitution, ethnicity, employment and economic factors (Social Trends 2004). The present study is based on a subsample of 1088 adolescent twin pairs with information relating to conduct problems (parent and/or teacher report in 1996) and marijuana use in 2004. For clarity, these assessments are referred to as Time 1 (1996) and Time 2 (2004), respectively from this point forward. Of this sample, there was complete information for 895 twin pairs. At Time 1 (1996), the sub-sample of twin pairs were aged 5–13 years (mean = 9.02 years old; SD = 1.98, range). At the Time 2 (2004), twin pairs were aged 11–20 years old (mean = 16.10 years old; SD = 1.94). There were approximately equal numbers of participating boys (n = 960; 44.1%) and girls (n = 1216; 55.9%). The sample comprised 425 monozygotic twins (177 male, 248 female pairs) and 663 dizygotic twins (132 male, 189 female, 342 opposite sex pairs). The majority of twins lived with their biological mother and father (66.2%) with smaller proportions living with one biological parent and a stepparent (10.4%), a single parent (18.7%) and ‘other’ (4.6%). Twins classified as living with ‘other’ included those who were living with another relative (e.g. grandparents) and twins who lived apart or who were away at university for part of the year. Analyses conducted in which this last group were excluded resulted in a similar pattern of findings to those reported below. The majority of parent questionnaires (Time 1) were completed by mothers (93.6%) with smaller numbers completed by fathers (5.3%) and others (1.1% e.g. stepparent; grandparent). There was no significant difference in mean levels of conduct problems as a function of the reporter of parent information. Tests indicated that twins who did not respond at the Time 2 follow-up had higher levels of Time 1 conduct problems than twins who did respond (t = 4.809, P < 0.001). This may indicate a possible bias, whereby adolescents with higher conduct problems who were less likely to respond at follow-up were also those adolescents more likely to use substances, including marijuana. Measures Conduct problems Parent and teacher-reported conduct problems were assessed using five items from the Strengths and Difficulties Questionnaire (Goodman 1997; Goodman and Scott 1999). Items included, ‘Often tells lies or cheats’ ‘Steals things’, ‘Fights or bullies other children’, ‘Often has temper tantrums or hot tempers’ and ‘Generally obedient, usually does what adults request’ (recoded to reflect disobedient behaviour). Each item was rated, 0 ‘Doesn’t apply’, 1 ‘Applies somewhat’ or 2 ‘Certainly applies’. These items are broadly equivalent to symptoms of conduct disorder as defined by the DSM-IV (American Psychological Association 1994). Parents and teachers reported on behaviour over the last six months. Recent evidence indicates that different informants provide related and unique information about children’s antisocial behaviour (Arseneault et al. 2003). Moreover, because children’s behaviour can vary between settings, the most valid measurement is that which includes data from more than one informant and from more than one setting or context (Scourfield et al. 2004). Consistent with this argument, conduct problem scores were combined for parent and teacher reports by counting symptoms reported by either parents or teachers as present, thereby capturing each of the five behaviours occurring in both the home and school context. The item-level correlations between parent and teacher reports of conduct problems ranged from r = 0.17 (stealing) to 0.56 (disobedience), P < 0.001. The highest rating given by either parent or teacher was taken as the score for that symptom, in other words the occurrence of the behaviour in one context (e.g. the home), was sufficient for it to be counted as present. The internal consistency estimate for the present sample was acceptable (α = 0.76) and the five items were added to give a total conduct problems score. Using criteria outlined by Goodman (1997), conduct problems ranging from 0 to 2 were classified as ‘normal’, a score of 3–4 was classified as ‘borderline’ and scores ranging from 5 to 10 were classified as ‘abnormal’. Of respondents with conduct information at Time 1, 585 (73%) monozygotic twins were classified as normal, 154 (19%) as borderline and 67 (8%) as abnormal. Of the dizygotic twins, 877 (69%) were classified as normal, 227 (18%) as borderline and 166 (13%) as abnormal. Conduct problems were classified on two dimensions: (1) the presence of conduct problems was indexed as either ‘0’ for ‘normal range’ or ‘1’ for borderline or abnormal conduct problems (2) the second dimension labelled ‘borderline/abnormal’, distinguished individuals in the borderline and abnormal range for conduct problems. Children in the normal range were coded as missing for borderline/abnormal problems, borderline conduct problems was coded as ‘0’ and abnormal conduct problems were coded ‘1’. Marijuana use The frequency of lifetime marijuana use was assessed using the following item from the Add Health questionnaire (Resnick et al. 1997), ‘During your life, how many times have you used marijuana?’ The six response options ranged from ‘Never used marijuana in my life’ to ‘More than 30 times’. Responses were collapsed to create two binary variables assessing initiation of use and frequency of use. Initiation was indexed as ‘0’ for never having used marijuana and ‘1’ for use. Frequency was indexed as light and heavy use with ‘0’ representing use of marijuana 1–5 times and ‘1’ representing use of marijuana six or more times. Individuals who had never used marijuana were coded as missing for frequency of use. Statistical analysis The software package Mx (Neale 1997) was used for genetic model fitting. Analyses were conducted using a ‘causal common contingent’ model which facilitates the expression of marijuana use as a two-stage process incorporating an initiation stage (‘upstream’, for example whether the individual has ever tried marijuana) that necessarily precedes a progression stage (‘downstream’, for example, whether the individual uses the marijuana frequently; see Fig. 1; Agrawal et al. 2005; Neale et al. 2006). The model estimates the magnitude of the relationship between initiation and progression by means of a beta pathway between these two stages (see Fig. 1). If the beta coefficient is estimated to be zero, this suggests that the initiation and progression stages for a substance are entirely unrelated processes, i.e. genetic and environmental risk factors for initiation are completely independent from those for progression. Alternatively, if the beta coefficient is estimated to be 1, this indicates that initiation and progression are entirely overlapping dimensions with identical genetic and environmental risk factors. The 95% confidence intervals around the beta coefficient provide further information on the degree of overlap between the two stages. Lower limits closer to zero (or below) support independent liabilities and upper limits approaching 1 provide support for identical liabilities. The model also allows the estimation of: (1) additive genetic effects (a2), (2) common environmental effects (c2), and (3) unique environmental effects (e2), on both initiation and progression of substance use. An important feature of this model is that it is uniquely suited to analysis of data from an adolescent age group, where individuals may have not yet engaged in marijuana use but will go on to become frequent users. The model takes into account the fact that some individuals may be above the liability threshold for progression of marijuana use (i.e. will become frequent users) but because they are not past the age of risk, have not yet initiated marijuana use. As such, their position on the liability distribution of progression is unknown. These individuals are treated as a special case of missing data for progression using the maximum likelihood approach for dealing with missing data in Mx (Neale 1997; Neale et al. 2006). Given the likelihood of an association between age and level of substance use, an age correction was also employed which adjusts the threshold for each twin according to his or her age at the time of questionnaire completion on the distribution of liability to conduct problems and marijuana use. Specifically, the threshold is modeled as a simple linear function: where t is the population baseline threshold (for individuals of age zero), ta models the regression of the threshold on age, and ageiis the age in years of the individual i at assessment (Neale et al. 2006). Models were estimated using full information maximum likelihood (FIML) estimation with raw ordinal data, which included zygosity, twin age, and initiation and progression information relating to conduct problems and/or marijuana use for each twin. The significance of parameters was evaluated using 95% confidence intervals (CIs), calculated using Mx (Neale and Miller 1997). Results The rate of lifetime marijuana use for the total sample of adolescents was 21.6%. Examining the prevalence of marijuana use for younger and older adolescents highlighted an age-related difference in levels of use. Approximately 11% of 11–15 year olds had used marijuana compared with 32.5% of adolescents aged 16–20 years old. The average age of initiation of marijuana use was 14 years (mean = 14.73, SD = 1.67, range: 9–19 years). There was no difference in the prevalence of marijuana use between MZ (19.7%) and DZ twins (22.9%; χ2 = 2.987, P = 0.084). Tests indicated greater variance in marijuana use for DZ twins compared to MZ twins (F = 4.220, P = 0.040). A mean difference for a combined estimate of parent and teacher-reported conduct problems between MZ and DZ twins was also found (MZ, mean = 1.68, SD = 1.92; DZ, mean = 1.89, SD = 2.13; t = 2.27, P = 0.024). Tests revealed greater variation around DZ conduct problems in comparison with MZ twins (F = 9.938, P = 0.002). The higher DZ variances for a parent and teacher rated measure of conduct problems may indicate contrast effects whereby one twin is rated as having more behaviour problems than the other twin. On the other hand, the greater variances for DZ twin marijuana use may indicate sibling interaction, for example where one twin is less likely to engage in substance use in response to the other twins’ substance using behaviour. However, this is somewhat counterintuitive because research indicates that sibling deviance, and drug and alcohol use specifically, predicts increased substance use (e.g. Stormshak et al. 2004). Tests of sex differences revealed no sex differences in levels of marijuana initiation or progression (initiation: males, mean = 0.22, SE = 0.01; females, mean = 0.21, SE = 0.02; t = 0.55, P = 0.583; progression: males, mean = 0.32, SE = 0.03; females, mean = 0.41, SE = 0.04; t = 1.75, P = 0.081) or for the presence and severity of conduct problems (presence: males, mean = 0.29, SE = 0.02; females, mean = 0.31, SE = 0.02; t = 0.78, P = 0.437; severity: males, mean = 0.38, SE = 0.03; females, mean = 0.37, SE = 0.03; t = 0.26, P = 0.792). All figures calculated for pooled twin data were conducted using the survey commands in STATA 9.0 (StataCorp 2005), appropriate for use with twin data when there is non-independence of observations. Preliminary analyses were also conducted to investigate the relationship between a continuously assessed index of conduct problems (1996) and marijuana use (2004). The phenotypic relationship between conduct problems and marijuana use 8 years later was significant (β = 0.19, P < 0.01, R2 = 0.09). The magnitude of association between conduct problems and marijuana use for dizygotic twins and monozygotic twins was similar, suggesting common environmental influence (MZ twins, r = 0.18; DZ twins, r = 0.19). Examining the tetrachoric correlations using PRELIS 2.50 (Joreskog and Sorbom 1996) for each of the four constructs (initiation and progression of conduct problems and marijuana use, respectively) suggested genetic and shared environmental influence. The correlations for presence of conduct problems correlations were r = 0.77 for MZ twins and 0.46 for DZ twins indicating genetic and environmental influence, while the correlations for the borderline/abnormal conduct problems construct were r = 0.21 for MZ twins and 0.30 for DZ twins, indicating stronger environmental influence. The results for initiation of marijuana use indicated genetic and shared environmental effects (MZ r = 0.80; DZ r = 0.70), while progression of marijuana use appeared to be more strongly genetically influenced (MZ r = 0.68; DZ r = 0.13). The relationship between the liability to initiation and progression of conduct problems and marijuana use The results of model tests for conduct problems and marijuana use, respectively, are presented in Table 1. Table 1CCC model tests for conduct problems and marijuana useInitiation Progression a2c2e2βa2c2e2Time 1Conduct problems0.69 (0.41, 0.86)0.08 (0.02, 0.32)0.22 (0.14, 0.32)0.74 (0.22, 0.95)0.00 (0.00, 0.00)0.27 (0.00, 0.37)0.73 (0.05, 0.76)(0.41, 0.86)(0.02, 0.32)(0.14, 0.32)(0.22, 0.95)(0.00, 0.00)(0.00, 0.37)(0.05, 0.76)Time 2Marijuana use0.35 (0.05, 0.63)0.47 (0.24, 0.71)0.18 (0.10, 0.36)0.88 (0.38, 0.99)0.64 (0.00, 0.65)0.00 (0.00, 0.00)0.36 (0.00, 0.48)(0.05, 0.63)(0.24, 0.71)(0.10, 0.36)(0.38, 0.99)(0.00, 0.65)(0.00, 0.00)(0.00, 0.48) Conduct problems To facilitate later multivariate tests of the relationship between earlier conduct problems (Time 1) and later marijuana use (Time 2), a CCC model was estimated for conduct problems. Given the differences found between MZ and DZ twins in levels of conduct problems, model tests were conducted in which thresholds for the different twin groups were allowed to vary. This provided a better fit to the data than a model in which thresholds were estimated to be the same between twin groups and these results are reported (χ2 = 7.853, df = 2, P < 0.05). Substantively however, the pattern of results was the same. The liability to any conduct problems accounted for a large proportion, but not all, of the variance in the liability to be classified as borderline/abnormal for conduct problems (β = 0.74; CI = 0.22, 0.95). The presence of conduct problems was mainly influenced by genetic (69%) and non-shared environmental factors (22%). Borderline/abnormal conduct problems were influenced by common environmental (27%) and non-shared environmental (73%) factors. The threshold estimates were 0.47 for presence and 1.10 for severity. A comparison of goodness-of-fit indices indicated that modelling thresholds as a function of age did not significantly improve the fit of the model (χ2 = 4.302, df = 2, P > 0.10). Marijuana use The results for marijuana use indicated that the initiation of use was explained by a heritable component (35%), common environment (47%) and non-shared environment (18%). In contrast, the frequency of marijuana use was explained by a heritable component (64%) and a non-shared environment component (36%). The threshold estimates were 0.57 for initiation and 1.17 for frequency. The beta value (β = 0.88; CI = 0.38, 0.99) represents the genetic and environmental influences on initiation that are, in turn, transmitted to the frequency of marijuana use. This value, which is less than unity, indicated that while the liabilities for the initiation and frequency of marijuana use were not independent, neither could these dimensions be assumed to reflect a single liability of risk. In other words, the liability to initiate use of marijuana accounted for a substantial proportion (approximately 77%), but not all, of the variance in the liability to more frequent use of marijuana. The CI’s around beta (0.38–0.99) further indicated that the two liabilities were moderately to strongly related but not identical. Goodness-of-fit statistics indicated that a model in which age-corrected thresholds were estimated provided a better fit to the data than a model that did not include age-corrected estimates (χ2 = 56.155, df = 2, P < 0.001), corroborating our findings of a higher prevalence of increased marijuana use in older adolescents. Multivariate analyses Analyses were conducted to examine whether the liability to conduct problems during childhood and early adolescence was a risk factor for the liability to initiate and use marijuana 8 years later. The model was again estimated using full information maximum likelihood (FIML) estimation with raw ordinal data, which included zygosity, twin age, and ‘initiation’ and ‘progression’ information relating to conduct problems and marijuana use for each twin. In addition to paths estimated from each initiation variable to the progression variable, paths were estimated from presence of and borderline/abnormal conduct problems at Time 1 to marijuana initiation and progression at Time 2. Figure 2 presents the results for the full model, in which all six pathways between conduct problems (Time 1) and marijuana use (Time 2) were estimated. This model provided similar estimates of the relationship between each classification of conduct problems and the initiation and frequency of marijuana use, respectively. Minor fluctuations were found between models (bivariate model to the full multivariate model) in the genetic and environmental estimates for conduct problems, which can be expected to be a result of the model estimation. Fixing estimates in the full model to their bivariate values would not change the substantive interpretation of the multivariate results, nor would it yield a significant difference in fit. However, the multivariate results should generally be regarded as superior, because they use more information. Fig. 2A causal covariance contingency model of the longitudinal relationship between conduct problems and marijuana use. Note: Superscript i refers to factors for initiation. Superscript p relates to factors specific to progression. 95% confidence intervals appear in brackets The strongest beta-paths were observed within traits, between initiation and progression variables for both conduct problems and marijuana use. No significant effects were observed between conduct problems and later marijuana use (β range = 0.04–0.17). In the context of strong effects from initiation to frequency of marijuana use (β = 0.75), associations between conduct problems and the frequency of marijuana use were relatively weak. As the bivariate analysis described above suggested however, there were also relatively high levels of covariation in the liabilities for presence and borderline/abnormal conduct problems. This may have affected the power of either variable to predict initiation of marijuana use. To investigate this possibility, nested models were analysed to test the effect of dropping pathways between initiation and progression constructs and between conduct problems and marijuana use. Models were fitted whereby the paths between liability to conduct problems, severity of conduct problems, liability to marijuana initiation and frequency of use were dropped in turn and the deterioration in chi-square fit compared to a model in which all possible path coefficients from Time 1 conduct problems to Time 2 marijuana use were estimated (see Table 2 for model fitting results). These results showed that when the path from liability to conduct problems and severity of conduct problems was dropped there was a significant reduction in model fit, compared to a full model. This was also the case when the path from liability to marijuana initiation and frequency of use was dropped. When the path from borderline/abnormal conduct problems to initiation of marijuana use was dropped, the pathway from presence of conduct problems to the initiation of marijuana use became statistically significant (β = 0.23, CI = 0.13, 0.33). Likewise, when the path from presence of conduct problems to initiation of marijuana use was dropped, the pathway from borderline/abnormal conduct problems to the initiation of marijuana use also became statistically significant (β = 0.24, CI = 0.22, 0.35). Inspecting the deterioration in model fit when each of these two paths was dropped indicated that when the pathway from presence of conduct problems to the initiation of marijuana use was dropped, there was a trend toward a significant reduction in model fit, (χ2 = 3.02, df = 1, P < 0.10). In contrast, when the pathway from borderline/abnormal conduct problems to the initiation of marijuana use was dropped, a significant drop in model fit was not observed (χ2 = 0.17, df = 1, P > 0.10). Table 2Multivariate model fitting for relations between conduct problems and marijuana useEstimated modelBeta coefficient set at zero-2LLdfχ2Model 1-Full theoretical model–5433.9455124–Model 2β 2 15438.72051254.78*Model 3β 3 15436.96951253.02Model 4β 4 15433.84751250.10Model 5β 3 25433.77151250.17Model 6β 4 25433.82551250.12Model 7β 4 35442.25851258.31**Note: * P < 0.05, ** P < 0.01 Discussion This study is among the first to investigate the relationship between the initiation and progression dimensions of substance use with an adolescent twin sample and highlights that the initiation and progression of marijuana use, although strongly related, cannot be assumed to have a single liability of risk. The findings of this study also provide insight into the relationship between conduct problems and adolescent substance use. Specifically, the results indicate that in a UK sample, the longitudinal association between the liability to conduct problems during childhood and early adolescence and the liability to initiate and progress marijuana use in adolescence and early adulthood are not strongly related. Thus, other risk factors and the mechanisms through which they exert effects on the initiation and progression of marijuana use in adolescence should also be investigated. Consistent with previous research examining the relationship between initiation and progression (or use and abuse) of substances in adult populations, additive genetic, common and non-shared environmental influences were found for initiation of marijuana while factors specific to progression were influenced by genetic and non-shared environment with no role for common environment (e.g. Agrawal et al. 2005; Heath et al. 1997; Kendler and Prescott 1998; Koopmans et al. 1999). These findings concur with the view that genetically influenced biological mechanisms appear to play a more important role in the aetiology of problem use and substance dependence (Kendler et al. 1999; van den Bree 2005). Interestingly, when conduct problems were introduced as predictors of marijuana initiation and progression in the multivariate model, there was a slight reduction in the genetic estimate and an increase in the environmental influence. This was particularly the case for initiation of marijuana use. It is also interesting to note that while not independent, the initiation and frequency of marijuana use, to some degree, represent different liabilities of risk. Conceptually, this indicates that the use of marijuana by adolescents does not always and inevitably lead to more frequent use and that there may be different risk factors underlying initiation and progression to more frequent marijuana use. Studies into risk factors that predispose individuals to continued use of marijuana after first experimentation with the drug will be helpful in increasing insight into which adolescents are at greatest risk for problem use. For example, van den Bree and Pickworth (2005) found that some risk factors predict both initiation of experimental marijuana use and progression to regular use, while others are specific to each stage. The results for conduct problems indicated that while the presence or absence of conduct problems were largely heritable, the relative severity of conduct problems, categorised as ‘borderline’ and ‘abnormal’ appeared to be more strongly environmentally influenced. However, we cannot rule out the possibility of greater measurement error at the upper end of the distribution contributing to a large non-shared environment estimate. The beta coefficient between the two dimensions suggested that they mostly, but not entirely overlap (β = 0.74; see Table 1), with wide 95% CIs (0.22, 0.95). Conceptually and epidemiologically, recent evidence (e.g. Pickles et al. 2001; van den Oord et al. 2003) indicates that conduct problems may be best regarded as a single liability of risk where there is no demarcation between normality and psychopathology (Rutter 2003). The categorization of conduct problems into dichotomous variables may have affected the power to predict later marijuana use. Preliminary analyses indicated that conduct problems assessed as a continuous variable predicted later marijuana use, although the strength of association was not large (β = 0.19; P < 0.01). Likewise, model tests in which presence and severity of conduct problems were assessed separately indicated small but significant effects from conduct problems to the initiation of marijuana use. Although the trend toward a significant drop in model fit when the path from severity of conduct problems to initiation of marijuana use was removed suggests that it is the presence rather than severity of conduct problems that is important in the prediction of marijuana use initiation, this is speculative and should be replicated in independent samples of larger number. Moreover, it will be interesting in the future (as these models become available) to repeat these analyses including conduct problems as a dimensional measure with a single liability. The findings of the present study also suggest that this modelling technique could be applied to questions relating to the transition from symptoms to diagnosis, particularly when longitudinal data are available to examine this relationship over time (see Neale et al. 2006). A limitation of the present study was that the results of both the bivariate and multivariate model tests had wide confidence intervals around the progression variables (borderline/abnormal conduct problems and frequency of marijuana use). These possibly reflect the low frequencies of adolescents with high levels of conduct problems and who had initiated marijuana use. For instance, preliminary analyses indicated that adolescents who did not respond at Time 2 (2004) had higher levels of conduct problems at Time 1 (1996). Thus, the present analyses may represent a conservative estimate of the influence of conduct problems on later initiation and progression of marijuana use. In addition, heterogeneity in the relationship between conduct problems and marijuana use as a function of the wide age range of the sample, together with a reliance on a single item to index marijuana use, may have affected the power to detect an association. In addition, in light of the strong relations between the initiation and progression variables, the cross-trait paths are relatively small and larger sample sizes than we currently have available are needed to obtain more conclusive nested model fitting results for the cross-trait paths. Finally, it should be noted that while the present study provides insights regarding the phenotypic relationship between conduct problems and the initiation and progression of marijuana use in adolescence together with sources of genetic and environmental influence on each index of conduct problems and marijuana use, it does not assess genetic and environmental sources of covariance between these behaviours (see Neale et al. 2006). In addition to addressing the caveats outlined above therefore, an important direction for further research is to assess these influences. Previous research found sex differences in the prevalence of marijuana use (greater use among males; Johnston et al. 2005) and in the genetic and environmental influences on adolescent male and female marijuana initiation (e.g. Rhee et al. 2003). Recent research has also identified sex differences in the pattern of relations between the severity of conduct problems and the later initiation of marijuana use (Pedersen et al. 2001). The binary nature of the study variables together with the sample size did not permit model tests incorporating sex differences. Nevertheless, an important direction for future research is to test causal common contingent models for sex differences in the pattern of relations between conduct problems and marijuana use in an adolescent age group. Notwithstanding these limitations, the present study extends previous research by investigating longitudinal relationships between conduct problems and marijuana use during adolescence using a modelling approach that enabled age appropriate adjustments to estimates of the relationship between the initiation and progression dimensions of substance use. The findings are also consistent with previous research indicating stronger associations between conduct problems and substance use within than across-time (e.g. Miles et al. 2002; Silberg et al. 2003). As such, they suggest that interventions aimed at helping children with conduct problems in the years preceding the onset of substance use are unlikely to reduce the risk of marijuana use in adolescence and young adulthood. Other risk factors need to be investigated. An important direction for future research will be to develop models that can test these processes with greater precision. For example, models are required that permit the testing of risk factors assessed using continuous measures as predictors of initiation and progression of substance use and the investigation of moderators of the liability to initiate and progress in substance use when these reflect independent liabilities of risk. The findings of this prospective, longitudinal study represent a first step in examining the risk factors that influence the initiation of substance use and the progression to more frequent use during adolescence and young adulthood.

Mar_Biotechnol_(NY)-3-1-2100433

Understanding Marine Mussel Adhesion

In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are water-impervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion. Introduction to Biological Adhesion Biomimetics Biomimetic materials are designed to mimic compounds with a biological origin. The biomaterial development field is actively pursuing the use of various synthetic and natural protein- and carbohydrate-containing compounds. The production of ecologically friendly materials also is of importance as a result of a rapid increase in the world’s population and the subsequent environmental problems associated with product wastes (Arora and Arora 2004). The key interest in biomimetic adhesives is the interaction between the material and a surface, whether that surface is of biological or nonbiological origin. Scientific studies in the interdisciplinary field of biomaterial research include areas such as surface characterization, protein adsorption, and adhesion behavior (Kirkpatrick et al. 1997). Table 1 briefly describes biomolecules with adhesive properties used for biomimetic research. Many of the proteins are scleroproteins, which contribute mechanical strength to supporting structures in animals. Waite (1983a, b) has commented on the durability of these proteins, in terms of relative insolubility, resistance to proteolytic hydrolysis, or other chemical dissolution. Repetitive amino acid motifs and intermolecular and intramolecular cross-linking are common; DOPA-containing scleroproteins produced through posttranslational modification of tyrosine by various organisms leads to further stabilization or curing of scleroproteins involved in adhesion and/or protection of more flexible structural proteins. Table 1.Research targets for biomaterial developmentBiological TargetDescriptionKeratinA hard, durable insoluble, structural protein that is the primary component of horns, hoofs, feathers, skin, hair, and nails; a scleroproteinElastinAn insoluble protein found in connective tissue and known for its elasticity and similarity to collagen; a scleroproteinCollagenA tough, insoluble, inelastic protein with high tensile strength that serves as the support structure in skin, tendons, and bone; a scleroproteinSilksHigh tensile strength protein fibers that contain various proteins (fibroin, spidroin); most commonly from spiders and silkwormsFibrin (and other coagulation system proteins)A sticky, insoluble, clot-forming protein formed by constituents in the blood; a scleroproteinChitinA specialized carbohydrate containing nitrogen (nitrogenous polysaccharide); found in the cell walls of certain fungi and in the exoskeletons of arthropodsCelluloseAn insoluble complex carbohydrate (polysaccharide composed of linked glucose units); main constituent of the cell walls of plantsMucinA nitrogenous, conjugated protein (protein linked to a sugar) found in mucous secretions; acts as a lubricant and protects body surfaces The production of an underwater adhesive that mimics the properties of marine mussels is a challenge that has received considerable attention. Many of the mussel adhesive proteins identified to date are polyphenolic proteins. Polyphenolic proteins are nontoxic, biodegradable, and have low immunogenic qualities that make them highly attractive for environmental, medical, and industrial purposes. Biological Adhesion Naturally produced adhesives are common in many biological systems and are known for their superior strength and durability compared with man-made materials. Examples of specialized biological systems that generate a vast amount of adhesives research include bacteria, spiders, marine tubeworms, sea cucumbers, barnacles, and mussels. Many bacteria synthesize exopolysaccharides—extracellular protective adhesive matrixes. Bacteria can form layers, or biofilms, on a variety of surfaces by embedding cells in exopolysaccharides. The polysaccharide-based holdfast of the aquatic bacterium Caulobacter crescentus has been shown recently to demonstrate the strongest adhesive force measured in bacteria (Tsang et al. 2006). Spiders also express adhesive proteins by synthesizing different types of silk, all of which have high tensile strength, extensibility, and an energy-dissipative viscoelastic response that is not matched by synthetic polymers (Zhou et al. 2001; Vollrath 2000). Current research with spider silks involves the production of spider silk in other organisms, a technique known as recombinant protein expression (Piruzian et al. 2003). Many marine organisms have developed adhesive strategies to deal with the dynamic ocean environment, particularly at the tidal interface. Marine invertebrates attach permanently or temporarily to inanimate, and sometimes living, surfaces. Therefore, adhesive mechanisms have evolved to promote attachment. The marine polychaete Phragmatopoma californica is a tubeworm that builds protective “tubes” with secreted proteinaceous cement mixed with shells and sand particles from the sea floor. The cement adheres rapidly to a variety of materials in seawater (Stewart et al. 2004; Zhao et al. 2005). The sea cucumber (Holothuria forskali) reacts defensively through the discharge of Cuvierian tubules that ensnare their threat. Biochemical analysis of the tubules indicates a 3:2 protein to carbohydrate ratio, with a high proportion of highly insoluble protein. The soluble protein component appears to contain up to ten glycine and acidic amino acid-rich proteins ranging in size from 17 to 220 kilodaltons (DeMoor et al. 2003). Barnacles adhere directly and permanently to surfaces, such as underwater substrata (ship hulls, oil platforms, and pipelines). Initial attachment of barnacle cyprid larvae is via o-quinone cross-linking that resembles the dihydroxyphenylalanine (DOPA)-containing adhesive proteins of Mytilus spp. (Wiegemann 2005), but the adult barnacle cement is substantially different—comprised of three groups of proteins that contain high levels of the amino acids serine, threonine, glycine, and alanine (Kamino et al. 2000; Wiegemann 2005). A pattern of short, alternating regions of hydrophobic and hydrophilic residues throughout the largest of the three groups of proteins has been noted and suggested that these alternating motifs may have a role in assembly in seawater (Kamino et al. 2000). Marine and freshwater mussel adhesive proteins differ from barnacle cement proteins because of the presence of repetitive amino acid motifs (characterized by a high polyphenolic content), high levels of the modified amino acid 3,4-DOPA, and chemical modifications (hydroxylations) to specific amino acids (Burzio et al. 1997). At least ten different adhesive-related proteins from the marine mussel M. edulis have been identified, reflecting the greater ease of working with the adhesive structures of this organism and also its popularity as a target for biomimetic research. Marine mussels, such as the blue mussel, M. edulis, attach to a variety of surfaces in an aqueous environment by using a natural adhesive that is incredibly strong and durable. There are no synthetic glues that can be similarly applied in an aqueous environment and are impervious to water and turbulent forces. Previous research has shown that one of the proteins in the adhesive, Mytilus edulis foot protein 1 (Mefp-1), bonds to glass, plastic, wood, concrete, and Teflon®. Nine other adhesive-related proteins from M. edulis have been identified to date. A tenth is implicated but has not been isolated. The precise mechanism for assembly of the ten proteins-Mefp-1, -2, -3, -4, -5; collagens such as precollagen-D, -P (variant P22 and P33), and -NG; proximal matrix thread protein (PMTP-1 and -1a); and a polyphenol oxidase—is not understood. There also may be additional proteins involved in the formation of the adhesive. Figure 1 illustrates adhesion of M. edulis to seaweed, other mussels, and a stainless steel surface. Figure 1M. edulis attachment to (a) seaweed, (b) other mussels, and (c) a stainless steel surface. Adhesion Mechanisms in Mussels The mollusk byssus evolved to anchor postlarval organisms during metamorphosis. Some species of mussels have retained the byssus in the adult animal, permitting a transition from a bottom-dwelling existence to an epibenthic lifestyle. Within the Mytilidae, examples of mussels across this range of marine lifestyles can be observed, from the burrowing Geukensia demissa, to Mytilus edulis and Bathymodiolus childressi, which live entirely exposed attached to hard surfaces, and intermediate species, such as Modiulus modiolus, which can survive under mixed conditions (Brazee and Carrington 2006). Common features of the byssus in the Mytilidae include a root attached to the byssal retractor muscle, a stem extending from the root, and individual byssal threads which are attached to overlapping cuffs of the root. The strength of mussel adhesive scleroproteins is attributed to the introduction of cross-links between polymer chains of individual adhesive proteins (a process called “curing”). Curing, or hardening, of the polyphenolic mussel proteins is believed to require a catecholic precursor (e.g., DOPA) and the presence of a catechol oxidase (Waite 1990). Individual adhesive proteins from mussels are produced by the foot organ of the animal. The proteins are stockpiled in the foot then secreted or released into the byssal groove, which creates a template for thread and plaque formation to form strong attachments underwater. The reactive, oxidized form of DOPA, quinone, is thought to provide the moisture-resistance characteristic of mussel underwater adhesion (Yu and Deming 1998; Yu et al. 1999). DOPA is formed from the hydroxylation of tyrosine residues by a polyphenoloxidase (tyrosinase). DOPA can complex with metal ions and oxides (Fe3+, Mn3+) and semimetals, such as silicon, thus explaining its ability to adhere to rocks and glass (Sever et al. 2004). Sun and Waite (2005) have reported an incremental posttranslational modification of tyrosine to DOPA from the proximal end to distal tips of threads, coupled with a selective localization of metals (aluminum, calcium, iron, and silicon) and an associated iron gradient. The sequestering of iron occurs during feeding rather than after threads have been formed. Thus, iron is incorporated in a chemical gradient pattern similar to that of DOPA during secretion of byssal threads and plaques. The identification of 12 variants of the DOPA-rich adhesive foot protein 3 from Mytilus californianus (Mcfp-3) further supports the intricate role of DOPA in marine mussel adhesion (Zhao et al. 2006). The catecholic content of mussel adhesive proteins has been linked to hydrogen-bonding and metal-liganding (strong chelating) capabilities (Monahan and Wilker 2003; Deming 1999). Examples of proteins undergoing the o-quinone intermediate process (quinone tanning) include collagen (mussel byssal threads), cellulose, chitin, and mineral deposits (mussel shells). Figure 2 illustrates the chemical steps for hydroxylation of tyrosine and DOPA residues in M. edulis polyphenolic proteins. Figure 2Hydroxylation of tyrosine residues in M. edulis polyphenolic proteins. Other constituents of mussel adhesive proteins include lysine and glycine. Lysine may contribute to adhesion via ionic bonding to negatively charged surfaces, such as collagen and acidic polysaccharides (Suci and Geesey 2000; Olivieri et al. 2002), and intermolecular cross-linking with o-quinones. Glycine may contribute to adhesion through the open, extended conformation it imparts on protein structures. Histidine, an amino acid exhibiting a gradient pattern and present in high concentrations in the distal region of byssal threads, has been correlated to transition metal content (zinc or copper) and adhesion in marine mussels (Waite et al. 2004). The exceptional strength of mussel adhesive proteins is undoubtedly the result of the repetitive nature of many of the individual proteins (decapeptide and hexapeptide repeats in Mefp-1 and hydroxyproline repeats in collagens), the modification of individual amino acids (e.g., hydroxylation of proline and tyrosine), and the gradient nature of byssal attachment devices. This gradient nature was first noted by Brown in 1952 (referenced by Brazee and Carrington 2006), who observed corrugated proximal (to the organism) and smooth distal regions. The development of our understanding of the transition of mechanical properties along the thread length, from the more flexible proximal region close to the soft tissues of the mussel, through the collagenous main body of the thread, to the stiffer, less extensible distal region, and the exquisite fine tuning of protein components to impart these properties, has been concisely reviewed recently (Brazee and Carrington 2006). Total mussel adhesive strength is a function of the individual adhesive proteins secreted by the animal, their association with other proteins, their distribution along the thread, and their proximity to materials of differing moduli. Details of the individual adhesive protein components are described in the next section. Components of Mussel Adhesion Attachment in Mussels: The Byssus The byssus, an exogenous attachment structure, was first described in 1711 (Brown 1952). High concentrations of polyphenolic proteins (e.g., DOPA), the presence of collagen, and the presence of a catechol oxidase were among the first biochemical observations of byssal attachments. Environmental factors, such as salinity, temperature, pH, season, and substratum choice, as well as biological factors, such as age and metabolic state of the animal, affect the efficiency and strength of byssal attachment to surfaces (Van Winkle 1970; Crisp et al. 1985; Carrington 2002). The stem is rooted in the byssal retractor muscles at the base of the foot organ. The byssal threads, flexible structures of variable dimensions (∼0.1 mm diameter, 2–4 cm length) and strength, originate from the stem. A byssal thread consists of a flexible, collagenous inner core surrounded by a hardened, cured polyphenolic protein. Numerous researchers photographed the collagen core in the 1930s (Brown 1952), well before three unique collagenous proteins were identified and characterized by Qin and Waite (1998, 1995). The outer polyphenolic protein, believed to undergo a quinone tanning-type reaction with a specialized catechol oxidase enzyme, is Mefp-1. Designation of this byssal thread polyphenolic adhesive protein, as well as subsequent adhesive proteins identified in Mytilus edulis, is preceded by the genus and species. The byssal structure culminates in a polyphasic plaque of varying size, dependent on both the size of the animal and the age of the byssus (Crisp et al. 1985). Plaques are commonly only ∼0.15 mm in diameter where they meet the thread and ∼2- to 3-mm diameter at the substrate interface. Plaque formation occurs from the deposition of proteins that originate in the foot organ. To date, five specialized adhesive proteins have been identified in byssal plaques from M. edulis: Mefp-1, -2, -3, -4, and -5. Figure 3 illustrates the anatomy of M. edulis and the associated byssus structures. Figure 4 illustrates the location of the adhesive-related proteins identified in the byssus of M. edulis. Figure 3Anatomy of M. edulis mussel and byssus structures.Figure 4Location of adhesive-related proteins identified in the byssus of M. edulis. Mechanical Properties of Mussels The most extensive research about the adhesive mechanical properties of mussels has been with M. edulis. The breaking energy of intact byssal threads is reported to be 12.5 × 106 Jm−3 versus tendon (2 × 106 Jm−3 to 5 × 106 Jm−3) and silk (50 × 106 Jm−3 to 180 × 106 Jm−3) (Denny 1988). Bond strengths are between 0.1 to 10 × 106 Nm−2, depending on the surface for adhesion (Waite 1999). Byssal thread strength at the distal portion of threads is as strong as a vertebrate tendon but three to five times more extensible (Qin and Waite 1998). Byssal thread strength at the proximal portion of threads is weaker but 15 to 20 times more extensible. Strain energy density of threads approaches that of silk, approximately six times tougher than a tendon (Smeathers and Vincent 1979; Coyne et al. 1997). Byssal threads can recover initial length and stiffness if given sufficient relaxation time (Bell and Gosline 1996). Mefp-1, the most studied mussel adhesive protein, has adhesive properties comparable to synthetic cyanoacrylate and epoxy resins. These resin adhesives are popular because of their high bond strengths, quick polymerization, and ability to bond to a number of substrates (metals, glass, ceramic) (Savla 1977; Coover and McIntire 1977). Research related to the mechanical properties of individual M. edulis adhesive proteins is limited because of the difficulty in obtaining large quantities of isolated proteins for adhesive testing. Byssal Thread Proteins Byssal Thread Polyphenolic Protein: Mefp-1 Mefp-1 was the first polyphenolic protein to be identified in the mussel byssus (Waite and Tanzer 1981; Waite 1983b). The primary location of Mefp-1 is in the byssal threads, cross-linked via a polyphenol oxidase to form a hardened sheath around the flexible, collagenous inner core. Byssal plaques contain ∼5% of Mefp-1 as well. Mefp-1 is a large (897 amino acids), basic hydrophilic protein with very little secondary structure and a molecular mass of 115 kDa, based on mass spectroscopy (Filpula et al. 1990). Decapeptide and hexapeptide repeats containing numerous posttranslational modifications (∼60–70% of the amino acid residues are hydroxylated) provided the first indication of an adhesive-related protein unlike any others identified in nature. The hexapeptide repeat is made up of the amino acids AKPTYK. The major decapeptide consensus repeat, consisting of the amino acids AKPSYP’P’’TYK (in which P represents proline, Y represents DOPA, P’ represents trans-2,3-cis-3,4-dihydroxyproline, and P’’ represents trans-4-hydroxy-L-proline) occurs ∼80 times in Mefp-1. DOPA residues constitute 10–15% of the protein (Waite 1983b). The open conformation of the protein is believed to allow functional groups full accessibility for cross-linking interactions with other proteins and a variety of surfaces, including glass, Teflon®, and metals. Mepf-1 has been characterized as possessing random coil conformations with helix-like decapeptide segments under physiologic conditions (Haemers et al. 2005). Our laboratory recently has deduced complementary DNA (cDNA) sequences for Mefp-1 (GenBank Accession number: AY845258 and AY845259). Other Mytilus mussel species contain a protein analogous to Mefp-1, with differences in the decapeptide repeat frequency, residue composition, and nonrepetitive regions; these species include M. galloprovincialis (Mgfp-1) (Inoue and Odo 1994); M. coruscus (Mcfp-1) (Inoue et al. 1996b); M. trossulus (Inoue et al. 1995b); M. californianus Conrad, California mussel (Waite 1986); M. chilensis (Pardo et al. 1990); and M. sp. JHX-2002 (Wang et al. 2004). Mefp-1–like adhesive proteins from mussels outside of the Mytilus genus also have been identified: Dreissena polymorpha, zebra mussel (Dpfp-1) (Rzepecki and Waite 1993; Anderson and Waite 2000); Dreissena bugensis, quagga mussel (Dbfp-1) (Anderson and Waite 2002); Perna viridis, green mussel (Pvfp-1) (Ohkawa et al. 2004); Perna canaliculus, green shell mussel (Pcfp-1) (Zhao and Waite 2005); Guekensia demissa, ribbed mussel (Waite et al. 1989); Limnoperna fortunei, Asian freshwater mussel (Lffp-1) (Ohkawa et al. 1999); Aulacomya ater (Saez et al. 1991); and Choromytilus chorus (Pardo et al. 1990). Polyphenolic proteins from other marine mussels also have been compared with Mefp-1 (Rzepecki et al. 1991). Mefp-1 requires oxidization of tyrosine residues by catechol oxidase, tyrosinase, or sodium periodate for conversion to the reactive DOPA residues required for strong adhesion. The enzyme catalyzing oxidation may serve as an oxidative agent and as a copolymer. Molecular oxygen can be used to further oxidize DOPA to a quinone. Possible cross-linking agents are oxygen, polyvalent metal ions (Fe3+ and Al3+), aldehydes, and many types of bi/polyfunctional cross-linkers. The hydroxylation of DOPA from incubation with tyrosinase has been shown to form 3-, 4-, 5-trihydroxyphenylalanine (TOPA), in a decapeptide related to Mefp-1 (Burzio and Waite 2002). The prospective functions of TOPA isomers have included metal binding, wound healing, and adhesion. Mefp-1 has been previously commercialized as a source for mussel adhesive protein. Companies supplying Mefp-1 have obtained adhesive protein from the byssal structures by using protein extraction techniques (Sigma-Aldrich: “Adhesive Protein”; Swedish BioScience Laboratory: “MAP”; BD Biosiences Clontech: Cell-Tak™) and recombinant protein techniques using synthetic gene constructs (Genex Corp.: “AdheraCell”). However, currently there are no commercial sources for pure Mefp-1 because of the high cost of extraction methods and inconsistencies in quality of protein from recombinant protein techniques (Cell-Tak™ Cell and Tissue Adhesive is a formulation of multiple polyphenolic proteins from Mytilus edulis). The laboratory-prepared products have not demonstrated comparable strength to the natural protein. Our laboratory is currently conducting research on the large-scale production of recombinant Mefp-1 protein (Silverman and Roberto 2006a). Byssal Thread Polyphenol Oxidase The Enzyme Commission of the International Union of Biochemistry classifies each enzyme into six major groups according to the reactions catalyzed. Enzymes are given specific nomenclature, with the letters “EC” prefacing the specific subclass with its major grouping (http://www.chem.qmul.ac.uk/iubmb/enzyme/). Polyphenol oxidases fall under the broad enzyme grouping of “oxidoreductase.” They are oxygen transferring, copper metalloproteins having both catecholase (act on diphenols) and cresolase (act on monophenols) activity. Polyphenol oxidases catalyze the orthohydroxylation of phenols to catechols and the dehydrogenation of catechols to orthoquinones. A monophenol monooxygenase (EC 1.14.18.1), more traditionally called tyrosinase, can oxidize tyrosine to L-DOPA and L-DOPA to o-quinone (Worthington 1993). A catechol oxidase (EC 1.10.3.1) can oxidize L-DOPA to o-quinone. The enzymatic formation of o-quinone is a browning/tanning/curing reaction that occurs in plants, bacteria, and animals (Waite 1990). The adhesive properties of marine mussels have been demonstrated by metal complex formation of DOPA and the intermolecular cross-linking of o-quinone with lysine residues. Very little definitive information exists about the polyphenol oxidase(s) present in M. edulis that is responsible for tyrosine and DOPA conversions in byssus formation. A cytochemical study of the enzyme gland in the foot organ of M. edulis demonstrated phenol oxidase activity in the Golgi complex but not the secretory granules (Zuccarello 1981). Waite (1985) measured catechol oxidase activity from enzyme gland extractions and whole byssus structures. The native enzyme was shown to prefer diphenols over monophenols as substrates. A single, purified protein was not obtained, and the enzyme was estimated to have subunits and a molecular weight of 120 kDa. Burzio (1996) extracted two catechol oxidases from M. edulis byssal threads, plaques, and feet. Both the byssal catechol oxidase-high molecular weight (BCO-H, 174 kDa) and byssal catechol oxidase-low molecular weight (BCO-L, 60 kDa) isozymes only oxidized catechols to o-quinones, and their amino acid composition was similar to other catechol oxidases found in nature. Again, homogenous purification was not possible. Recently, Hellio et al. (2000) purified a 34 kDa phenol oxidase monomer from mussel feet. The substrate specificity of the enzyme was greatest toward dihydroxyphenols (L-DOPA and catechol), followed by trihydroxyphenols, suggesting that there may be at least two forms of phenol oxidase involved in mussel adhesion. Extremely low relative activity was observed with monohydroxyphenols. Thus, the specific identification of the polyphenol oxidase enzyme(s) involved in byssus formation continues to elude researchers. Various compounds have been found to inhibit the activity of the M. edulis phenol oxidases identified above: oxygen competitors (cyanide and nitrogen), metal chelators (diethyldithiocarbamate (DETC) and ethylenediaminetetraacetic acid (EDTA)), a reducing agent (L-ascorbic acid), and the competitive inhibitors benzoic acid and L-DOPA, with respect to 4-methylcatechol and L-DOPA (Waite 1985; Burzio 1996; Hellio et al. 2000). There is currently extensive research in the area of anti-fouling methods to prevent attachment of marine and/or freshwater mussels to surfaces. These studies rely on understanding the biochemical properties of mussel adhesive proteins, including polyphenol oxidase, and the surface chemistry of paints, varnishes, and other materials. Byssal Thread Collagens All collagens in nature contain a triple-helical domain with the sequence repeat (Gly-X-Y)n, in which Gly is glycine, X often is proline, and Y is usually hydroxyproline. This triple-helical configuration provides mechanical strength but on its own offers little flexibility and elasticity. The collagens present in M. edulis byssal threads contain additional sequence domains that make the byssal thread approximately six times tougher than the human Achilles tendon (Smeathers and Vincent 1979; Coyne et al. 1997). Two M. edulis collagens appear in a gradient fashion in byssal threads, and a third is distributed evenly throughout the thread. Analogous byssal collagens have been identified in M. galloprovincialis (Lucas et al. 2002; Hassenkam et al. 2004). Proximal Collagen (Col-P) The proximal region of a byssal thread (the region closest to the animal) consists of a unique, natural co-polymer, termed proximal collagen (Col-P) (Coyne et al. 1997; Qin and Waite 1995). This protein is an incredible shock absorber with 160% extensibility. The precursor collagen (PreCol-P) has a molecular mass of 95 kDa and consists of seven domains: representative amino and carboxyl termini; a large, central collagenous domain (40 kDa) flanked by elastin-like domains (11 kDa each); and small, histidine-rich domains (5 kDa) that flank the elastin-like domains. A small acidic patch is present between the collagen and elastin-like domains at the carboxy end of the protein. The collagen domain, coupled with the presence of the two, elastin-like domains, provides for an incredibly tough and extensible proximal region in byssal threads. Histidine-rich domains are believed to function in metal binding. The transition metal zinc has been detected in M. edulis byssi, supporting theories of Zn2+-mediated cross-linking in the terminal domains of Col-P. Two variants of PreCol-P have been implicated in molecular dovetailing between the proximal byssal thread and the byssal stem (P22 and P33) (Coyne and Waite 2000). The presence of these variants suggests a mechanism in which mussels can mitigate stresses by overlapping different proteins, a phenomenon that occurs between tendon and bone (Fukuta et al. 1998) and in spiders from frame silk (web frame and radial supports) to viscid (glue-covered) silks (Guerette et al. 1996). Distal Collagen (Col-D) The distal region of a byssal thread contains another natural co-polymer: distal collagen (Col-D). The Col-D is stiffer than the Col-P. The precursor collagen (PreCol-D) (molecular mass 97 kDa) also contains seven separate domains: representative amino and carboxyl termini; a central, collagenous domain larger than PreCol-P (45.5 kDa); silk fibroin-like domains flanking the collagen domain (5 and 15 kDa); and small, histidine-rich domains (7 and 5 kDa) that flank the silk-fibroin domains. A large collagen domain (175 repeats of Gly-X-Y), a small, acidic patch, and the histidine-rich domains are similar to the PreCol-P structure. The presence of silk-fibroin domains allow for extensibility in PreCol-D, as is the case for elastin in PreCol-P. However, the distal region of byssal threads is localized in straight bundles (strong and stiff), whereas the collagen fibers are coiled in the proximal region (pliable and elastic) (Waite et al. 1998). Before the characterization of PreCol-D, silk fibroin domains had been previously reported only in arthropods. Pepsin-resistant Nongradient Collagen (Col-NG) The entire length of a byssal thread contains a third block copolymer-like protein, pepsin-resistant nongradient collagen (Col-NG) (Qin and Waite 1998). The precursor pepsin-resistant nongradient collagen (PreCol-NG) (molecular mass 76 kDa) is believed to function as a mediator between PreCol-D and PreCol-P. It consists of a central collagenous domain (∼38 kDa), an acidic patch, and histidine-rich termini, similar to PreCol-D and PreCol-P. The regions flanking the collagen domain contain (X-Glyn)m repeats (which are similar motifs found in plant cell walls) in addition to the familiar silk fibroin-like domains that are present in PreCol-D. There is a larger distribution of tyrosine in the histidine domain of the N- and C- terminal sequences of PreCol-NG compared to the other two collagens. The elastic domains of PreCol-P, the silk fibroin-like domains of PreCol-D, and the plant cell wall-like domains of PreCol-NG characterize the unique, collagenous block copolymers found in the byssal threads of M. edulis. Proximal Thread Matrix Protein (PTMP) The proximal portion of each byssal thread contains a water-soluble, noncollagenous protein designated proximal thread matrix protein (PTMP; Sun et al. 2002). This ∼50 kDa matrix protein has a capacity for collagen binding and resembles the von Willebrand factor in amino acid sequence, antigenicity, and its stiffening effect. Two variants have been identified in M. edulis (PTMP1a and PTMP1b). A similar PTMP1-like cDNA has been found in M. galloprovincialis. Byssal Plaque Polyphenolic Proteins Mefp-2 Mefp-2 is found exclusively in byssal plaques, constituting from 25% to 40% of the total plaque proteins. Unlike Mefp-1, Mefp-2 is a smaller adhesive protein (molecular mass 42–47 kDa) with only 2 to 3 mol% DOPA and no hydroxylation of proline to trans-2,3-cis-3,4-dihydroxyproline or trans-4-hydroxy-L-proline. The DOPA residues occur primarily in the N- and C- terminal regions of the protein. Mefp-2 contains considerable secondary structure and is relatively resistant to a variety of proteases (compared with Mefp-1), an important characteristic for integrity of the byssal plaque. The high cysteine content (6–7 mol%), coupled with tandem, repetitive motifs similar to epidermal growth factor, suggests that Mefp-2 is an adhesive protein with a stabilization role in the byssus (Inoue et al. 1995a). A Mefp-2 multigene family may exist, based on evidence that at least three different repetitive motifs have been identified in the primary protein sequence (Rzepecki et al. 1992). A published full-length gene sequence for Mefp-2 is not available. However, an investigation of Mefp-2 cDNA sequences identified by our laboratory supports the multigene family premise (GenBank Submission numbers: AY845260, AY845261, and AY845262). Our laboratory is currently conducting research on the large-scale production of recombinant Mefp-2 protein (Silverman and Roberto 2006b). As is the case with Mefp-1, other mussel species contain proteins analogous to Mefp-2: M. galloprovincialis (Mgfp-2) (Inoue et al. 1995b); M. coruscus (Mcfp-2) (Inoue et al. 2000); and D. polymorpha (Dpfp-2) (Rzepecki and Waite 1993). Stewart et al. (2004) reported that the tube cement of Phragmatopoma californica, a marine polychaete, forms solid foam (similar to Mefp-2) via cross-linking with DOPA. Mefp-3 Mefp-3 is the smallest byssal adhesive protein identified to date, with a molecular mass of ∼5 to 7 kDa (Papov et al. 1995; Warner and Waite 1999). Mefp-3 contains no repeats, 20 to 25 mol% DOPA, and a prevalence of 4-hydroxyarginine and tryptophan residues. Warner and Waite (1999) identified 20 gene variants (∼0.3 kB) of Mefp-3 in the foot organ; however, only four or five proteins have actually been detected in plaques deposited on glass or plastic. The presence of a gene family for Mefp-3 supports the primer-like function proposed for the protein in adhering to substrata. One hypothesis has been that deposition of a specific Mefp-3 variant is dependent on the surface used for attachment. However, Floriolli et al. (2000) reported no correlation between Mefp-3 expression and surface type (stainless steel, glass, polyethylene) in an individual mussel. Differences in cDNA transcripts were found between animals. Zhao et al. (2006) reported 12 different Mcfp-3 variants from a mussel population—broken into “fast” or “slow” electrophoretic characteristics—upon exposure to glass coverslips. The molecular mechanism(s) for the numerous Mefp-3 and Mcfp-3 variants is not known. Variables, such as exposure time to surfaces, water temperature, and the age of the animals, could influence expression levels. As is the case with Mefp-1 and Mefp-2, other mussel species contain proteins analogous to Mefp-3 variants: M. galloprovincialis (Mgfp-3A and Mgfp-3B) (Inoue et al. 1996a) and M. californianus (Mcfp-3–12 variants, mentioned above) (Zhao et al. 2006). Mefp-4 Mefp-4 is another protein identified in byssal plaques, with a molecular mass of 79 kDa (Weaver 1998; Vreeland et al. 1998; Warner and Waite 1999). Mefp-4 contains elevated levels of glycine, arginine, and histidine, as well as 4 mol% DOPA. A unique tyrosine-rich octapeptide is present, with variations in residue substitutions giving rise to a family of proteins. This very large protein most likely serves as a coupling agent in the thread-plaque junction designated by the precollagens and the byssal plaque protein Mefp-2. A gene sequence for Mefp-4 has not been identified; however, two-foot protein variants from M. californianus have recently been identified (Mcfp-4; Zhao and Waite 2006a). Mefp-5 Mefp-5 is the most recent adhesive-related byssal plaque protein identified from Mytilus edulis (Waite and Qin 2001). Mefp-5 is a relatively small protein with a molecular mass of 9.5 kDa, a 27 mol% DOPA content, and the presence of phosphoserine. Phosphoserine is known to occur in acidic mineral-binding motifs of proteins that bind calcareous materials (e.g., osteopontin); therefore, its presence in byssal plaques may aid in adhesion of one animal to a neighboring mussel’s shell. Mefp-5 was formerly associated with the Mefp-3 family of variants, and similarly, plays an interfacial role as a primer for substrate adhesion. An adhesive protein analogous to Mefp-5 has recently been reported to be present in M. galloprovincialis (Mgfp-5) (Hwang et al. 2004) and M. californianus (Mcfp-5-2 variants) (Zhao and Waite 2006b). Discovery of Additional Foot Proteins: Mcfp-6 Mcfp-6 was identified along with Mcfp-5 by Zhao and Waite (2006b; 3 variants). This small protein (11.6 kDa) contains a relatively large amount of tyrosine and a small amount of DOPA. Its suggested role in adhesion may be to provide a link between the DOPA-rich proteins and the plaque proteins present in byssal attachment plaques. Adhesive Testing of Mussel Proteins A wide variety of adhesive tests have been applied to intact byssal threads, plaque, portions of threads, or materials bonded or coated with individual adhesive proteins. Gross comparative tests on byssal threads were described earlier in this review. Mussels seem to prefer surfaces with higher critical surface energy, a phenomenon also observed in barnacles (Crisp et al. 1985; Waite 1987). Plaques were shown to attach more strongly to slate and glass than plastic acetal (acetate), paraffin wax, and polytetrafluoroethylene (PTFE). The high-energy, hydrophilic surfaces (glass, slate) had smaller plaque surfaces attached to substrates and smaller contact angles compared with the low-energy, hydrophobic surfaces (wax, PTFE). Attachment Failure Byssal attachments can fail in a number of ways. A byssal plaque can cleanly peel away from a surface, the byssal thread itself can tear or fail, or the root can tear away from the animal. Failure of the thread is the most common cause of failure of byssal attachment. If byssal threads break or tear away from the animal, the true adhesive force is underestimated. Therefore, when analyzing adhesive forces, it is important to consider the type of failure. The surface for attachment, source of foot protein, age of animal, temperature, and season are important in evaluating the strength of byssal attachments (Crisp et al. 1985). Adhesive Techniques Numerous surfaces and techniques are used to evaluate the mechanical properties of mussel adhesive proteins. Atomic force microscopy (AFM; for surface topography), attenuated total reflection fourier transform infrared spectroscopy (ATR/FT-IR; for information on molecular composition, bonding, conformation and orientation with respect to interfaces), surface plasmon resonance (SPR; to study interactions of samples and surfaces), and tensometer methods (for tensile strength and elastic properties) are common techniques used in adhesives research. Table 2 provides examples of studies in which surfaces have been tested with mussel adhesive proteins or synthetic analogs containing repetitive motifs from mussel proteins Table 2.Examples of materials testing with mussel adhesive proteins or synthetic analogs containing repetitive motifs from mussel proteinsSurfaces TestedMussel Protein or Synthetic AnalogReferencesSlateSynthetic recombinant Mefp-1Crisp et al. 1985Silica (glass)Plastic acetal (acetate)Paraffin waxPolytetrafluoroethylene (PTFE)PolystyreneRecombinant Mefp-1Filpula et al. 1990Limestone/dolomite cobbleD. bugensis and D. polymorphaAckerman et al. 1992Mild steelStainless steelMarine concreteAckerman et al. 1995Marine plywoodPolyvinyl chloridePolymethylmethacrylate (Plexiglas®)Ackerman et al. 1996AluminumTeflon®D. bugensis and D. polymorphaDormon et al. 1997ConcreteMild steelPolyvinyl chlorideStainless steelSiliconeSynthetic recombinant Mefp-1Kitamura et al. 1999SilicaPolyethylene terephthalate (PET)Teflon®AluminumSynthetic polypeptide mimics of marine adhesivesYu and Deming 1998SteelSilicaPlasticsMicroporous apatite surfaceMefp-1Shirkhanzadeh 1998SilicaSynthetic homo- and copolypeptides of marine and related adhesive proteins (M. edulis, M. californianus, A. ater, G. demissa, liver fluke, pearl oyster)Yamamoto et al. 1999Polytetrafluoroethylene (PTFE)Teflon®NylonIronSoda glassMethyl- and oligo (ethylene oxide)-terminated, self-assembled monolayersMefp-1 (Cell-Tak™) and fibrinogenHarder et al. 2000Germanium (oxide)Mefp-1, Mefp-2, and polylysineSuci and Geesey 2000PolystyrenePoly octadecyl methacrylateSuci and Geesey 2001SilicaMefp-1Frank and Belfort 2001Porcine skinM. edulis mussel feet extractNinan et al. 2003SilicaRecombinant Mgfp-5 and Mefp-1 (Cell-Tak™)Hwang et al. 2004Polymethylmethacrylate (Plexiglas®)PolystyreneAluminum Brazee and Carrington (2006) recently provided an elegant and detailed study of whole byssal thread material properties comparing threads from a range of Mytilidae. This study also compared their results to those of previous studies, along with statistics to quantify the variation in the results. Quasi-static tensile strength tests and dynamic testing of elastic properties (extensibility, modulus, resilience, and recovery) were performed and correlated with morphometric measurements, including shell weight and dimensions, and thread morphometry. Several interesting findings were reported, including the elliptical cross-section (rather than a regular, round section) of the byssal thread, novel behavior of Modiolus modiolus threads (“double yield” under strain), the surprising strength of Dreissena polymorpha threads (although they lack a collagen core), and the overall greater strength of Mytilis californianus threads (suggested to be a factor in its success in living in the strong tidal environment of the Pacific coast). The application of atomic force microscopy (AFM) is revolutionizing our understanding of the interactions of mussel adhesive proteins with surfaces and with other adhesive proteins that make up the byssal thread. It is now possible to measure adhesion energy and shear forces of individual adhesive proteins, such as has been done in comparing M. edulis foot proteins 1 and 3 (Lin et al. 2007). Even more remarkably, the cantilevers of an AFM can be coated with DOPA residues to perform single molecule measurements and quantify the differences in bond strength that might be expected between unmodified and DOPA-modified adhesives (Lee et al. 2006). As our ability to perform sensitive measurements with small amounts of protein improves, along with the increased resolution of the techniques used, we can anticipate that our understanding of the interactions of adhesive proteins necessary to achieve robust adhesion will increase. Future Supply of Adhesive Proteins by Recombinant Approaches Background An impediment to further understanding the unique adhesion system of M. edulis is the lack of availability of the individual protein components. Large quantities of M. edulis (or other mussel) adhesive proteins are needed to perform research and development for commercial adhesives. Current methods for obtaining the adhesive proteins of the mussel byssus rely largely on excision of the byssus followed by extraction of the proteins under acidic conditions. However, the chemical extraction process does not always yield pure or individual adhesive proteins. Approximately 10,000 M. edulis mussels are needed to produce 1 g of Mefp-1 adhesive from byssal structures (Strausberg and Link 1990; Hwang et al. 2007a). At least 100 mg of material is needed for performing small, conventional adhesive tests, such as tensile strength or wall-jet analyses. The sacrifice of such a large number of animals is neither environmentally friendly nor economically practical. Therefore, an alternate approach to obtain large amounts of adhesive protein components is necessary for further conventional adhesive testing formats and adhesive formulation development. Molecular and microbiology techniques can be used for the directed production of large quantities of many different proteins. Prokaryotes and eukaryotes are used as hosts in recombinant protein expression systems. Common prokaryote hosts include bacteria (Escherichia coli), Saccharomyces cerevisiae (baker’s yeast), Pichia pastoris (a methylotrophic yeast), or Kluyveromyces lactis (a lactic-acid–producing yeast). Eukaryote hosts include plants (tobacco, potatoes), trees (loblolly-pine), and mammals (rabbit, mouse, goat). Cell culture techniques use insect, plant, and mammalian cells or tissue cultures rather than whole organisms. Variables important for successful recombinant protein expression systems include a codon-usage–compatible host; promoter, transcriptional, and translational regulators; fine-tuned cultivation methods; targeted recombinant protein purification methodologies; posttranslational modification ability; and three-dimensional configuration of the recombinant protein. Codon usage refers to the predominant DNA bases that code for specific amino acids. Codon analysis can be performed for the source organism across all its known proteins or only for the specific protein or protein class of interest. Possible choices for mussel adhesive recombinant protein expression include a eukaryotic host, a special bacterial line (E. coli BL21 strains), or a plant source, such as tobacco (because plant cell wall proteins are repetitive proteins with hydroxylations). Promoter and transcriptional regulators are DNA sequences that direct gene expression. They can be native to the host, artificially added to the host DNA, or incorporated in vector systems. Translational stop signals can be incorporated into the DNA genes or are present on an expression vector. Prokaryotes and eukaryotes use different regulators for gene expression. Cultivation factors, such as energy sources, aeration (oxygen), temperature, and induction protocols are specific to the host organism or cell type and are dependent on the quantity used for production. Small-scale cultivation generally involves flasks or petri dishes and volumes less than 1 L. Larger-scale cultivation in the research laboratory setting can use bioreactors with volumes as large as 100 L. Automated monitoring and control of cultivation variables is used in large-scale recombinant protein production. Heterologous recombinant protein production occurs simultaneously with the host’s production of its own proteins. Thus, harvesting of a recombinant protein involves its identification and separation from the host’s native proteins. Special tags that can be incorporated with a recombinant protein include specific amino acid repeats (histidine), fluorescent molecules (green fluorescent protein), biotinylation, or antibody recognition sequences (epitopes). The specific chemical properties of proteins also can be used for isolation and purification. For M. edulis adhesive proteins Mefp-1 and Mefp-2, acidic conditions in purification steps will exclude many of the host’s native proteins while maintaining the integrity of the mussel proteins. One caveat in heterologous protein production, however, is that correctly folded or modified proteins are not always produced. Prokaryotes and eukaryotes can modify their proteins following translation with specific disulfide bond formation for correct protein folding, cleavage of precursor protein forms to yield functional proteins, glycosylation of amino acid residues for protein stability, and other modifications to amino acids, such as phosphorylation, acetylation, or hydroxylation (as in the case with many M. edulis adhesive proteins). Posttranslational modifications do not always occur naturally in heterologous systems; therefore, in vitro methods often are required to obtain properly modified proteins. For example, heterologously produced, unhydroxylated Mefp-1 requires treatment with mushroom or bacterial tyrosinase to produce hydroxylated tyrosine (DOPA) at positions two and nine of the decapeptide repeat (Strausberg and Link 1990; Filpula et al. 1990; Kitamura et al. 1999). Other considerations for biologically functional, recombinant proteins include hyperglycosylation, inclusion bodies, and loss of expression plasmids with a scale-up in cultivation volumes. The Recombinant Protein Approach The recombinant expression of cDNAs or synthetic adhesive-related proteins modeled from organisms other than mussels has been reported: human blood coagulation protein Factor XIIIa in the yeast S. cerevisiae (Broker et al. 1991) and collagen and spider silk proteins in bacteria, yeast, insect, and mammalian cells and plants (Fahnestock et al. 2000; Kieliszewski and Lamport 1994; Scheller et al. 2001). Researchers have, to some extent, successfully produced Mytilus mussel adhesive proteins in bacteria, yeast, mammalian cells, and plants. Filpula et al. (1990) used a hybrid combination of promoters to direct the production of Mefp-1-like tandem repeats in S. cerevisiae. A single copy of the genomic cDNA clone for Mefp-1 encoded the carboxy terminus, including 19 decapeptide and one hexapeptide repeat sequences. Synthetic gene constructs carrying between one and four copies of a portion of the genomic sequence for Mefp-1 were shown to express proteins between 20 to 100 kDa. Amino acid analyses of the four different recombinant proteins expressed in the yeast system were similar to unhydroxylated natural Mefp-1. The recombinant protein constituted 2% to 5% of the total cell protein, and adhesive tests showed water-resistant bonding after in vitro modification of tyrosine residues to DOPA and then quinone. Salerno and Goldberg (1993) expressed a synthetic mussel adhesive protein analog in E. coli using a T7 promoter system and a repetitive gene cassette that encoded the consensus decapeptide repeat of Mefp-1. The synthetic polydecapeptide was produced from a 600 base pair gene (20 decapeptide repeats) and constituted up to 60% of total cell protein. Kitamura et al. (1999) also expressed a synthetic mussel adhesive protein analog in E. coli with a T7 promoter system and a repetitive gene cassette that encoded six repeats of the consensus decapeptide of Mefp-1. The researchers were able to obtain 10 mg of the model peptide per liter of growth medium. Surface strength and contact-angle were measured and used to calculate the value of work of adhesion (WA) for both the unmodified and mushroom-tyrosinase–treated peptide. Adhesion to silicone, glass, polyethylene terephthalate, and Teflon® substrates was found to be dependent on sodium chloride concentration (e.g., sea water). Our laboratory has patented a technique for cloning and expressing recombinant Mefp-1 and Mefp-2 in S. cerevisiae from cDNA clones (Silverman and Roberto 2006a, b). We are currently producing rMefp-1, rMefp-2, and rMefp-3 in 20-L fermentation batches (unpublished data). Purification of each individual recombinant protein is in progress. The purified products will be analyzed by mass spectrometry (MS) for total intact mass followed by MS analysis (post-protease digestion). Adhesive studies will be performed in-house and/or through interested parties after the confirmation of rMefp-1, rMefp-2, and rMefp-3. Recombinant mussel adhesive proteins from M. galloprovincialis also have been produced. Takeuchi et al. (1999) cultured mussel foot cells from M. galloprovincialis in Petri dishes. Gene-specific probes were used to identify the stage of development for different adhesive proteins. Probes for the genes encoding Mgfp-1, -2, and -3 were used to demonstrate that the expression of the byssal protein genes in M. galloprovincialis is morphologically programmed (Takeuchi et al. 1997). The findings suggested that a cell culture system for in vitro expression of byssal adhesive proteins might be a plausible alternative to yeast- or bacterial-based expression systems. Hwang et al. (2004) produced a soluble, recombinant Mgfp-5 fused with a histidine tag in E. coli. A purification yield of ∼7% was obtained from a 3 L working volume because of difficulties in removing the recombinant protein from the chromatographic resin used to bind histidine proteins. The adhesive properties of recombinant Mgfp-5 were compared to Cell-Tak™ by using atomic force microscopy (AFM) , material surface coating (on glass, polymethylmethacrylate, polystyrene, a silicone-based antifouling agent-coated slide, and aluminum), and quartz crystal microbalance (QCM) techniques. Recombinant Mgfp-5 was shown to have adhesive abilities comparable to—and sometimes exceeding—those of Cell-Tak™. The adhesion force of tyrosinase-treated Mgfp-5 (∼981 nN) was higher than both tyrosinase-treated Cell-Tak™ (∼302 nN) and untreated Cell-Tak™ (∼624 nN). The adhesion ability of recombinant Mgfp-5 to a glass slide and a poly-(methyl methacrylate) plate was comparable to Cell-Tak™ but better than Cell-Tak™ on a silicone-based antifouling agent coated slide. And, adsorption of Mgfp-5 to a gold surface was comparable to Cell-Tak™. Hwang et al. (2005) also produced recombinant Mgfp-3 (variant A) with a histidine tag in E. coli. The purification yield and solubility exceeded that of the above-mentioned recombinant Mgfp-5. The adsorption and adhesion force were comparable to Cell-Tak™ but less than recombinant Mgfp-5 (e.g. ∼230 nN for Mgfp-3, ∼240 nN for Cell-Tak™, and ∼550 nN for Mgfp-5). Hwang and colleagues noted the low production yields, low purification yields, and high levels of insolubility of rMgfp-3 and rMgfp-5 and sought to improve the technique for recombinant protein expression by designing novel fusion proteins (Hwang et al. 2007a, b). The first hybrid MAP mussel bioadhesive—fp-151—was produced from a fusion protein containing six Mgfp-1 decapeptide repeats at both the N- and C-termini of Mgfp-5 (Hwang et al. 2007a). The second hybrid MAP mussel bioadhesive—fp-151-RGD—was a fusion of the GRGDSP residues found in fibronectin (designated RGD) to the C-terminus of fp-151 (Hwang et al. 2007b). The authors reported greater production yields, easier purification, and improved solubility for both novel fusion proteins fp-151 and fp-151-RGD. In addition, the cell adhesion and spreading abilities of fp-151-RGD were superior to Cell-Tak™, poly-L-lysine (PLL), a noncoated surface, and fp-151—regardless of mammalian cell type tested (human or hamster). The first non-Mytilus recombinant mussel adhesive protein, the Mefp-1–like adhesive protein Dpfp-1 from the zebra mussel D. polymorpha, was expressed by Anderson and Waite (2000). Recombinant Dpfp-1 was produced as a maltose-fusion protein in E. coli. The recombinant protein was then used as an antigen for polyclonal antibody production. Immunologic studies definitively revealed the presence of Dpfp-1 in foot organs and byssal threads of D. polymorpha. Whole plants have been used to synthesize foreign/recombinant proteins, such as antibodies, vaccines, and industrial enzymes (Doran 2000). An alternative to the use of whole plants is the use of plant tissue cultures. Tissue cultures provide a technology that may gain momentum because of the ease in manipulation of culture conditions for greater foreign protein levels. There is considerable expense in cell culture techniques, indicating that this scheme would not be optimal for industrial production. Tobacco and rice are the most commonly used plant cell cultures used to date. Correct posttranslational modification is an issue when using plants as hosts, similar to bacterial or yeast expression systems. However, plants may be very good hosts for production of the repetitive mussel adhesive proteins. Plant cell walls contain large, repetitive proteins similar to foot protein-1 analogs and the diverse collagens found in byssal threads. Dr. Simon McQueen-Mason (Department of Biology, University of York, United Kingdom) has investigated the ability of transgenic tobacco plants to produce recombinant spider silks and Mgfp-1 because of their similarity to the repetitive plant cell wall proteins expansin and extensin (http://www.cnap.org.uk/). His group continues to research expansins and pectins from various plant species (Jones et al. 2005). Recently, Patel et al. (2007) reported the use of elastin-like polypeptide fusions (an ELP tag) to enhance the accumulation of a range of different recombinant proteins (human interleukin-10, murine interleukin-4, and the native major ampullate spidroin protein 2 gene from the spider Nephilla clavipes) in the leaves of a transgenic tobacco plant. The fusion protein concentrations in the plant leaves were significantly higher than the target recombinant proteins alone in all instances, suggesting that this type of fusion tag may be beneficial for producing large, repetitive recombinant proteins in plant hosts. Current and Future Research Areas Related to Mussel Adhesion and Adhesives Commercialization of Mussel Adhesive Proteins Numerous economic factors are important in the production and synthesis of foreign proteins, whether using microbial cell culture, animal cell culture, plant tissue culture, transgenic plants, or transgenic animals. Production costs (yield for cost comparisons), safety issues (for therapeutic use), and stability of the product (the potential for the protein to degrade or lose function during extraction/purification procedures) are a few issues that require careful analysis before the method is chosen. Regulatory issues relevant to Good Manufacturing Practice (GMP) for production of therapeutic proteins must be followed. Any product containing recombinant mussel adhesive protein will require extensive testing and validation from health, environmental, and adhesives industries before commercialization. The underwater adhesion of the marine mussel, M. edulis, has intrigued scientists for decades. Extensive progress has been made in elucidating the mechanisms responsible for adhesion, in understanding adhesion to a variety of substrates, and in producing recombinant proteins and materials that mimic the natural mussel adhesive. A selection of current and future research areas related to mussel adhesives is discussed below. Biofouling In 1987, mussel biofouling cost the Navy more than $200M in the areas of hull scraping and excess fuel consumption from unnecessary drag on large vessels (Morgan 1990). Understandably, the Office of Naval Research and other organizations have invested considerably in the areas of antifouling and foul-release. Approaches to antifouling and bivalve control can generally be classified as chemical or physical (hull scraping) methodologies. Chemical methods have received the most attention. Until recently, bis(tributyltin)oxide was a popular antifouling component of boat paints. However, heavy metal-based and organotin coatings are now restricted because they are toxic to marine and freshwater organisms. Synthetic and natural biodegradable compounds are currently being investigated for their efficacy as antifouling agents. Imides (special types of amino acid-based compounds), terpenoids (multicyclic structures with basic carbon skeletons) from brown alga species and a marine sponge, proteases from a marine bacterium, phloroglucinol compounds (white, crystalline phenols) from Eucalyptus, and a variety of chemicals have all been shown to inhibit mussel attachment to surfaces (Zentz et al. 2001; Hellio et al. 2001; Sera et al. 1999; Venkateswaran and Dohmoto 2000; Cope et al. 1997). Experimentation with low-adhesion surfaces has included modified glass (Yamamoto et al. 1997), polyethylene glycol (PEG) (Dalsin et al. 2003), and various other polymeric surfaces (Frank and Belfort 2001). Antifouling and detachment research related to mussels and other sessile organisms is vitally important in addressing the economical problems associated with biofouling (Taylor 2006; Bellas 2006; Bellas et al. 2005). Mussels from Extreme Environments Hydrothermal vents and cold seeps from the Gulf of Mexico and the Japan Trench are home to unique combinations of sea life. New classes of animals, plants, and bacteria are rapidly being identified with the aid of special underwater submersibles (ALVIN, the Johnson Sea Link) and advances in molecular biology and microbiology. Bathymodiolinae is a subfamily of mussels that contains species harboring methanotrophic, chemoautotrophic, and/or both types of bacterial endosymbionts within the mussel’s gill tissue. Geological forces have caused cracks in the continental slope, allowing oil, natural gases, such as methane and hydrogen sulfide, and related substances to seep into the ocean environments. Bathymodiolinae mussels and other organisms from the Gulf of Mexico have been able to survive under anoxic, high-pressure, and high-saline (brine) environments for centuries. Figure 5 illustrates the large shells and byssal plaques and threads from Bathymodiolus childressi mussels obtained by Dr. Charles Fisher from Brine Pool NR1 in the Gulf of Mexico (MacDonald and Fisher 1996) compared with M. edulis (Figure 5a and c). The foot organs of M. edulis and B. childressi and M. edulis (Figure 5b and d) also are shown for comparison. The morphologic differences in the foot organ, shell, and gill characteristics between the two marine mussel species, as well as the vast differences between the environmental conditions in which the two reside, may be important variables affecting byssus characteristics (material properties, adhesion mechanisms, individual proteins, etc.; Brazee and Carrington 2006). The identification of adhesive proteins from extreme mussels is an interesting task for prospective research. Figure 5Gross appearance and internal organs of B. childressi and M. edulis mussels: (a) B. childressi shell, thread, and plaques, (b) B. childressi foot organ, (c) M. edulis shell, thread, and plaques, (d) M. edulis foot organ. Novel Applications for Mussel Adhesive Collagens and Polyphenol Oxidase Immobilization of mussel adhesive proteins (Mefp class and collagens) on solid supports may be exploited for uses in the design of biosensors, immunosensors, or artificial tissue scaffoldings and constructs: Mefp-1 with glucose (Saby and Luong 1998; Newman and Setford 2006), Mefp-1 with human chorionic gonadotrophin (Burzio et al. 1996), collagens as supports (BD™ Three-Dimensional Collagen Composite Scaffold; BD Biosciences; San Jose, CA). The identification and subsequent use of the polyphenol oxidase derived from M. edulis byssal structures also could be exploited as a thickening agent for numerous industries/applications (Yamada et al. 2000). Medical and Dental Adhesives Current adhesives approved for medical use in the United States include fibrin sealants, albumin-based compounds, glutaraldehyde glues, cyanoacrylates, hydrogels, and collagen-based compounds. Fibrin sealants are the most prevalent. They consist primarily of thrombin and fibrinogen, clotting factors in blood. Fibrin sealants have a wide range of uses and are bioabsorbed by the body. However, risks of products isolated from blood, albeit minimal, are a concern with this class of tissue adhesives. One albumin-based compound approved for use in the United States is BioGlue® Surgical Adhesive (CryoLife Inc.; Kennesaw, GA). It is glutaraldehyde-based, or gelatin-resorcinol-formaldehyde-glutaraldehyde-like, glue (without the formaldehyde) that has limited U.S. Food and Drug Administration approval because of known immunoreactivity problems. The cyanoacrylates approved for use in the United States are stronger than the fibrin sealants but not bioabsorbable. Their use is restricted to external or temporary applications because of associations with carcinogenicity, inflammation, and infection. Hydrogels are water-soluble polyethylene glycol polymers that are activated by light. Both protein-based and DOPA-based hydrogel formulations are being researched (Nowak et al. 2002; Mo et al. 2006; Liu and Li 2002). Currently, their use is limited because of the time required for application, setting, and bioabsorption. Collagen-based adhesives are the newest type of tissue adhesive available (Koob and Hernandez 2002). So far they work well and are inexpensive, thus adding an alternative to the use of fibrin sealants. Dr. Robin L. Garrell (Organic Chemistry Department, University of California, Los Angeles [UCLA]) has worked on elucidating the chemical mechanisms for adhesion and cohesion in Mefp-1 and other marine biopolymers to develop artificial tissues and bioreactors (Ooka and Garrell 2000; http://www.chem.ucla.edu/dept/Organic/garrell.html). Future dental and medical adhesives may contain mussel adhesive proteins and/or domains from mussel adhesives (Tay and Pashley 2002; Ninan et al. 2003; Fulkerson et al. 1990; Robin et al. 1988; Schmidt et al. 1994). Conclusions Scientists and laymen alike have been fascinated for years with the ability of mussels to cling to surfaces under water. The ability to adhere in an aqueous environment, withstand numerous environmental forces, and resist conventional approaches to detachment are factors that continue to intrigue researchers today. During the last two decades, considerable time and effort has been spent in identifying the proteins that contribute to underwater adhesion by marine mussels. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to develop formulations for adhesives in which there exist endless applications for the commercialization of water-impervious, ecologically safe adhesives derived from mussels.

Bioinformation-1-6-1891690

DoD2006: molecular biology database update

DoD2006, an updated version of Database of Databases is an online resource maintained collectively by ProGene Biosciences and Department of Inorganic and Analytical Chemistry, Andhra University. It links to all molecular biology databases that appeared in Nucleic Acids Research 2006 database issue. DoD2006 includes 873 databases, of which, 858 are derived from Nucleic Acids Research database issue and 15 are collected from In Silico Biology, Bioinformation journals and Google Scholar search. Each database has a search option, keyword help and a brief description with direct link to the database home page. The database is freely available online at http://www.progenebio.in/DoD/index.htm Background Online availability of databases is increasing due to their utility by structural and molecular biologists. These databases are generated using a variety of programming languages and they differ in content and access methods. [1] The information explosion in biology resulted in database proliferation and this necessitates the importance of data integration, storage and retrieval. Many new databases tend to appear every year, with ever increasing information or knowledge from biology, driven by genome sequencing projects and easy internet access. [2] On the contrary, fund crunching is a limitation for data collection, constant updates, infrastructure facilities and maintenance of databases. Biological databases with infrastructural funding are hosted by academic, governmental organizations and industry as well as non-profit organizations. [3] Collection and analysis of data from various databases is essential for computational biologists. But it is time consuming and proportional with increasing number of databases. In this context, there is a need to integrate all databases at a single interface, usually with one search engine to query all databases. Therefore, we partially address the issue by maintaining the molecular biology databases at a single interface, with links to their respective pages. In practice, there are both legal and technical impediments to cross-database communication which include digital locks, firewalls and the inability of search engines to recognize the data. [4] Methodology DoD2006 Content DoD2006 is an updated version of DoD. [5] It is a cross-database interface with 858 databases collected from Nucleic Acids Research [6], Bioinformatics [7], BMC Bioinformatics [8] and other journals [9] and 15 databases collected from In Silico Biology [10], Bioinformation [11] journals and Google Scholar [12] search. The current update with 873 databases against 719 reported in 2005 [5] represents additional databases that continue to grow despite the demise of a few. [13] A small number of inaccessible databases (MitoPD, ISSD [14]) reported in 2005 are unchanged in DoD2006 so as to enable the users to know the type of database that once existed. The complete list of databases is given in the website [15] and their categorization is given in Table 1. During the search, results are displayed in a separate window as this helps in further database scan without losing the home page. The screen-shot of nucleotide database is given in Figure 1. A hyperlink to ‘Keyword help’ lists keywords used to scan respective databases and the description of different links is given in our earlier publication. [5] Features of DoD2006 Newly appended databases in DoD2006 are distinguished from the existing ones with a ‘+’ sign and inaccessible ones are recognized by a ‘*’ sign before the name. All databases in each category are given alphabetically. The appended databases are colored blue and the remaining in green color. Those who wish to have their database to be listed can contact the authors. DoD2006 provides an excellent and essential resource for molecular and computational biologists. Research through computational tools and software using databases has been recognized as a front-line research coupled with experimental design. Our database suits to deliver the wide biological content through the cross-database interface. Updates to DoD shall be made on yearly basis.

Photosynth_Res-3-1-2117338

Chlorophylls, ligands and assembly of light-harvesting complexes in chloroplasts

Chlorophyll (Chl) b serves an essential function in accumulation of light-harvesting complexes (LHCs) in plants. In this article, this role of Chl b is explored by considering the properties of Chls and the ligands with which they interact in the complexes. The overall properties of the Chls, not only their spectral features, are altered as consequences of chemical modifications on the periphery of the molecules. Important modifications are introduction of oxygen atoms at specific locations and reduction or desaturation of sidechains. These modifications influence formation of coordination bonds by which the central Mg atom, the Lewis acid, of Chl molecules interacts with amino acid sidechains, as the Lewis base, in proteins. Chl a is a versatile Lewis acid and interacts principally with imidazole groups but also with sidechain amides and water. The 7-formyl group on Chl b withdraws electron density toward the periphery of the molecule and consequently the positive Mg is less shielded by the molecular electron cloud than in Chl a. Chl b thus tends to form electrostatic bonds with Lewis bases with a fixed dipole, such as water and, in particular, peptide backbone carbonyl groups. The coordination bonds are enhanced by H-bonds between the protein and the 7-formyl group. These additional strong interactions with Chl b are necessary to achieve assembly of stable LHCs. Introduction The dramatic developmental transformation performed by the chloroplast has attracted broad interest over the past several decades (see Hoober and Argyroudi-Akoyunoglou 2004; Wise and Hoober 2006, for reviews). Although the organelle displays a variety of features among different organisms, as revealed by electron microscopy, its monophyletic origin by endosymbiosis of an ancient cyanobacterium has received increasingly strong support (Palmer 2003; Bhattacharya and Medlin 2004; Rodriguez-Ezpeleta et al. 2005). Descendents of the primary endosymbiotic event branched into the glaucophytes, green algae, and plants, which contain chlorophyll (Chl) a and Chl b, and the red algae, which contain only Chl a. Tomitani et al. (1999) provided evidence that the genes for chlorophyllide a oxygenase (CAO), the enzyme that catalyzes conversion of chlorophyllide (Chlide) a to Chlide b, also have a common origin. The evolutionary relationship of CAO in plants with the enzyme in the cyanobacterial prochlorophytes indicates that the original ancestor of plastids contained Chl b and that modern cyanobacteria, along with the red algae, lost this ability. Divergence from a secondary endosymbiotic event, also apparently singular, of a red alga gave rise to four major groups of chromophyte algae, the dinoflagellates, heterokonts, haptophytes, and cryptophytes, that contain Chl c as a major pigment in addition to Chl a (Bachvaroff et al. 2005; Shalchian-Tabrizi et al. 2006). This lineage suggests that the same fundamental mechanisms should underlie processes in chloroplast development in all plant species. Among these are expected to be the mechanisms that guide the interactions of Chls, proteins, and lipids during assembly of the thylakoid membrane. Even with the extensive studies already done on these processes, our understanding of many details of these mechanisms remains clouded. This article will consider physicochemical factors that are likely fundamental in the assembly of light-harvesting complexes (LHCs) in the plastids of eukaryotic organisms. Particular emphasis is given to the properties of Chls a, b, and c and their interactions with ligands. Chls b and c occur essentially exclusively in LHCs. Whereas Chl a is ubiquitous, it alone is not sufficient for LHC assembly. Chl c seems to play the same role in LHC assembly in chromophyte algae as Chl b does in green algae and plants (Durnford et al. 1999; De Martino et al. 2000; Goss et al. 2000). Thus a principal question is the step in LHC assembly for which synthesis of Chl b is required. It is likely that the plastids derived from the secondary endosymbiotic event solved this problem by finding a pathway to Chl c. The Chl-binding proteins in these organisms are evolutionarily related to those in green algae and plants (Schmitt et al. 1994; Green and Durnford 1996). The relatively simple LHCs are well-defined structures and consequently are excellent systems to search for basic mechanisms. It is useful for illustration purposes to include Chl d, the most recently characterized member of the Chl family (Miyashita et al. 1997; Akiyama et al. 2002) found as the major Chl in the cyanobacterium Acaryochloris marina, which functions in core complexes in an analogous fashion to Chl a. Bacteriochlorophyll (BChl) a is also briefly mentioned for comparison. Summary of differences in chlorophylls The spectral differences between the species of Chls in chloroplasts expand the ability of photosynthetic organisms to harvest light. However, the spectral range of Chl a alone is broadened by various micro-environments within complexes with proteins (Nishigaki et al. 2001; Croce et al. 2002; Linnanto et al. 2006), which argues against spectral differences per se as the raison d’être for occurrence of the “secondary” Chls, in particular, Chls b and c. Overall properties, not only their spectral features, are altered as consequences of chemical modifications on the periphery of the molecules. Important modifications are desaturation of sidechains and introduction of oxygen atoms at specific locations. Oxygen is the most electronegative atom commonly found in biological systems and exerts significant effects on the electronic distribution in the Chl molecule. We previously (Hoober and Eggink 1999; Eggink et al. 2001, 2004; Chen et al. 2005) proposed that modifications at the periphery of the Chl molecule influence the coordination chemistry of the central Mg atom and that this effect plays an important, if not major, role in the interaction of Chl b with LHC apoproteins (LHCPs) and thus in the assembly of LHCs. In particular, we proposed that, as a Lewis acid, the Mg atom in Chl b favors axial coordination bonds with harder Lewis bases than does Chl a. This proposal was supported experimentally by direct measurement of equilibrium constants of various tetrapyrrole derivatives with specific ligands (Tamiaki et al. 1998). In an unbiased chemical context, Chls a and b should then prefer different ligands. Recent evidence demonstrated, however, that Chl a interacts with a broad range of ligands, from the imidazole group of histidine to water. Chl b, on the other hand, is found only with ligands containing an oxygen atom. Chlorophylls a and d Conversion of 3,8-divinyl-Chl a to 3-monovinyl-Chl a by reduction of the 8-vinyl group to an ethyl group is the final step in Chl a biosynthesis and yields the predominant form of Chl a (Nagata et al. 2005). As a result, Chl a has electron-donating methyl and ethyl groups at positions 7 and 8, respectively (Fig. 1). Along with reduction of the C17–C18 double bond to a single bond, which converts the porphyrin precursor protochlorophyllide (Pchlide) to the chlorin ring system, these groups impose an electron density, from opposite sides of the molecule along the X axis, on the pyrrole nitrogens, which partially shields the positive charge of the central Mg atom. In addition, the 3-vinyl and 131-keto groups exert weak electron withdrawing effects on opposite ends of the Y axis. Fig. 1Structures of the major Chls. Except for the oxidation of the 7-methyl group in Chl a to the formyl group in Chl b, Chls a and b are identical. Chl d contains a formyl group at position 3. Chls a, b, and d include the 20-carbon isoprene alcohol, phytol (Ph), esterified to the carboxyl group at position 173. This carboxyl group remains unesterified in Chl c, which also contains double bonds in the sidechain between positions 171 and 172 and in the macrocycle between carbons 17 and 18. These additional double bonds extend conjugation of the macrocyclic π system to the free carboxyl group. Chl c species differ at positions 7 and 8; c1: 7, –CH3, 8, –C2H5; c2: 7, –CH3, 8, –C2H3; c3: 7, –COOCH3, 8, –C2H3 (shown in figure) The geometrical coordinates of the molecular framework for Chl a are shown in Fig. 1. The primary X axis transects the molecule from the position of C17 to C7. The Y axis transects the molecule from C2 to C12. The experimentally determined, functional Qy transition-moment direction is a vector 70° clockwise from the X axis (Fragata et al. 1988; Simonetto et al. 1999; Sundholm 2003; Cai et al. 2006). This displacement from the geometrical Y axis places the functional Qy vector from near C1 to near C11. The functional Qx vector is within a few degrees of the geometrical X axis. Chl d is synthesized by oxidation of the 3-vinyl group to a formyl group, whose electron-withdrawing character further extends the Qy vector but should also cause the transition direction to align more closely with the molecular Y axis. As a result, the lowest energy absorption band shifts from 665 nm (in methanol) for Chl a to a longer wavelength (lower energy) maximum of 697 nm for Chl d and increases the dipole strength, which is proportional to the molar absorption coefficient. The transition dipole is strengthened further in BChl a, which has an electron-withdrawing acetyl group on C3 and a single bond between C7 and C8. The Qy absorption maximum is shifted to 772 nm (in methanol), with a dipole of 7.2 Debye (D) (at a refractive index for the environment of 1.35) (Knox and Spring 2003). The electronic distribution in BChl a is more symmetrically aligned along the Y axis, with an elliptical electron density, and the Qy transition-moment direction is essentially perpendicular to the X axis, i.e., from C2 to C12 (Sundholm 2003). Chlorophyll b Synthesis of Chl b involves incorporation of the electronegative oxygen atom to generate the 7-formyl group, which, as an aromatic aldehyde, is expected to have a dipole moment for the group of approximately 3.0 D (Desyatnyk et al. 2005). The oxygen provides a significant pull on electrons away from the core of the molecule along the X axis, which weakens the Qy dipole strength of the molecule from 5.33 D for Chl a to 4.41 D in Chl b (at a refractive index for the environment of 1.35) (Knox and Spring 2003). Since the dipole strength determines the magnitude of the absorption coefficients, among other properties, the absorption coefficient of Chl b is only 50–62% (depending on solvent) of the Qy absorption peak of Chl a and is shifted to higher energy, with a maximum at 652 nm (in methanol). The Qy transition-moment direction is displaced further from the Y axis than in Chl a and is at an angle of only 61° clockwise from the X axis (Simonetto et al. 1999), described as a transect from near C20 to near C10. Intuitively, the Qx transition moment of Chl b should be stronger than that of Chl a. Computational analysis of molecular orbitals suggest that the Qx oscillator strength of Chl b is 2-fold greater than for Chl a, with a maximum at 538 nm (Linnanto and Korppi-Tommola 2004). The magnetic circular dichroism (MCD) spectrum of Chl b contains a weak negative transition at 540 nm and an intense negative transition at 625 nm (Frackowiak et al. 1987). From the argument that a stronger molecular dipole is consistent with a longer wavelength absorption maximum, the longer-wavelength negative transition in the MCD spectrum of Chl b may correspond to the Qx transition. The molecular orbital calculations suggest that, with weaker Qy and stronger Qx transitions, the electronic distribution in the Chl b molecule is essentially circular (Hoff and Amesz 1991). The lessened electron density around the pyrrole nitrogen atoms lowers the pK values for these atoms by about two pH units (Phillips 1963; Smith KM 1975). The withdrawal of electron density from the pyrrole nitrogens results in less shielding of the Mg atom of Chl b and allows it to more strongly express its positive point charge. Molecular orbital calculations give the Mg in Chl an atomic charge with a value of +0.7 to +1.3 (Linnanto and Korppi-Tommola 2004). Chlorophyll c Chl c is found with Chl a in chromophyte algae, where it functions as a light-harvesting pigment. Whereas most of these algae do not contain Chl b, some Prasinophycean algae, such as Montoniella squamata, accumulate a Chl c, 3,8-divinyl-Pchlide, along with Chls a and b (Schmitt et al. 1994; Green and Durnford 1996). The red alga that contributed its plastid in the secondary endosymbiotic event apparently lacked Chl c. Ability to make Chl c was the solution these organisms found to solve the problem that was accomplished in chlorophytes with Chl b. As shown in Fig. 1, the structure of Chl c3 has several unique features. The c-type Chls characteristically retain the C17–C18 double bond that occurs in the porphyrin precursor, Pchlide a. In addition, a trans double bond is introduced between C171 and C172 in the sidechain, which extends conjugation of the ring π system to the usually unesterified, electronegative C173 carboxyl group. Conversion of the propionate sidechain of Pchlide a to the acrylate sidechain of Chl c inhibits the ability of NADPH:Pchlide oxidoreductase to reduce the C17–C18 double bond as occurs in Chl a synthesis (Helfrich et al. 2003), which suggests that sidechain desaturation occurs prior to potential interaction of Pchlide a with the oxidoreductase. Thus Chl c retains the porphyrin ring system. The three major sub-types of Chl c (c1, c2 and c3) occur as the result of oxidation of the C7 and/or C8 substituents. The C7 methyl group is modified to a methylcarboxylate (–COOCH3) in Chl c3 and the C8 vinyl group remains unreduced (Porra 1997). These remarkable modifications all lie on the X axis of the molecule. Absorbance spectra of the series (c1, c2 and c3) suggest that the functional Qx transition moment increasingly dominates the long-wavelength absorption peaks (Jeffrey and Wright 1987; Helfrich et al. 2003), further reduces the absorption coefficient and blue-shifts the Qy absorbance maximum to 630 nm for Chl c1 (in acetone). Additional modifications, such as esterification of the acrylate sidechain with galactosyl diacylglycerol, increase the number of minor forms of Chl c (Garrido et al. 2000). A summary of ligands of chlorophylls in light-harvesting complexes Coordination bonds are formed between Lewis acids and bases. A Lewis acid has an unfilled orbital that can accept a pair of electrons. A Lewis base (ligand) has a pair of unshared electrons that are available for donation to the Lewis acid to form a donor-acceptor complex. Lewis acids and bases are characterized as “soft” or “hard” according to their chemical properties (Jensen 1978). Soft species tend to bond by short-range orbital interactions, while hard species interact primarily by electrostatic forces. The central Mg atom of Chl molecules, as the Lewis acid, interacts with proteins by formation of coordination bonds with an amino acid sidechain as the Lewis base. Compression of the electron cloud toward the Y axis of the Chl a molecule, as when the C17–C18 double-bond and C8 vinyl group are reduced, tends to shield the Mg atom and effectively reduces the electronegativity of the metal. This results in weaker interaction with the negative end of a fixed dipole or even repulsion of negatively charged groups. In contrast, in Chl b the Mg atom is less shielded and more strongly expresses its positive charge. Electron density in Chl b is also pulled outward by H-bonds between the 7-formyl group and other structures, which further enhances the Qx transition moment. Substituting the central Mg in BChl a with other metals (e.g., Ni) of greater electronegativity strongly influences the Qx but not the Qy transition energies of the tetrapyrrole molecule (Hartwich et al. 1998). In Chls a and b the metal is the same, but the argument can be applied in reverse, in which substitution of peripheral groups on the Qx axis alters the environment of the central Mg ion and thus its effective electronegativity. If H+ is considered as a Lewis acid, the availability of electrons in a Lewis base should be reflected in its pK value (Jensen 1978) (pK values vary dramatically from those obtained in aqueous media when the ionizable group is located in a nonpolar micro-environment (Mehler er al. 2002)). The electron pair available on an amine nitrogen atom binds H+ strongly (pK ∼9). Although the nonpolar micro-environment within a protein molecule may lower the pK of an amino group of lysine (Gunner et al. 2000, 2006), the amine is usually protonated under physiological conditions, thus positively charged, and the electron pair is not available for coordination with Chl. Lysine amino groups are not ligands in membrane-spanning regions of Chl-binding proteins (Balaban et al. 2002; Ferreira et al. 2004; Liu et al. 2004). The strength of other amino acid sidechains as Lewis bases should then decrease in the order of decreasing pK, e.g., imidazole (pK 7) > carboxylate (pK 4–5) > peptide backbone amide (pK −0.42) > sidechain amide (pK −0.62) > water (pK −1.74). However, formation of Chl–ligand complexes does not follow this pattern. Chl a interacts as expected with imidazole, its predominant partner, but also with amide sidechains and water. Chl b does not interact significantly with imidazole, whereas the peptide backbone carbonyl group and water are favorable ligands. Orbital interactions may play a greater role in coordination bonds with ligands containing accessible electron pairs (i.e., high pK values) such as imidazole, whereas electrostatic interactions are more likely with ligands having a low pK value such as carbonyl groups. This comparison suggests that interactions of the Chls with ligands involve more than simply the availability of non-bonded electrons on the ligand. In addition to differences in the properties of the Lewis bases, the more exposed positively charged Mg ion in Chl b is more electronegative and acts as a harder Lewis acid than the metal in Chl a. Thus Chl b should favor electrostatic bonds with groups containing a strong, fixed dipole. With the exception of the imidazole group, ligands to Chl contain oxygen. Lewis bases that contain oxygen atoms are polar, with a “fixed” dipole, and tend to be “hard” Lewis bases. The structures listed in Table 1, which are common or potential ligands of Chl, are ordered according to increasing dipole moment of the monomeric molecule, as calculated by ab initio methods, to emphasize the importance of this parameter. When these molecules interact by H-bonding with other molecules, as in a solution or a crystal structure, the dipole moments increase (Spackman 1992; Abramov et al. 1999; Whitfield et al. 2006). This effect is unlikely to be significant when ligands are isolated within the nonpolar environment in a membrane, although the dipole of a “polarizable” ligand is affected by its interaction with the Mg of Chl. The dipole moment of an alcohol is too weak to effectively compete with water and thus hydroxyl groups are not common ligands. Each productive ligand is discussed in more detail in the following. Table 1Values of dipole moments selected from the literature for potential ligands of ChlLigandpKaSolution Dipole (D)ab initio Dipole (D)Carboxyl group    Formic acid3–51.41 (University of Southern Maine website 2006)1.52 (Dudev et al. 1999)Alcohol∼ −2 (Herschlag and Jencks 1989; Smith and March 2001)1.70 (University of Southern Maine website 2006)1.94 (Dudev et al. 1999)Water−1.74 (Herschlag and Jencks 1989; Smith and March 2001)2.70 (Gregory et al. 1997)1.868 (Gregory et al. 1997)1.855 (Dyke and Muenter 1973)Imidazole6–73.96 (Spackman 1992)3.66 (Spackman 1992)4.80b (Spackman 1992)Amide    Formamide3.84 (Spackman 1992)4.13 (Dudev et al. 1999)4.83b (Spackman 1992)3.72 (Spackman 1992)    Acetamide−0.62 (Grant et al. 1983)3.87 (Spackman 1992)3.69 (Spackman 1992)4.95b (Spackman 1992)    Sidechain3.46 (Antoine et al. 2002)    Peptide bond4.2 (Gunner et al. 2000)    N-Methylacetamide−0.42 (Grant et al. 1983)4.2 (Whitfield et al. 2006)3.73 (Whitfield et al. 2006)∼6 (Whitfield et al. 2006)Urea0.053 (Grant et al. 1983)5.15 (Abramov et al. 1999)4.56 (Spackman 1992)7.04b (Abramov et al. 1999)a pK for the conjugate acid reaction: AH(+) ↔ A(−) + H+b Crystal formReferences are indicated in parenthesis Water Water is the Lewis base that seems to be a “regulatory” ligand because of its strong interaction with Chl b and its weaker interaction with Chl a (Ballschmitter et al. 1969). In solution, where water is fully H-bonded (dielectric constant, 81), its dipole moment is 2.70 D (Table 1); in ice, this value is 3.09 D (Batista et al. 1998). In an environment in which the dielectric constant is 2–4, as occurs in a protein or membrane, the dipole of a water molecule is likely nearer to that in the gas phase, 1.85 D (Dyke and Muenter 1973). However, when associated with a positive charge such as the Mg in Chl, the dipole moment is probably near the H-bonded value. The charge at the negative end of the dipole of water provides an electrostatic contribution to the interaction. For a functional group in a protein to form a coordination bond with the Mg atom in Chl, a water ligand, which is likely present throughout the latter steps in the biosynthetic pathway from Mg-protoporphyrin IX onward, must be displaced. It is interesting that three Chl b molecules retain water as a ligand and connect with the protein via a water bridge (see below). Imidazole The imidazole sidechain of histidine in the unprotonated form has an unshared pair of electrons on N(3) (designated as Nε2 by Standfuss et al. 2005). H+ binds to the electron pair with a pK value that lies within the range of 5–8, depending upon the environment. Nonpolar environments stabilize the unprotonated form, and thus the electron-rich imidazole group is available for coordination with the Mg of Chl a within a membrane. The dipole moment for imidazole is between 3.66 D (gas phase) and 4.80 D (crystal structure), with the predominant contribution to the dipole provided by the N(1)-H bond (Spackman 1992). When the N(1) hydrogen is replaced with the electron-donating methyl group, the resulting coordination bond at N(3) is stronger (van Gammeren et al. 2004). In aqueous solution, the dipole moment is enhanced to a value of 3.96 D by H-bonding (Table 1). Both N atoms have a small negative charge, and the electron density is distributed nearly symmetrically (Fig. 2). The aromatic character of imidazole allows the dipole to reorganize in response to interaction with another structure. Fig. 2(A) The structure of the imidazole group of histidine and (B) its electronic charge density, determined by X-ray diffraction at 103 K for the projection in (A) (adapted from Epstein et al. 1982). In (A), R = remainder of the histidine molecule His120, at the lumenal end of helix-2 in LHCII (see Fig. 3), is not a ligand to Chl a, possibly because of its exposure to the thylakoid lumen where competition with water is greater than within the membrane. Also, the imidazole group may be protonated at the pH of the lumen during active photosynthesis, estimated to be near pH 5 (Kramer et al. 1999; Sacksteder et al. 2000). His212, a ligand to Chl a, is also near the lumenal surface of the membrane but is likely shielded from the aqueous lumen by helix-4 of the LHC protein. Fig. 3Model of the association of Chls with Lhcb1. The arrangement of the protein in thylakoid membranes is illustrated according to Green and Durnford (1996). The symbols designating the chlorin rings of the six Chl b molecules are filled (green). A water ligand for four Chl molecules is indicated by a central blue dot. The Chl a molecules are numbered 1–8 and the Chl b molecules 9–14, as designated by Standfuss et al. (2005) The imidazole group provides a good example of the attractive/repulsive forces that limit the strength of the coordination bond. A repulsive force should exist between the π clouds of Chl and the ligand. However, the pair of electrons on N3 is an attractive force that satisfies the needs of the Lewis acid, Mg. The concept of electronegativity equalization (Noy et al. 2000) suggests that a partial charge (about 0.3e) is transferred from imidazole to the metal ion upon coordination (analyzed with Ni-BChl a). This shift in electron density should reduce the role of the dipole in the coordination bond and generate a partial positive charge on the ligand that is accommodated by the electron cloud of the conjugated π system of Chl a. In contrast, the more exposed positive charge on Mg in Chl b likely repels the positive charge that develops on the ligand. Thus the attractive force is limited by the repulsive force created between Mg and the induced positive charge on the ligand. Carboxyl group Two ligands for Chl a in LHCII are charge-compensated ion-pairs formed by electrostatic interaction between the sidechain carboxylate of glutamic acid and the guanidinium group of arginine. Although at pH 7 the carboxyl group has a negative charge and multiple pairs of unbonded electrons, these electrons are distributed between the two electronegative oxygen atoms in a resonance structure and are less available for protonation than in the imidazole group. This property is reflected in the higher-proton concentration (pH 3–5) required to protonate the carboxylate in solution. In proteins, its pK varies from a low of about 2, to a high of nearly 9 (Gunner et al. 2000; Georgescu et al. 2002; Laurents et al. 2003; Li et al. 2004). The higher pK values occur when the protonated carboxyl group is stabilized in a nonpolar environment (Mehler et al. 2002). The protonated carboxyl group has a relatively low ab initio dipole moment of 1.52 D (Table 1). When ionized, the negative charge is likely repelled by the π electron cloud of Chl a. As the LHC apoprotein folds, the approach of a positively charged guanidinium group attracts electron density from the carboxylate group and the ion-pair is stabilized by the nonpolar environment. The resulting glutamate in this ion-pair is a soft Lewis base, with a dipole moment probably near that of a protonated carboxyl group. The dipole moment of the ion-pair is possibly too low to displace a water molecule from Chl b. With its electron cloud pulled away from the central Mg (∼+1 charge), Chl b is expected to coordinate more readily with a carboxyl group (∼−1 charge) and thus form a bond with largely electrostatic character. In LHCII, Chl b is coordinated with the sidechain of Glu139 near the stromal end of helix-2, which nevertheless is sufficiently near Arg142 for at least partial charge-compensation (Standfuss et al. 2005). The ability of Chls to bind to imidazole and glutamate/arginine ion-pair ligands was assayed experimentally by interaction with a synthetic peptide that mimicked helix-1 in LHCPs. Chls a and d bound with nearly equal affinity, assayed by Förster resonance energy transfer from a tryptophan residue next to arginine (Chen et al. 2005). In contrast, Chls b and c did not interact significantly with the peptide. A theoretical analysis of the interaction of the Chls with the peptide (Chen and Cai 2007) strongly supports the experimental data and indicates that bonding of Chls b and c with these ligands is thermodynamically unfavorable (Table 2). In the presence of water, however, complex formation with Chl b is more favorable, which, as shown by molecular modeling, is the result of a water molecule bridging Chl b and the ligand (Chen and Cai 2007). Table 2Heat of formation of Chl-peptide complexes calculated by molecular modelingSpeciesHeat of formation (kcal/mol) In vacuumIn waterChl a−706−870Chl b770−133Chl c11,001160Chl c21,115310Chl c387784Chl d−806−949BChl a−892−1047The parametric method 5 was used to calculate the association of each of the species of Chl with a 16-mer maquette of helix-1 of Lhcb1 (Eggink and Hoober 2000) as described by Chen and Cai (2007). The more negative the value, the more thermodynamically stable the complex Amide group The pK of an amide is −0.62 (Grant et al. 1983), an indication that electrons on the oxygen or nitrogen are not readily available for bonding with H+. However, the group exhibits a relatively strong dipole, with the negative end on the oxygen atom. Sidechain amides in proteins have a dipole moment of 3.46 D (Table 1). The dipole moments of the model compounds, formamide and acetamide, are about 3.8 D in solution. The dipole is sufficiently strong to displace a coordinated water molecule from Chl a and should also allow effective competition with water for Chl b within the environment of a membrane. In reconstituted complexes, several sites, including Gln197, have mixed occupancy (Bassi et al. 1999; Remelli et al. 1999), which suggests competition during folding. Yet the sidechain amide groups of Asn183 and Gln197 are ligands for only Chl a in LHCII in vivo, which suggests that Chl a is more abundant during folding and competes effectively by mass action. Peptide bond Backbone amides are usually H-bonded within helical or β-sheet structures in proteins and therefore unavailable for interaction with Chls. However, proline residues occur at conserved regions within Chl-binding proteins, particularly in the N-terminal region of LHCPs (Jansson 1999), which preclude formation of H-bonds to nearby backbone carbonyl groups. The pK value for the model compound N-methylacetamide, an analog of the peptide bond, is −0.42 (Grant et al. 1983), slightly more basic than a sidechain amide group. The electron-donating methyl group bonded to the nitrogen provides a larger “pool” of electrons for the carbonyl oxygen to draw from, which is reflected in the slightly higher pK value than of the sidechain amide group. This effect also increases the dipole moment of the carbonyl group. N-Methylacetamide has a calculated dipole of 3.73 D in the gas phase and a monomeric dipole moment of 4.2 D in liquid, which is the same as the calculated dipole moment of the peptide bond in proteins (Table 1). The ab initio dipole moment of the fully H-bonded N-methylacetamide in liquid is calculated to have an average of 6 D, with a spread from 4 to 8 D (Whitfield et al. 2006). Urea has an even stronger dipole moment than N-methylacetamide and also illustrates the effect of H-bonding. Its molecular dipole moment is 5.15 D and is increased to 7.04 D in the crystal form (Abramov et al. 1999). An electrophilic center such as the Mg atom of Chl should have a similar effect to that of H-bonding. As expected from the strong dipole moment of urea, the negative point charge on its carbonyl oxygen may be repulsed by the electron density enclosing the Mg of Chl a, which prevents it from being an effective ligand. Urea does not seem to compete with imidazole or the glutamate/arginine ion-pair, the most favorable ligands of Chl a (Eggink and Hoober 2000). However, it should effectively compete with other ligands for binding to Chl b. A peptide bond carbonyl in association with a polarizing, positively charged Mg should have a dipole moment at least as large as H-bonded N-methylacetamide and therefore should form a strong electrostatic bond with Chl b. A free backbone carbonyl group in helix-1 in Lhcb1 occurs at Gly78, because of nearby Pro82, within the interior of the membrane. This group should displace water from a Chl molecule but is probably sterically hindered from coordinating directly with the Mg atom by the adjacent, bulky amino acid sidechains of leucine and phenylalanine (see Fig. 3). As a result, this ligand is bridged by a water molecule to Chl a6 in LHCII (Standfuss et al. 2005). That this position is occupied by Chl a rather than Chl b may be determined by the availability and order of binding of the Chls during assembly of the complex. Repulsion of the strong dipole by the electron cloud of Chl a possibly limits its direct interaction with backbone carbonyl groups. Of particular interest is the finding that backbone carbonyls of proline residues provide ligands to Chl a in a water-soluble Chl-binding protein from Lepidium virginicum (Horigome et al. 2007). In the complex, four Chl a molecules are bound in a solvent-excluded pocket at the interfaces of the tetrameric protein. This observation is evidence that Chl a can fulfill the full range of ligand coordination with sufficient support from the local environment. However, this arrangement is quite different from the interaction of monomeric Chls with LHCPs during LHC assembly. Phosphatidyl glycerol An oxygen of the phosphodiester linkage in a phosphatidyl glycerol molecule serves as the ligand in LHCII to Chl a7, which resides near the stromal surface of the thylakoid membrane (Liu et al. 2004). This rare ligand is also found in photosystem (PS) I (Jordan et al. 2001). The side of the phosphodiester group opposite the Chl a molecule is H-bonded to sidechains of tyrosine and lysine residues (Liu et al. 2004), which probably reduces the electron density on the ligand oxygen. Approach of the negative end of a strong dipole toward the Mg in Chl a should again be hindered by the electron density surrounding the metal. Implications for assembly of light-harvesting complexes The concept of ligand preference was developed largely because Chl b is found only in LHCs, at specific sites, and with rare exceptions is not found in core complexes. X-ray diffraction studies of crystallized reaction centers (Fromme et al. 2001; Jordan et al. 2001; Loll et al. 2005) and LHCII (Liu et al. 2004; Standfuss et al. 2005) revealed unambiguous Chl-ligand pairs, without mixed occupancy. Whereas a nitrogen atom of the imidazole group of histidine is the most common ligand, sidechain amide groups, water molecules and even a few carboxylate groups occur as ligands of Chl a in reaction centers of PS I and PS II (Jordan et al. 2001; Balaban 2005; Balaban et al. 2002; Oba and Tamiaki 2002, 2005; Ferreira et al. 2004). Although Chl b expresses more strongly a positive charge on the central Mg than Chl a, and consequently interacts electrostatically more readily with hard Lewis bases, the above analysis indicates that Chl a can also bind to these ligands. However, a stable LHCII cannot be reconstituted in vitro with only Chl a. Conversely, stable LHCII was reconstituted with only Chl b, and the number of Chl b molecules (13.5) was the same as when both Chls were present (13.7) (Kleima et al. 1999; Reinsberg et al. 2001; Schmid et al. 2001). Thus sites normally occupied by Chl a can be occupied by Chl b. Whether Chl b molecules indeed interact directly with ligands of Chl a or are bridged by water molecules remains to be determined. As shown in Table 2, calculated ΔH of formation of complexes with a synthetic peptide containing the glutamate/arginine ion-pair and a histidine residue indicated that complex formation with Chl b is thermodynamically unfavorable. However, insertion of a water molecule to bridge Chl b and the ligand dramatically increased thermodynamic stability (Chen and Cai 2007). The overlap of ligand characteristics is substantial and too much may have been made about ligand selectivity with Chls. Therefore, unambiguous occupancy of specific binding sites as found in vivo must involve more than these properties. Two recent publications shed light on how the LHCII complex is assembled in vivo. Reinbothe et al. (2006) showed that LHCPs are not imported at a detectable rate into plastids purified from a Chl b-less mutant of Arabidopsisthaliana. These authors also confirmed the localization of CAO on the inner membrane of the chloroplast envelope as reported by Eggink et al. (2004). An alternate site for CAO activity was achieved by Hirashima et al. (2006), who transformed the Chl b-less mutant of A. thaliana ch1-1 with the gene for CAO from the cyanobacterium Prochlorothrix hollandica to achieve active CAO on thylakoid membranes. In these plants, the higher Chl b content resulted in a Chl a/b ratio that approached 1, much lower than the ratio of 3–4 in wild-type plants. In LHCII, the ratio was 0.8 in contrast to 1.3 in the complex from wild-type plants. Chl b was recovered in purified PSI and PSII core complexes, which normally lack Chl b, as well as in LHCs. Because of the widespread distribution of Chl b in the transformed plants, Hirashima et al. (2006) concluded that the restrictive distribution of the Chls in wild-type plants is not the result of discriminatory binding affinities of Chl a and Chl b to ligands. Because the P. hollandica CAO on thylakoid membranes led to a widespread distribution of Chl b, the conclusion emerges that active CAO only on the envelope of chloroplasts leads to restriction of Chl b to LHCs. To achieve incorporation of Chl b selectively into LHCs, as found in wild-type plants, assembly of LHCs should therefore occur during import at the level of the envelope. In Chl b-less mutants of higher plants, only a few of the apoproteins for LHCI and LHCII accumulate in the organelle in vivo (Król et al. 1995; Bossmann et al. 1997; Espineda et al. 1999). This observation has traditionally been interpreted as an indication that the proteins are rapidly degraded upon entry into the chloroplast stroma unless Chl b is present to allow stable integration into the thylakoid membrane. However, as noted above, chloroplasts from a Chl b-less mutant of A. thaliana lacked the ability to import LHCPs. Accumulation of LHCPs in vivo into the plastid of the alga Chlamydomonas reinhardtii was markedly reduced in the Chl b-less strain, cbn1-113 (Park and Hoober, 1997). Mature-sized proteins were detected in the cytosol, which indicated that import was aborted (White et al. 1996; Park and Hoober 1997). In the absence of Chl, a condition achieved because the mutant strain was unable to synthesize Chl in the dark, accumulation of LHCPs in the plastid was not detected. However, the proteins were synthesized at the same rate as in cells greening in the light but accumulated in the cytosol and vacuoles. These results point to a requirement of Chl, and particularly of Chl b, for import and/or retention of LHCPs in the organelle. Even in wild-type cells, excess LHCPs were shunted to vacuoles when the rate of Chl synthesis was insufficient to accommodate the rate of synthesis of the Chl-binding proteins (White et al. 1996). Model of LHCII assembly Folding of a thylakoid membrane protein of cytosolic origin is a complex process, made more so by the environmental sensing of domains as the protein is threaded through the translocon in the chloroplast envelope. Popot and Engelman (2000) and Bowie (2005) described two steps of the process of folding of a membrane protein. The first involves achieving the correct location and topology by the initially inserted segments. Second is the folding and condensation of the protein from this starting point. The evidence indicates that LHCP precursors achieve location and topology as they are guided by the transit sequence through translocons on the outer and inner envelope membranes. The transit sequence is removed soon after the N-terminal domain gains access to the stroma (Soll and Schleiff 2004; Vothknecht and Soll 2006). Membrane-spanning, nonpolar sequences that serve as stop-transfer domains are minimally 14–16 amino acids in length (Davis and Model 1985; Adams and Rose 1985; Popot and Engelman 2000). In this respect, assembly of LHCs presents an interesting problem. Inspection of helix-1 of most LHCPs reveals that the length of the nonpolar sequence in the first membrane-spanning domain is only 10–12 amino acids long, which is on the short side of a significant stop-transfer signal. The nonpolar sequence is within the lagging half of the span, terminated by several charged amino acids (Green and Durnford 1996; Jansson 1999). Binding of Chl to amino acids in the leading half of the helix, which is untypically polar and charged for a membrane-spanning segment of a protein (see Fig. 3), should increase the probability that this domain remains in the membrane. It seems plausible to consider that as the N-terminal domain of LHCPs traverses the envelope inner membrane, sidechains of glutamate and arginine within the conserved sequence –EVIHSR– in helix-1 form a looped ion-pair ligand for Chl a. The histidine residue provides a second ligand for Chl a, as described by Eggink and Hoober (2000). Kohorn (1990) showed that mutation of this sequence, to replace histidine with alanine, eliminated the ability of the chloroplast to import a LHCP precursor. Binding of Chl to these sidechains may allow this polar sequence to diffuse more readily into the nonpolar phase of the membrane. However, these interactions are insufficient to retain the protein in the membrane in the absence of Chl b. As shown by Chen et al. (2005), Chls b and c bind poorly to imidazole or glutamate/arginine ion pairs in vitro. A possible ligand for the Chl b molecule that is necessary for retention of the protein in the plastid was suggested by the crystal structure of LHCII. Several backbone carbonyls near the N-terminus are precluded from H-bonding and formation of an α-helix because of the richness of proline residues in this region of the LHCP. The carbonyl of tyrosine (Tyr24 in spinach Lhcb1) resides three positions distant in the amino acid sequence from a proline residue and is thus free to form a coordination bond with Chl b (Liu et al. 2004; Standfuss et al. 2005). The unusual abundance of proline in the N-terminal domain also extends to an iron-deficiency-induced (Tidi) protein, a homolog of the light-harvesting Chl a/b proteins, in Dunaliella, which increases the probability of interaction with Chl b during this stress condition that leads to chlorosis (Varsano et al. 2006). The strong electrostatic bond formed by further polarization of the carbonyl dipole through interaction with Chl b may be essential to anchor a LHCP in the envelope membrane sufficiently long for the remainder of the protein to be transported from the cytosol to complete assembly (Fig. 4). Fig. 4Model of LHCII assembly in the chloroplast envelope and the proposed role of Chl b. Several proposed intermediates are shown in the sequence, left to right. After synthesis in the cytosol, a LHCP precursor is imported sufficiently into the chloroplast stroma for removal of the transit sequence from the N-terminus and for the first membrane-spanning region to engage the inner membrane. Chl a (dark green rectangles) binds to ligands in the motif provided by the ion-pair of the sidechains of glutamate and arginine and the imidazole group of histidine (dotted line, a). However, binding to these sites is not sufficient to retain the protein in the envelope. Without Chl b the protein slips back into the cytosol for transfer to vacuoles and subsequent degradation. Chl b (light green rectangles) forms a strong coordination bond with the peptide bond carbonyl of Tyr24, near the N-terminus, and provides an additional hold on the protein (solid line, a + b). Along with the Chl a molecules that bind to the motifs in membrane-spanning helix 1, Chl b binds to Try24 and the peptide carbonyl of Val119 at the lumenal end of helix-2. These Chls retain the protein in the membrane sufficiently long for the remainder of the protein, including the conserved motif in membrane-spanning helix-3, to enter the membrane, bind additional Chl and xanthophylls molecules, and complete assembly (LHC). Other proteins in the membrane and stroma apparently assist assembly of the complete complex (see text) As the remainder of LHCP is transferred through the translocon, a second Chl b possibly coordinates with the backbone carbonyl of Val119 (Liu et al. 2004; Standfuss et al. 2005). Helices 2 and 3 are then transferred through the membrane, which would complete the first step described above by Bowie (2005). The rather weak hydrophobic character of helix-2, and the short nonpolar sequence (again the lagging half) in helix-3, suggest that these domains may enter the membrane largely unassisted, as found for other membrane proteins containing transmembrane sequences that are only moderately hydrophobic (Brambillasca et al. 2006). Other factors are also required at this step, probably to prevent helix-3 from escaping the membrane into the stroma (Fig. 4). One of these factors is the chloroplast signal-recognition particle. The ability of this complex to bind with high affinity to the loop between helices 2 and 3 (Tu et al. 2000) suggests that it plays an important role in this process (Schünemann 2003). Other proteins such as Albino3 have been identified as important in the integration of LHCPs into the membrane (Moore et al. 2000; Bellafiore et al. 2002), although their specific actions are not known. Gerdes et al. (2006) found that Alb3 mutants were defective in chloroplast biogenesis but not in accumulation of LHCPs. With the overall disposition of the protein now achieved in the membrane, the glutamate/arginine ion-pairs between helices 1 and 3 can now form, which stabilizes the protein. Although the pK values vary widely, the magnitude of the dipole moments of the ligands that selectively coordinate with Chl a or Chl b are not substantially different, except for the backbone carbonyl group. Therefore, as Hirashima et al. (2006) conclude, highly specific ligand selectivity should not be expected. In vitro reconstitution of LHCs has been remarkably successful in reflecting the innate stability of Chl-ligand pairs (Bassi et al. 1999; Remelli et al. 1999; Rogl and Kühlbrandt 1999; Horn and Paulsen 2004) but these experiments did not fully achieve the selectivity of interaction that is found in complexes that are assembled in vivo. Ligand selection can reasonably be considered by taking into account (i) the unique Lewis acid properties of Chl b, (ii) the preference of Chl b to form electrostatic bonds with hard ligands containing a fixed dipole (i.e., an oxygen atom), (iii) the micro-environments in which the interactions occur (i.e., the dielectric constant), and (iv) the order of addition of the pigment molecules, which is determined partly by the local concentrations of the two Chls. Interaction of Chl b with sidechain amide groups would be expected from the properties of the ligands, and both Chl a and b are found with these ligands after in vitro reconstitution of LHCs (Remelli et al. 1999). Yet these groups are not ligands of Chl b in the crystal structure of LHCII. Chl a is probably more abundant within the membrane and competes favorably for amide ligands by mass action during assembly. Thus, basing theoretical proposals for a specific Chl in each binding site on only the first three factors above is not sufficient. The lack of ambiguity, or mixed sites, found in complexes isolated from plants after in vivo assembly is most likely achieved also by the order in which Chls bind, determined by the relative concentrations of each. An important aspect in the interaction of Chls and ligands is the location of the ligand within the protein structure. Tyr24 is near the stromal surface of LHCII, in a region expected to have a membrane interface dielectric constant of 5–10 (Tanizaki and Feig 2005). As suggested below, association of Chl b9 with this residue may form prior to reaching this position in the membrane. Likewise, the carbonyl of Val119, non-H-bonded because of Pro116, is at the lumenal end of helix-2 in LHCII, also a region of relatively high dielectric constant. The backbone carbonyl group of Val119 coordinates with Chl b14. The Chl b molecules are possibly protected from the aqueous environment by the protein strand. H-Bonding of the 7-formyl group to Gln122 and Ser123 (Liu et al., 2004) would increase the Lewis acid strength of Chl b14 and strengthen this bond. These two Chl b molecules possibly stabilize an intermediate in the assembly pathway of LHCII (Fig. 4). Four Chl b molecules interact with helix-2 of Lhcb1. Chl b12 forms a coordination bond with Glu139, which as discussed above is likely charge-compensated by Arg142. The other three Chl b molecules (b10, b11 and b13) retain water ligands (Fig. 3). H-Bonding of these Chl b molecules through the 7-formyl group would further enhance the electrostatic character of the Mg atom (Liu et al. 2004). The 7-formyl group of Chl b11 is H-bonded to the peptide bond N of Leu148, and along with Chl b12 may help to retain the stromal end of helix-2 in the membrane. The 7-formyl group of Chl b13 is H-bonded to the water ligand of Chl b10, which is in turn H-bonded via its 7-formyl group to the amide N of Gln131. These Chl b molecules, as an aggregate, may fill a void in the protein between helices 2 and 3 as the protein folds. Such an aggregate of Chl b molecules would be considerably more stable than a similar complex composed of Chl a. These molecules would then enter the structure late in assembly, as Horn and Paulsen (2004) and Horn et al. (2007) found during studies of the kinetics of reconstitution of the complex in vitro. The argument is then reduced to one or two critical Chl b molecules that are required for retention of LHCPs in the chloroplast envelop during assembly–one that interacts with a backbone carbonyl near the N-terminus and possibly the second that binds to the lumenal end of helix-2. Since the catalytic center of CAO is on the envelope inner membrane facing the intermembrane space (Reinbothe et al. 2006), the N-terminal domain of LHCP possibly binds to Chl b on that side of the membrane. As the protein is transported through the membrane, the bound Chl b would approach the stromal surface while the motif –ExxHxR– in helix-1 enters the interior of the membrane and binds Chl a (Fig. 4). These Chl molecules may then hold the N-terminal domain in the inner membrane sufficiently long for the remainder of the protein to be transported across the outer membrane of the envelope and become integrated into the inner membrane. This scenario ensures that Chl b enters the complex from the surface of the inner membrane that faces the outer membrane and thus occurs only in peripheral LHCs that are assembled with apoproteins synthesized in the cytosol.

Qual_Life_Res-4-1-2358934

Individual quality of life: adaptive conjoint analysis as an alternative for direct weighting?

In the schedule for the evaluation of individual quality of life (SEIQoL) the weights for five individualized quality of life domains have been derived by judgment analysis and direct weighting (DW). We studied the feasibility and validity of adaptive conjoint analysis (ACA) as an alternative method to derive weights in 27 cancer patients and 20 patients with rheumatoid arthritis. Further, we assessed the convergence between direct weights and weights derived by ACA, and their correlation with global quality-of-life scores. All respondents finished the ACA task, but one in five respondents were upset about the ACA task. Further, the task was vulnerable to judgment ‘errors’, such as inconsistent answers. The agreement between the two weights was low. Both weighted index scores were strongly correlated to the unweighted index score. The relationships between the index score and scores on a visual analogue scale for global individual quality of life and global quality of life were similar whether or not the index score was calculated with DW weights, with ACA weights, or without using weights. We conclude that, because weights did not improve the correlation between the index score and global quality of life scores, it seems sufficient to use the unweighted index score as a measure for global individual quality of life. Introduction Quality of life (QoL) instruments have traditionally been based on a needs model of QoL, with the same domains, assessment criteria, and weighting applied to all respondents [1]. Individual quality of life (iQoL) measures recognize that people define life domains in different ways, use different criteria to evaluate the domains, and place differing emphasis on their importance to overall quality of life [2, 3]. The schedule for the evaluation of individual quality of life (SEIQoL) is a method of assessing iQoL. It has been used in many contexts [3–7]. The SEIQoL consists of three stages of administration. The first is a semistructured interview, in which respondents are asked to nominate five areas of life (domains) that they consider most important to the overall quality of their lives [1]. In the second stage the respondents rate their functioning on each of these domains, and in the third the relative weights of the domains are quantified. In the original version of the SEIQoL a method called judgment analysis (JA), based on social judgment theory, was used for stage 3 [2, 3]. In social judgment theory, linear models are used to explain the impact of important factors, or cues, and their weights, on judgments. For the SEIQoL, to quantify the relative weights, subjects were presented with 30 randomly generated profiles of hypothetical situations labeled with the five chosen domains [3]. Respondents were asked to rate the QoL they associated with each profile on a VAS. Ten of the 30 scenarios were replicates to allow for a measure of consistency of judgments. The judgments were modeled using multiple regression to produce five relative weights summing to 100 [2]. The authors stated that JA was a successful aid to the evaluation of QoL [2, 3, 8], but from a theoretical viewpoint the number of 20 scenarios was small. Thirty to 50 scenarios are recommended to model the five weights [9]. McGee reported the use of 40 scenarios in the first study on the SEIQoL [8], but in later studies only 30 scenarios were used, of which ten were replicates [1–3]. Further, the time required to administer the JA to respondents is long and the task cumbersome, especially for older and cognitively impaired people. It requires compensatory decision-making processes, i.e., the ability to make an overall judgment on the basis of weighted information. These drawbacks make JA unsuitable for application in most clinical practices as well as for frequent assessment over a short period of time [1]. Because of these problems with JA, Browne et al. [1] developed a more easy to administer version of the SEIQoL, namely SEIQoL-direct weighting (SEIQoL-DW). Respondents are asked to fill in a pie chart in which the relative size of each sector of the pie represents the weight the respondent attaches to a QoL domain. The validity and reliability of the DW in comparison with the JA-based SEIQoL have only been investigated in two small studies. The first was done in a sample of 40 healthy volunteers [1]. The mean absolute difference between the 200 weights derived for each method, that is, 40 times five weights, was 7.8 at the first measurement, and 7.2 at the second measurement. When weights derived from the two methods were converted to ranks and compared for agreement, the κ value was moderate at both measurements (0.40 and 0.44, respectively). The reliability of the weights was moderate for the DW (κ = 0.51), and only fair for JA (κ = 0.31) [1]. In a later study, Waldron et al. compared the psychometric characteristics of the SEIQoL-DW with the SEIQoL-JA among 80 patients with advanced incurable cancer. The index scores generated by the two methods fell within a range of 14.9 [10]. These differences are large in clinical terms [3]. Therefore, the authors [10] concluded that the two methods are not interchangeable. Despite this lack of data on the DW, and its demonstrated lack of agreement with the JA, it has become the standard method for eliciting weights of the SEIQoL, due to it being simple to administer. However, a fundamental distinction in cognitive psychology is that between explicit and implicit thought [1], and previous evidence with weighting methods suggests that respondents may be unable to provide accurate implicit weights through a method such as the DW [1]. Indirect methods such as JA are based on more basic and simple judgmental tasks, such as paired comparisons, and thereby may reduce possible biases that may play a role in direct judgments. Further, indirect methods are more likely to avoid the social desirability effect according to which respondents bias their response toward the perceived values of the researcher/clinician [11]. Another advantage is that indirect methods can provide measures of internal reliability and validity for individual interviews [1]. An alternative indirect weighting method might be conjoint analysis, which was developed in mathematical psychology and, like JA, has a strong theoretical basis [12–14]. As in JA, conjoint analysis is based on the premises that any treatment or health state can be described by its characteristics (or attributes) and that the extent to which an individual values a treatment or health state depends on the levels of these characteristics. The method can be used to estimate the relative importance of these attributes, and may therefore be suitable for eliciting the weights of domains of iQoL, since the domains can be seen as attributes of iQoL. In conjoint analysis the number of paired comparisons needed to estimate the weights may be as high as the number of scenarios needed in JA, but nowadays software, adaptive conjoint analysis, is available that is adaptive to respondent’s answers and can minimize the number of paired comparisons. As an indirect method, it may provide a feasible alternative to JA. ACA has been used to derive treatment preferences in patients with lupus nephritis, with HIV medication, and with cancer [15–17]. This study aims to assess the feasibility and the validity of the ACA to derive weights for iQoL domains. Furthermore, agreement of the weighting procedures performed by the ACA and the DW will be assessed. Because it would not be feasible to use the JA as well, the ACA was only compared with the DW. Since JA is rarely used and the scientific community has overwhelmingly embraced the DW, despite the lack of data on its validity, we wished to compare ACA and DW. Further, relationships of the resulting iQoL index scores with scores on a VAS for QoL and for iQoL may give more insight into the validity of both weighting methods. Methods Patients To assess the feasibility and validity of the ACA to derive iQoL weights, a convenience sample of outpatients with rheumatoid arthritis or cancer who were treated at the Leiden University Medical Center were asked to participate in the study. We selected patients with rheumatoid arthritis (RA) who received multidisciplinary day treatment or had an appointment with the specialized nurse consultant about their treatment. Patients with cancer were selected if they received curative radiotherapy at the time of the study or had received curative radiotherapy in the 6 months before. The latter patients received a letter at home in which the head of the Department of Radiotherapy asked them to participate in the study. These groups were selected because they presented at the clinic with symptoms (RA) or were known to have side effects (cancer) impacting their quality of life. All patients were only included in the study after they had given their informed consent. The Medical Ethical Committee of the Leiden University Medical Center approved the research protocol. Interview and questionnaire Patients were interviewed either at the hospital or at home. First, patients had to rate their current global QoL on a horizontal VAS anchored at the two extremes by the terms ‘best imaginable quality of life’ and ‘worst imaginable quality of life’. Next, iQoL was assessed by the SEIQoL. If patients were unable to nominate five areas of life, they were presented with a list with predetermined domains, such as family, health, finances, living conditions, work, social, and leisure activities, to help them make a choice [3]. Next, patients rated their functioning on each of these domains on five adjacent 0–100 mm vertical VAS scales anchored at the two extremes by the terms ‘best possible’ and ‘worst possible’. Further, patients had to rate their global iQoL, given their ratings on the five domains, on a VAS anchored at the two extremes by the terms ‘the best life I can imagine’ and ‘the worst life I can imagine’. In this article, the score for global iQoL on the VAS is named as SEIQoL-VAS. Finally, the weights of the IQoL domains were determined from the DW and the ACA. The DW-pie consists of five stacked, centrally mounted, interlocking laminated discs. Each disc has a different color and is labeled with one of the five domains nominated by the individual. The discs can be rotated over each other to produce a dynamic pie chart where the relative size of each sector represents the weight the respondent attaches to a QoL domain [1]. The ACA is a computerized questionnaire [18]. At the start of the ACA survey, respondents were asked to indicate, using the mouse or key strokes, how important they considered the difference between the best and the worst level of functioning on each of the five SEIQoL domains on a seven-point Likert scale, with adjectives at the first (not important), third (somewhat important), fifth (quite important), and seventh (very important) radio button. Next they were presented with a series of paired comparisons. The pairs consisted of two scenarios, one on the left-hand side, and one on the right-hand side (see appendix). The scenarios differed with respect to the level of functioning on two or three domains of QoL. Although the use of scenarios with four or five domains would have provided for more precise estimates, we had to balance this against feasibility. We decided such scenarios were too difficult for this first experience using the ACA to assess SEIQoL weights. Each domain had four levels of functioning: very well, fairly reasonable, rather bad, and very bad. Respondents had to indicate their preference for the alternative on the left or right on a nine-point Likert scale. The number of pairs presented was based on the formula 3×(N − n − 1) − N, where N is the number of levels across all domains and n is the number of domains, resulting in 3×(20 − 5 − 1) − 20 = 22 pairs [19]. This formula presents a rule of thumb leading to three times the number of observations as parameters available (for simulations on accuracy of prediction with various numbers of pairs, see [20]). We presented 25 paired comparisons, where scenarios were defined using two (pairs 1–15) or three (pairs 16–25) domains. Finally, patients filled out a questionnaire that addressed demographic factors such as age, sex, marital status, education, and religion. Computation of iQoL weights and index scores For the DW, the relative weight of a domain is equal to the proportion of the pie chart that its sector represents, which can be read from a 100-point scale on the circumference. Relative weights for the ACA are calculated as follows. First, patient utilities for all levels of functioning on the domains are derived by ordinary least-squares regression analysis, from participants' answers to the pairwise comparisons, assuming a linear main effects additive model (for details see [18]). Next, the relative weights for the domains are calculated by dividing the range of each domain (utility of highest level – utility of lowest level) by the sum of ranges of all domains, and multiplying by 100 [16, 19]. The relative weights are expressed as percentages (the five weights add up to 100%) and reflect the extent to which the difference between the best and worst levels of each domain drives the decision to choose a specific scenario [16]. The index score for iQoL is a weighted score, calculated by multiplying the functioning scores for the domains with their corresponding weights as derived by the DW and the ACA method, and summing these. Further, an unweighted index score was calculated by simply summing up the functioning scores and dividing by 5. Feasibility and validity The feasibility of the ACA was assessed by measuring the percentage of patients that were able to finish the task, by measuring the administration time, and by asking the patients how they evaluated the ACA with respect to difficulty and acceptability. We asked patients two quantitative items about the method being confronting (very, somewhat, not) or being unpleasant versus fun (1 = very unpleasant, 5 = much fun). Further, we also coded qualitative statements about the ACA being upsetting (comments such as ‘nasty’, ‘mean’, ‘suicide questions’, ‘I felt like a prisoner’). As a measure of difficulty we also assessed how often patients chose the worst option in a dominant pair, a pair in which one of the scenarios was on all domains better than the other. The validity of the ACA was first studied by assessing the number of inconsistencies in the rank ordering of utilities, that is, the number of pairs in which the utilities for two levels of functioning were ranked opposite to the direction of the levels of functioning. We analyzed whether age, health status, and level of education were related to answers to dominant pairs and the number of inconsistencies by Pearson’s correlation and analysis of variance. Next, we assessed whether patients were willing to trade off a decrease from the best to the second-best functioning level on their most important domain with the largest improvement on their second important domain. This was done by computing the ratio between the difference in utilities for the largest benefit in the second important domain and the difference in utilities for the two highest functioning levels of the most important domain. A value smaller than 1 was taken as indicating that the patient was not willing to trade off decline in the most important domain for any benefit in the second important domain. We similarly assessed whether patients were willing to trade off a decrease from the second to the third functioning level on their most important domain with the highest improvement on their second important domain. Agreement The absolute differences between the weights assigned in the DW and in the ACA were described by means and standard deviations. Agreement between the weights was assessed by the intraclass correlation coefficient and Pearson’s correlation coefficient. Pearson’s correlation represents the linear association between two measures and is not strictly a measure of agreement. However, a Pearson’s correlation which is much larger than the intraclass correlation would provide some indication of a systematic change between measures, as might occur if there were learning effects [21]. Correlations between the weighted scores and the unweighted score on the one hand, and the VAS scores for QoL and iQoL on the other hand, were assessed using Pearson’s correlation coefficient. We used SPSS version 12.0 for Windows. Results Patients Of the 71 patients approached, 27 patients with cancer and 20 patients with rheumatoid arthritis were willing to participate (response rate 66%). Twenty cancer patients and four patients with rheumatoid arthritis declined to participate. Reasons not to participate were: too burdensome (n = 5), patients too busy with treatment or work (n = 3), and other (n = 3). In 13 cases the reason was unknown: two patients did not give a reason and 11 patients did not respond at all. Characteristics of the patients are described in Table 1. Patients were, on average, 61 years old (SD 10 years). In 32 cases (68%), the interview was held at the hospital, the other interviews were held at home. Table 1Characteristics of patients (N = 47)N (%)Sex    Female24 (51) Living arrangement    With partner41 (87) Education    Lowa19 (40) Religion    Religious25 (53) Diagnosis    Rheumatoid arthritis20 (43)     Breast cancer11 (23)     Prostate cancer11 (23)     Rectal cancer5 (11) Place of interview    Hospital32 (68)    At home15 (32)aLow education: lower vocational, lower secondary general education, or primary school; high education: intermediate vocational, higher secondary general education, higher vocational education, or university ACA: feasibility and validity All patients nominated five areas which they considered most important to their QoL, of whom two patients had to consult the list with predetermined domains. Domains of life mentioned most frequently were own health, relationships with partner and children, social contacts and friendships, hobbies and recreation, and work (Table 2). Table 2Nominated cues during the first stage of the SEIQoL (N = 47 patients)1N (%)Partner22 (47)Children12 (26)Partner and children13 (28)Family18 (38)Own health30 (64)Health of partner5 (11)Social contacts and friendship21 (45)Transportation10 (21)Independence5 (11)Hobbies and relaxation23 (49)Work21 (45)Feelings5 (11)Activities of daily life5 (11)Sports and holidays11 (23)Other34 (72)Total2351Only domains that were mentioned by five or more patients are reported, the others (such as sexuality and income) are grouped together in the final row The ACA survey took on average 20 min (range 10–37 min). All patients were able to finish the ACA. Five patients (11%) were in some sense upset about the ACA survey, and a further three (6%) judged the questions as very confronting. An additional patient found it very unpleasant. For example, when a patient had nominated own health and relationship with the partner as domains, the ACA could offer one scenario in which the patient’s health was very good whereas the relationship with the partner was poor, and another scenario in which the patient’s health was very poor whereas the relationship with the partner was very good. Some patients became upset when they had to make a choice between such options. Patients were offered, on average, 2.9 dominant pairs (range 0–6). In such pairs, four times (3%) the worst option was chosen and once (1%) the patient had no preference. The four patients who chose the worst option had the same level of education and were of the same age as the other patients. When the utility assigned to a higher level of functioning on a particular domain is lower than that assigned to a lower level of functioning on that same domain, this is inconsistent. Because each domain had four functioning levels, six different pairs of levels [(4×3)/2] can be constructed for each domain, that is a total of 30 pairs of utilities per person. On average, patients’ utilities were rank-ordered opposite to the level of functioning in 3.9 out of these 30 pairs (13%; Table 3). For the most important domain, the mean number of inconsistencies was 0.2, whereas this was 1.7 for the least important domain, and the correlation between the inconsistencies and the domain weights was r = −0.50 (P = 0.000, n = 235, 47 × 5 weights). The number of inconsistencies was lower in patients with a higher education level (Spearman’s rho –0.30; P = 0.04), whereas it was not related to age or health status. Table 3Consistency in utilities for levels of functioning of individual quality of life domainsaMost importantLeast importantTotal N = 47Domain 1Domain 2Domain 3Domain 4Domain 5Number of inconsistencies in rank order utilities out of six pairs per domainMean (SD)0.2 (0.6)0.2 (0.5)0.8 (1.0)1.0 (1.2)1.7 (1.1)3.9 (2.1)N(%)N(%)N(%)N(%)N(%)N(%)No inconsistencies41 (87)41 (87)25 (53)23 (49)8 (17)138 (59)One inconsistency2 (4)4 (9)7 (15)9 (19)11 (23)33 (14)Two inconsistencies4 (9)2 (4)13 (28)10 (21)17 (36)46 (20)Three inconsistencies2 (4)4 (9)9 (19)15 (6)Four inconsistencies1 (2)2 (4)3 (1)aThe content of the domains may vary between patients, according to what an individual patient evaluates as most important One out of 43 patients was not willing to trade off a decline from the best to second-best functioning level on the most important domain for the largest benefit on the second important domain. Further, all patients were willing to trade off a decline from the second to the third level of the most important domain for the largest benefit on the second important domain. Agreement between DW weights and ACA weights The mean absolute difference between the DW weights and the ACA weights varied from 4.4 to 7.5 for the five domains of iQoL, on a scale from 0–100 (Table 4). For all five domains together, 36 pairs (15%) differed by more than 10 points. For the most important domain according to the ACA weighting, 17 patients (36%) had a difference of more than 10 points between their DW weight and ACA weight. Table 4Absolute differences between DW weights and ACA weightsMost important aLeast important aTotal (N = 235)Domain 1Domain 2Domain 3Domain 4Domain 5Mean (SD)7.5 (5.3)4.7 (4.2)4.4 (3.5)4.6 (4.1)5.9 (4.9)P = 0.003Absolute difference between ACA and DW N (%)N (%)N (%)N (%)N (%)N (%)Less than 5 points17 (36)31 (66)28 (60)31 (66)26 (55)133 (57)5–10 points13 (28)12 (26)16 (34)13 (28)12 (26)66 (28)More than 10 points17 (36)4 (8)3 (6)3 (6)9 (19)36 (15)a Importance based on adaptive conjoint analysis procedure The correlation between the DW weights and the ACA weights varied from 0.22 to 0.43, and the intraclass correlation coefficient varied from 0.18 to 0.33, both indicating a low agreement between the two weighting methods (Table 5). Table 5Agreement between DW weights and ACA weightsACA–DW linear correlationACA–DW intraclass agreementPearson rPICCbPDomain of individual quality of lifea    Most important domain0.270.060.230.06    Domain 20.300.040.280.03    Domain 30.430.0030.330.01    Domain 40.330.020.280.03    Least important domain0.220.140.180.11ACA, adaptive conjoint analysis; DW, direct weightingaRank ordered according to weighting derived by the method of adaptive conjoint analysisbTwo-way mixed effects model where people effects are random and measure effects are fixed; f-test with true value 0 Consequences of weighting method The index score for iQoL calculated with the weights derived by the ACA (SEIQoL-ACA) was slightly higher than the index score of the SEIQoL-DW [mean score 71.6 (SD 11.5) versus 70.1 (SD 12.2); P = 0.02] and both were higher than the unweighted index (mean 67.2, SD 12.4). The SEIQoL-VAS score correlated strongly and almost equally with the SEIQoL-ACA, the SEIQoL-DW, and the unweighted score. The three index scores were also positively related to the global QoL VAS (Table 6). Table 6Impact of weighting procedure on index score for individual quality of life, and on correlations with global quality of life DW indexACA indexUnweighted indexrrrIndex score for individual quality of lifeDW-index score1.00.95**0.92**ACA-index score0.95**1.00.89**Unweighted index score0.92**0.89**1.0SEIQoL VAS0.62**0.54**0.63**QoL VAS0.40*0.36*0.33**P < 0.05; **P < 0.001DW, direct weighting; ACA, adaptive conjoint analysis; SEIQoL, schedule for evaluation of individual quality of life; VAS, visual analogue scale; QoL, quality of life Discussion We explored whether the ACA was a feasible and valid method to derive weights for iQoL domains. The iQoL community seems to have fully embraced the DW, despite its lack of agreement with the original JA method. Contrary to JA, the DW has no theoretical underpinning. Further, DW is a direct method, and as such may not capture implicit or unconscious thoughts or preferences. Since respondents will generally not be explicitly aware of the contributions of their life domains to their overall QoL, indirect methods may be of more value. JA is not feasible, however, in most situations, and for this reason we piloted another theory-based indirect method, the ACA. ACA and JA are based on the same premises. However, JA was initially developed for assessing expert opinion [22, 23], respondents have to rate many scenarios, and the task is cumbersome. Due to its adaptive nature, ACA is much less cumbersome than a full-scale conjoint analysis, which is a nonparametric form of JA. In addition to being indirect, ACA can provide measures of internal reliability and validity (inconsistencies, willingness to trade) for individual interviews. The agreement between DW weights and ACA weights may give insight into the validity of the two methods. ACA was to some extent feasible, because the ACA took on average 20 min and all patients were able to finish it. However, one in five patients judged the ACA task as upsetting, very confronting, or very unpleasant. The paired comparison task, despite working well for some domains, turned out not to be appropriate for some others. Especially choosing between two domains that are dear to the patient turned out not to be feasible. Sometimes, the computer offered a dominant pair, mostly resulting from the fact that the utilities of the functioning levels were almost equal. Only seldom was the worst option chosen. This finding shows that almost all patients understood the task of paired comparisons and were able to make a valid choice. A limitation of our procedure was that patients did not rate scenarios with four or five domains. Although the use of such scenarios would have provided for more precise estimates, this had to be balanced against feasibility. Using all five attributes in the pairwise comparisons would also have allowed for the evaluation of full profiles (health states). The goal of this first study on the use of ACA for the SEIQoL was merely to assess its feasibility and to compare ACA weights with DW weights, not to assess full profiles. We therefore preferred to opt for the more feasible approach, which we deemed sufficiently difficult already. Many patients gave inconsistent answers, but these inconsistencies mostly occurred on domains 3 and lower, and especially on the least important domain. For the two most important domains, the large majority of patients had utilities ordered in the same direction as the corresponding levels of functioning. In the case of less important domains, the differences between utilities of successive functioning levels are probably small, which leads to inconsistent answers. A major premise of the ACA is that respondents are willing to use compensatory decision making. Our findings show that almost all people were willing to do so. The willingness to trade off the difference between a decline on the most important domain with the largest benefit on the second most important domain is consistent with the finding that, in patients with colorectal cancer, 95% were willing to trade off a 1% loss of survival [17]. The agreement between DW and ACA weights was low. Browne et al. reported similar findings for the comparison between JA and DW [1]. Further, the weights derived from each method were only poorly or moderately correlated to each other. The differing weights from the DW and the ACA suggest that the two methods are not interchangeable. Weighting had almost no effect on relationships with other global measures of QoL, and the DW index score correlated 0.95 with the ACA index score, and both weighted index scores were also highly correlated to the unweighted index score. Weighting or not weighting domains of QoL has received much attention in the literature. Some studies have shown that there is no effect of weighting [24–28]. For iQoL, the findings of Wettergren et al. suggest that the degree of importance is already built into the process of selection, when the participants themselves select the domains [28]. Further, it has been suggested that a satisfaction evaluation had incorporated the judgment of item importance [29]. Our findings indeed show that the weighted iQoL index scores were higher than the unweighted index score, which indicates that patients gave higher weights to domains with better functioning. Most likely, the patients already take into account the importance of a specific domain, first in the selection of the domain and next when they evaluate the functioning on that domain. Our study has some limitations. The number of patients was relatively small, due to the qualitative character and semistructured design of the SEIQoL interview. However, larger numbers would not have changed the conclusion of our paper. A problem in measuring the validity of the SEIQoL is that a gold standard for iQoL is lacking. JA has been considered the standard weighting method, but due to its complex nature has been replaced in the field by DW. Unfortunately, it was not possible to include both the JA and the ACA, because of the cognitive burden imposed on the patients. Despite the patients’ reluctance to perform the ACA, our study gave clear insight into the problems of deriving weights for iQoL domains. Our findings show that weighting has almost no effect on the association between the SEIQoL and global iQoL, although incorporating weights for domain functioning led to slightly higher iQoL index scores than the unweighted index score. Selecting and weighting domains are clearly confounded. Because of the high correlations between the weighted and unweighted index scores, it seems sufficient to use the unweighted index score as a measure for global iQoL.

J_Behav_Med-4-1-2346512

Internet-administered cognitive behavior therapy for health problems: a systematic review

Cognitive-behavioral interventions are the most extensively researched form of psychological treatment and are increasingly offered through the Internet. Internet-based interventions may save therapist time, reduce waiting-lists, cut traveling time, and reach populations with health problems who can not easily access other more traditional forms of treatments. We conducted a systematic review of twelve randomized controlled or comparative trials. Studies were identified through systematic searches in major bibliographical databases. Three studies focused on patients suffering from pain, three on headache, and six on other health problems. The effects found for Internet interventions targeting pain were comparable to the effects found for face-to-face treatments, and the same was true for interventions aimed at headache. The other interventions also showed some effects, although effects differed across target conditions. Internet-delivered cognitive-behavioral interventions are a promising addition and complement to existing treatments. The Internet will most likely assume a major role in the future delivery of cognitive-behavioral interventions to patients with health problems. Introduction Cognitive-behavioral interventions are probably the most extensively researched form of psychological treatment (Butler et al. 2006). Cognitive-behavioral interventions are aimed at challenging negative automatic thoughts and dysfunctional underlying beliefs, and at changing behavioral patterns which are related to the problem being targeted in the therapy. More than 300 published controlled outcome studies, and probably many more, have examined the effects of cognitive-behavioral therapy (CBT) for a wide range of disorders and health problems, ranging from mental health disorders, such as depression (Hollon et al. 2002), anxiety disorders (Barlow 2002), schizophrenia (Pilling et al. 2002), to health conditions such as chronic pain (Morley et al. 1999), sleep problems (Morin et al. 1999), headache (Holroyd 2002), cancer (Moorey and Greer 2002) and many others. Most of these studies have shown that CBT has positive effects on these and several other health conditions. CBT is not only the most extensively researched form of psychotherapy, but also the most widely applied type of psychotherapy (Norcross et al. 2005), and certainly the most widely applied ‘evidence-based’ type of psychological therapy. CBT is increasingly offered through the Internet. Internet-based interventions may have several advantages over other more traditional forms of delivery. They may save therapist time, reduce waiting-lists, allow patients to work at their own pace, abolish the need to schedule appointments with a therapist, save traveling time, reduce the stigma of going to a psychologist or therapist, and facilitate help for the hard-of-hearing as self-help treatments typically work with visual rather than auditory information (Marks et al. 2007). Furthermore, Internet-delivered self-help may be programmed to enhance patients’ motivation by presenting a wide range of attractive audiovisual information with voices giving instructions in whichever gender, age, accent, language and perhaps game format the client prefers. It can also quickly and automatically report patient progress and self-ratings. Internet-based interventions may reach populations with health problems, who can not be reached with other more traditional forms of treatments. For example, a considerable proportion of the patients with mental disorders are not reached with traditional forms of treatment (Bijl and Ravelli 2000) because of the stigma associated with mental disorder, prejudices about therapists, lack of willingness to talk to a stranger about personal problems, or because of physical obstacles like walking problems or long distances. For patients with somatic conditions there may be barriers to seek psychological treatment. Internet-guided interventions may reach a segment of this population who cannot be reached through traditional interventions. Internet-based psychological interventions for many health problems are commonly based on CBT techniques. One reason is that the effects of CBT have been shown in numerous trials. Another reason why CBT is often used in internet-interventions is that these techniques lend themselves to be operationalized in text. CBT interventions can very well be converted into a structured format, with psychoeducation, homework assignments and registration exercises presented via web pages (Ritterband et al. 2006). Delivering CBT through the Internet does not, however, only have advantages. An online programme may not be suitable for technophobic patients and illiterates, nor can it answer all the possible questions users may ask; it can not detect subtle nonverbal and verbal clues to clients’ misunderstandings; it may encourage clients to cherry-pick from a range of homework options presented; and not all clients find communicating via computers acceptable (Marks et al. 2007). However, subtle text nuances may be detected and somewhat surprisingly, Internet interventions has been found to generate good working alliance between the patient and the therapist (Knaevelsrud and Maercker 2006). Furthermore, it is not yet very clear whether CBT interventions which have been proven to be effective when delivered in traditional format, are also effective when delivered through the internet. Recent studies, however, indicate that this might be the case for at least some patients and some conditions (Carlbring et al. 2005). Whether or not an internet-based CBT is effective should be examined in randomized controlled trials, and can obviously not be based on the effects of traditionally delivered CBT (e.g., individual or group format). Since the Internet has become available to the broad public in many Western countries, several trials have examined the effects of cognitive behavioral interventions in randomized trials. In a recent meta-analysis, we examined the effects of internet-based treatments for depression and anxiety disorders, and found that these interventions had large effects (Cohen’s d = 1.00) compared to control conditions, when some kind of guidance was given to the patients receiving the treatment (Spek et al. 2007). This latter observation was also confirmed in a recent review in which a correlation of rho = .75, P < .005 was obtained between amount of contact spent with clients and the effect size (Palmqvist et al. 2007). These large effect sizes suggest that Internet-administered CBT is as effective as face-to-face CBT, and that the format in which CBT is delivered may not be related to the effect sizes found. It is not known, however, whether Internet-administered CBT is also equally effective when other health problems are targeted. In the current study, we will present the results of a systematic review of Internet-delivered CBT for health problems. In the review, we aim to establish for which health problems Internet-based CBT has been developed, and examined in randomized controlled or comparative trials, and whether these interventions were effective. We also examine the target groups and contents of these intervention, as well the quality of the studies. Methods Search strategy and selection of studies Studies were traced through several methods. First, we conducted a comprehensive literature search in bibliographical databases (from 1966 to February 2007). We examined 1,608 abstracts in Pubmed (295 abstracts), Psycinfo (109), Embase (330) and the Cochrane Central Register of Controlled Trials (374). In order to find unpublished studies, we also searched Digital Dissertations (500 abstracts). We searched these databases by combining terms indicative of effect studies (randomized trials, controlled trials, clinical trials) and Internet (both keywords and text words). Second, we examined the references of earlier reviews of Internet-based interventions (Griffiths et al. 2006; Wantland et al. 2004), and we reviewed the reference lists of retrieved papers. Studies were included if they met the following criteria: (a) randomized controlled or comparative trials (b) examining interventions that were conducted through the Internet (at least 50% of the intervention), (c) based on CBT techniques, (d) aimed at behavior change (e) in patients with an existing disorder or health problem. We excluded studies aimed at mental disorders, because another recent systematic review was published about these studies (Spek et al. 2007). We also excluded studies focusing on lifestyle (smoking, obesity, exercise, nutrition), because the character of these interventions differs strongly because the focus of these interventions is typically preventative. Quality assessment There are at least 25 scales available to assess the validity and quality of randomized controlled trials (Higgins and Green 2005). There is no evidence, however, that these scales provide more reliable assessments of validity. We preferred therefore to use a simple approach for assessing the validity of the studies, as suggested in the Cochrane Handbook (Higgins and Green 2005). In this context, the validity of a study can be defined as the extent to which its design and conduct are likely to prevent systematic errors (Moher 1995). Variation in validity can explain variation in the results of the studies included in a systematic review and may result in an erroneous conclusions that an intervention is effective if the less rigorous studies are biased toward overestimating an intervention’s effectiveness (Higgins and Green 2005). We assessed the validity of the studies using four basic criteria: allocation to conditions is conducted by an independent (third) party; adequacy of random allocation concealment to respondents; blinding of assessors of outcomes; and completeness of follow-up data. Analyses We examined the characteristics of the target populations, the interventions, and the design of the included studies. We also examined which main outcome measures were used for each study, and we calculated standardized effect sizes for each of the main outcome measures. These effect sizes (d) were calculated by subtracting (at post-test) the average score of the control group (Mc) from the average score of the experimental group (Me) and dividing the result by the average of the standard deviations of the experimental and control group (SDec). An effect size of 0.5 thus indicates that the mean of the experimental group is half a standard deviation larger than the mean of the control group. Effect sizes of 0.8 can be assumed to be large, while effect sizes of 0.5 are moderate, and effect sizes of 0.2 are small (Cohen 1988). When sufficient effect sizes were available (at least three effect sizes examining the same outcome measure in the same health problem), we calculated pooled mean effect sizes. For these analyses, we used the computer program Comprehensive Meta-analysis (version 2.2.021), developed for support in meta-analysis. As we expected considerable heterogeneity, we decided to calculate mean effect sizes with the random effects model. In the random effects model, it is assumed that the included studies are drawn from ‘populations’ of studies that differ from each other systematically. In these analyses, we tested whether there are genuine differences underlying the results of the studies (heterogeneity), or whether the variation in findings is compatible with chance alone (homogeneity; Higgins et al. 2003). As an indicator of homogeneity, we calculated the Q-statistic. We also calculated the I2-statistic which is an indicator of heterogeneity in percentages as well. A value of 0% indicates no observed heterogeneity, and larger values show increasing heterogeneity, with 25% as low, 50% as moderate, and 75% as high heterogeneity (Higgins et al. 2003). Results Included studies A total of 61 papers which possibly met our inclusion criteria were retrieved. Twelve studies (13 papers; two papers were published about the same study) met our inclusion criteria. The other studies were excluded because they did not examine a cognitive-behavioral intervention (30 papers), because they were not a randomized controlled or comparative trial (11 papers), or because they did not examine an Internet-based intervention (7 papers). Selected characteristics of the target groups, the intervention, and the general design of the twelve included studies are presented in Table 1. Table 1Selected characteristics of Internet-based cognitive behavioral interventions for health problemsParticipantsIntervention and conditionsStudyHealth conditionRecruitmentWomen (%)Age group (M)Conditions NInterventionContactPeriodMeasurementsDropout (%)CountryAndersson et al. 2003HeadacheCommunity8218–59 (40)1. I-CBT + telephone24Psychoeducation, applied relaxation; problem solving; cognitive restructuringE-mail (at request)6 weeksPre, post32Sweden2. I-CBT20Weekly telephone callsDevineni and Blanchard 2005Tension-type, migraine-only or mixed headache Community83NR (42)1. I-CBT39Progressive muscle relaxation + cognitive stress coping therapy (tension-type), or autogenic training + PMR (migraine/mixed)No therapist contact4 weeksPre, post38US2. Waiting-list47Ström et al. 2000 [33]Recurrent headacheCommunity68≥ 18 (37)1. I-CBT51Psychoeducation, applied relaxation; problem solving; cognitive restructuringE-mail (at request)6 weeksPre, post61Sweden2. Waiting-list51Brattberg 2006Chronic pain + burn-out in patients on long-term sick leave Community9018–65 (47)1. I-CBT30Films + texts; psychoeducation + cognitive self-treatment (changing, coping with shame and guilt, depression, identity, etc.)Real live introduction meeting + weekly online sessions20 weeksPre, post, 1 year8Sweden2. Waiting-list30Buhrman et al. 2004Chronic back pain Community6318–65 (45)1. I-CBT25Applied relaxation; physical exercise, coping strategiesE-mail (at request) weekly telephone calls8 weeksPre, post, 3 months8Sweden2. Waiting-list32Hicks et al. 2006Pediatric recurrent pain/headacheCommunity649–16 (12)1. I-CBT25Psychoeduation; relaxation; cognitive restructuringE-mail contact (5 times) + telephone contact (3 times)7 weeksPre, post, 3 months21Canada2. Waiting-list22Andersson et al. 2002TinnitusCommunity4718–70 (48)1. I-CBT53Psychoeducation; applied relaxation; positive imagery; advice on noise sensitivity; cognitive restructuring; behavioral sleep management.Weekly report on progress, weekly encouraging e-mail 6 weeksPre, post, 1 year41Sweden2. Waiting-list64Hopps et al. 2003People with physical disabilities who feel lonelyCommunity53≥ 18 (34)1. I-CBT11Psychoeducation on communication; self-observation, role-playing, confrontation, positive verbalizations; social skills and assertiveness.Weekly sessions on the Internet12 weeksPre, post, 4 months14Canada2. Waiting-list11Lorig et al. 2006Chronic diseases (heart, lung, or type 2 diabetes)Community71≥ 18 (57)1. I-CBT457Exercise programs; relaxation; cognitive restructuring; psychoeducation; physician-patient communication; healthy eating; fatigue management; problem solving Web-based bulletin board discussion groups (trained peer moderators)6 weeksPre, post, 1 year18US2. Care-as-Usual501Owen et al. 2005Early-stage breast cancerClinical100NR (52)1. I-CBT32Psychoeducation; coping advice for common physical symptoms such as pain and fatigue; structured coping-skills training exercisesBulletin board for asynchronous group discussion12 weeksPre, post15US2. Waiting-list30Ström et al. 2004InsomniaCommunity65≥ 18 (44) 1. I-CBT54Psychoeducation; sleep restriction, stimulus control, cognitive restructuringE-mail (at request)5 weeksPre, post24Sweden2. Waiting-list55Wade et al. 2006Pediatric brain injuryClinical385–16 (11)1. I-CBT20Problem solving; communication; behavior management skills; Videocontact with therapist14 weeksPre, post2US2. I-information20Abbreviations: I-CBT: Internet-CBT; NR: not reported In the twelve included studies, a total of 1,704 patients participated, 841 in the Internet-CBT conditions, and 863 in the control conditions (mean number of respondents per study: 142; standard deviation: 258.7). More than half of these 1,704 patients (56%) participated in one study (Lorig et al. 2006). In none of the other studies was the number of patients per condition larger than 64. Eleven studies compared an Internet-based CBT intervention to a control condition, while one study compared two types of Internet-based CBT to each other (one with and one without weekly telephone calls). Nine of the eleven controlled studies used a waiting list control group, while one study used a care-as-usual control group, and the other one used an information control group. In all studies, participants were randomized to one of two conditions. In none of the studies was Internet-based CBT compared to a face-to-face intervention of another treatment. Six studies only presented pre-post data, while the other six also had a follow-up measurement (mean length of follow-up in these six studies was 7.67 months; standard deviation 4.76). Six studies were conducted in Sweden, four in the United States, and two in Canada. All included studies were conducted in the year 2000 or later (one in 2000 and another one in 2002, two in 2003, in 2004, and in 2005, and four in 2006). The quality of studies varied. Three of the twelve studies reported that allocation to conditions was conducted by an independent party. Concealment of random allocation to respondents was not possible in any of the studies, while blinding of assessors was reported in none of the studies. Drop-out numbers ranged from 2% to 61%. In only one of the studies intention-to-treat analyses were conducted (Hicks et al. 2006; the other studies were limited to completers-only analyses). The target populations Three studies focused on patients suffering from pain, three on headache, and six on other health problems (tinnitus; physical disabilities; chronic diseases; breast cancer; insomnia; and pediatric brain injury). In ten of the twelve studies, patients were recruited through announcements on websites, referrals, and community recruitment. In the other two studies patients were recruited through screening of clinical samples. Ten studies were aimed at adults, two at children. None of the interventions were aimed at older adults, although three studies allowed older adults (≥70 years) to participate. The other studies on adults only included younger adults for participation or did not report that they used an age limit. The interventions The character of the interventions differed from each other. One group of interventions consisted of self-help materials on the Internet, with supporting e-mails or telephone calls (5 studies). In two studies the intervention consisted of self-help materials on the Internet, but without the supporting e-mails or calls. In the other studies, the core of the intervention consists of online contact between a therapist or moderator and the patients (individual or in groups). Most interventions contained psychoeducation on the specific problem, and different CBT modules such as cognitive restructuring, relaxation techniques, and social skills training. The duration of the interventions ranged between 4 and 20 weeks. Effects of the interventions The effects of the interventions on the main outcomes of each included study at post-test are presented in Table 2. Table 2Main outcomes of studies on Internet-based cognitive behavioral interventions for health problemsHealth conditionComparisonMain outcomeEffect size95% CIPainBrattberg 2006Chronic pain and burnoutRehabilitation course versus waiting list controlFunctional limitations0.48−0.03–0.99Buhrman et al. 2004Chronic back painInternet-guided self-help versus waiting list controlCoping with pain0.790.22–1.36Hicks et al. 2006 BPediatric recurrent pain/headacheInternet-guided self-help versus waiting list controlPain0.47−0.24–1.18POOLED0.58a0.25–0.92HeadacheAndersson et al. 2003HeadacheInternet self-help with support versus self-help onlyHeadache0.38−0.35–1.11Devineni and Blanchard 2005Chronic headacheInternet self-help versus waiting list controlHeadache0.560.13–0.99Ström et al. 2000Recurrent headacheInternet self-help versus waiting list controlHeadache0.19−0.40–0.78OtherAndersson et al. 2002TinnitusInternet CBT versus waiting list controlDistress from tinnitus0.26−0.23–0.75Hopps et al. 2003Physical disabilitiesGoal-oriented CBT chat-group teletherapy versus waiting list controlLoneliness0.46−0.45–1.37Lorig et al. 2006Chronic diseases Online CBT workshops versus care-as-usualHealth indicators (only 1 year FU)0.10−0.04–0.24Owen et al. 2005Early-stage breast cancerOnline CBT coping group versus waiting list controlHealth-related quality of life0.22−0.32–0.76Ström et al. 2004InsomniaInternet CBT versus waiting list controlWade et al. 2006Pediatric brain injuryOnline family problem solving therapy versus Internet resources Parental mental health0.700.05–1.35aPooled with the random effects model; Z = 3.40, P < 0.001; Q = 0.75, n.s.; I2 = 0 We pooled the three studies in which Internet-based CBT for pain was compared to control groups. The mean effect size on the main outcome measure was 0.58 (95% CI: 0.25–0.92; P < 0.001), indicating a moderate to large effect of the interventions compared to the control groups at post-test. Heterogeneity was very low (Q = 0.75, n.s.; I2 = 0). There were also three studies on headache, however one did not use a true control group (but compared two active interventions to each other). Therefore we did not pool the results of these studies. The effect sizes of these interventions were small (d = 0.19; Ström et al. 2000) to moderate (d = 56; Devineni et al. 2005). The effect sizes of the other studies were in the small to moderate range, varying from small (d = 0.10) for health indicators in chronic diseases at one-year follow-up, to somewhat larger for health-related quality of life in breast cancer patients (d = 0.22) and from tinnitus sufferers (d = 0.26), to moderate (loneliness in patients with physical disabilities; d = 0.46) and large (parental mental health in pediatric brain injury; d = 0.70). Most effect sizes did not significantly differ from zero because of the small sample sizes in the majority of the studies. Discussion This systematic review of controlled and comparative studies of Internet-based CBT for health problems showed that this field is developing fast. Since 2000, twelve randomized studies have examined interventions for pain, headache, and several other health problems. Half of these trials were published in 2005 and 2006, and it can be expected that the number of trials will rise sharply in the next few years. Overall, findings are promising but effects are slightly below the effect sizes found for Internet-delivered CBT for anxiety and depression (Spek et al. 2007). Although several health problems were targeted in these Internet-based studies, there are gaps in the literature in terms of treatments for health problems which have been found to improve by means of CBT. For example, several studies have examined the effects of CBT for chronic fatigue syndrome (Knoop et al. 2007), fibromyalgia (Garcia et al. 2006), incontinence (Garley and Unwin 2006), or multiple sclerosis (Thomas et al. 2006), but these have not yet been transformed into a web based intervention, although trials found positive effects of face-to-face CBT for these problems. Because the promising results of earlier studies, and because of the benefits of Internet-based interventions, we can expect development of new programs for these conditions in the future. Our review does not cover the whole field of internet interventions. While we focused on CBT for existing health problems, several other studies have examined CBT for mental health problems (Spek et al. 2007), on internet-based preventive interventions aimed at a healthy lifestyle (weight loss, smoking, exercise; e.g., Swartz et al. 2006; Muñoz et al. 2006; Oenema et al. 2001; Tate et al. 2006), and interventions using non-CBT methods (McMahon et al. 2005; Edwards et al. 2006; Gray et al. 2000). However, as was shown in this review, research on CBT interventions has been growing fast in the past few years. Because CBT interventions are very well suited to be used through the internet, it can be expected that research in this area will continue to grow further in the next years. The included studies do not yet allow us to draw definite conclusions about whether CBT through the Internet are as effective as face-to-face interventions. For most health problems we found only one study examining the effects of an Internet-based CBT study. In fact, it was only for pain and headache did we find more than one studies. However, the effects found for Internet-based interventions aimed at pain are comparable to the effects found for face-to-face treatments for pain (Morley et al. 1999), and the same is most likely true for the Internet-interventions aimed at headache (Bogaards and ter Kuile 1994). The other interventions also found some effects, although some effects were stronger than others. It does seem clear, however, that Internet-based CBT can have significant effects on some of the health problems described in this review. For at least one of the conditions—tinnitus—an effectiveness study has been published showing better results then the first controlled efficacy trial (Kaldo-Sandström et al. 2004). It has been suggested self-help interventions be used as one of the first steps in stepped-care programs (Scogin et al. 2003). Perhaps Internet-based interventions which are used in healthcare settings should also be placed within these stepped-care frameworks. In these cases, additional care is available if the Internet-based intervention does not reduce the problem of a patient sufficiently. On the other hand, Internet interventions can develop as well, and might at least for some patients be more suitable than face to face CBT. As many health conditions such as chronic pain and cancer require a multidisciplinary team approach for optimal treatment, we assume that future Internet interventions will take advantage of this possibility. There is no consensus yet among researchers about the way CBT should be presented on the Internet, although standards are emerging. Most interventions used a guided self-help format in which the treatment protocol is presented on the Internet and the patient works it through more or less independently. The patients are supported by brief contacts with therapists through e-mail or telephone. However, other studies use a more traditional format in that the patients go online at the same time as the therapist and have a more or less regular treatment session. Group treatments can also be delivered in such a way. Another difference between interventions concerns the additional elements on the Internet, apart from CBT. Some interventions have combined the cognitive behavioral interventions with other components, such as psychoeducation, films and texts to read, and a forum for users of the website. Other interventions do not provide such extras. Our review showed up several other important limitations of the current research in this area. First, most studies used waiting list control groups, and only very few used a care-as-usual or another control group. Subjects in waiting list control groups probably do not take constructive action to reduce their problems themselves during the waiting period, because they are expecting professional help in the future. This may result in an overestimation of the effects of an intervention, because there may be less spontaneous recovery. Second, most studies recruited participants through the community and through other websites. This is not a problem for interventions that target the general population. But when such an intervention is effective this does not automatically mean that it is also effective in clinical settings. Subjects who are responding to community recruitment are probably very motivated which may improve their results compared to subjects who receive treatment. Third, none of the twelve identified trials compared Internet-based treatments to face-to-face or other treatments. This is, however, an important issue, because only direct comparisons can give evidence about the comparative effects of Internet-based treatments compared to more traditional treatments and the type of patients who can benefit from it. Fourth, most studies were aimed at adults. Only two studies were aimed at children and adolescents, while these groups are probably the most familiar with the Internet. None of the studies were specifically aimed at older adults, while they suffer most from health conditions. Future research should focus on these limitations of current research. More studies are needed with care-as-usual or other control groups, clinical recruitment strategies, comparisons with face-to-face treatments, and children or older adults as target populations. More research is also needed to examine how CBT should be presented on the Internet, and to examine reasons and solutions to the relatively high drop-out rates in several studies. Finally, it is also important to study how Internet-administered CBT can be integrated in stepped-care models of care. This review has several limitations. First, the number of included studies is still very small. And the number of studies examining specific health problems is too small to integrate the results of these studies statistically into a meta-analysis. Second, the quality of the included studies is not optimal. Third, the drop-out rates reported are high in some studies. This is a concern for this type of intervention, as patients can very easily withdraw from the intervention. Remarkably, the studies in which more traditional therapies (live sessions with therapists) are delivered through the Internet have the lowest drop-out rates. Despite these limitations, however, there is no doubt that the number of studies in this area will increase considerably in the next few years, while the promising results of the studies in this review indicate that the Internet will assume a major role in the delivery of CBT to patients with health problems.

Knee_Surg_Sports_Traumatol_Arthrosc-3-1-1915597

The distal fascicle of the anterior inferior tibiofibular ligament as a cause of tibiotalar impingement syndrome: a current concepts review

Impingement syndromes of the ankle involve either osseous or soft tissue impingement and can be anterior, anterolateral, or posterior. Ankle impingement syndromes are painful conditions caused by the friction of joint tissues, which are both the cause and the effect of altered joint biomechanics. The distal fascicle of the anterior inferior tibiofibular ligament (AITFL) is possible cause of anterior impingement. The objective of this article was to review the literature concerning the anatomy, pathogenesis, symptoms and treatment of the AITFL impingement and finally to formulate treatment recommendations. The AITFL starts from the distal tibia, 5 mm in average above the articular surface, and descends obliquely between the adjacent margins of the tibia and fibula, anterior to the syndesmosis to the anterior aspect of the lateral malleolus. The incidence of the accessory fascicle differs very widely in the several studies. The presence of the distal fascicle of the AITFL and also the contact with the anterolateral talus is probably a normal finding. It may become pathological, due to anatomical variations and/or anterolateral instability of the ankle resulting from an anterior talofibular ligament injury. When observed during an ankle arthroscopy, the surgeon should look for the criteria described to decide whether it is pathological and considering resection of the distal fascicle. The presence of the AITFL and the contact with the talus is a normal finding. An impingement of the AITFL can result from an anatomical variant or anteroposterior instability of the ankle. The diagnosis of ligamentous impingement in the anterior aspect of the ankle should be considered in patients who have chronic ankle pain in the anterolateral aspect of the ankle after an inversion injury and have a stable ankle, normal plain radiographs, and isolated point tenderness on the anterolateral aspect of the talar dome and in the anteroinferior tibiofibular ligament. The impingement syndrome can be treated arthroscopically. Introduction Impingement syndromes of the ankle involve either osseous or soft tissue impingement and can be anterior, anterolateral, or posterior [5, 25]. Ankle impingement syndromes are painful conditions caused by the friction of joint tissues, which are both the cause and the effect of altered joint biomechanics [14]. The leading causes of impingement lesions are post-traumatic ankle injuries, usually ankle sprains, resulting in chronic ankle pain [33]. According to the ISAKOS definition, anterior ankle impingement is a pain syndrome characterised by anterior ankle pain on activity. Recurrent dorsiflexion is often the cause. On investigation, there is pain on palpation at the antero/medial and/or anterolateral aspect of the ankle joint. Some swelling and/or limitations of dorsiflexion are present [32]. The first reported impingement syndromes about the ankle have evolved osseous impingement and generally have been noted in athletes whose sports necessitated sudden acceleration, jumping, and extremes of dorsiflexion or plantar flexion. Impingement of the ankle was first described in the English language literature by Morris [20], in 1943, in five patients who had what he called the athlete’s ankle. This was later called footballer’s ankle in the report of McMurray [18]. Soft tissue impingement of the ankle is a common cause of chronic ankle pain that usually arises at the lateral and/or anterolateral compartment of the ankle joint following an inversion injury [8]. It is estimated that approximately 3% of ankle sprains may lead to anterolateral impingement [32]. Three types of intra-articular soft tissue lesions that lead to these complaints have been described: the meniscoid lesion, synovitis, and the distal fascicle of the anterior inferior tibiofibular ligament (AITFL) [4, 6, 8, 9, 12, 16, 19, 28]. The formation of tibiotalar spurs due to repetitive capsule traction therefore does not seem plausible reason for anterior ankle impingement [29]. Impinging soft tissue pathology in the ankle was first described by Wolin et al. as a “meniscoid mass or lesion” [34]. They described the lesion as a membrane shaped hyalinised scar tissue obstructing the lateral talofibular articulation in nine patients with persistent symptoms over the anterolateral corner of the ankle after an inversion trauma [34]. Some authors thought this lesion was formed from the torn ends of the anterior tibiofibular ligament [3, 21]. However there was no ligamentous tissue found on histologic examination of this lesion [9]. Guhl thought the lesion was of synovial origin [11]. Irritation of the joint by bleeding and a torn capsule or a torn anterior tibiofibular ligament are thought to be the underlying factors for developing post-traumatic synovitis in the ankle [17] which in unstable ankles may lead to formation of fibrosis and granulation tissue. Although synovitis and the meniscoid lesion have been referred frequently, impingement by a separated fascicle of the AITFL is a relatively new and unknown pathology. Bassett et al. [4] were the first who reported a ligamentous etiology of impingement of the anterior aspect of the ankle. The presence of the so-called accessory AITFL, as described by Nikolopoulos was confirmed by them [21]. However they called this accessory ligament, the distal fascicle of the normal AITFL [4]. The distal part of the fascicle rubs against the talus, and that this causes pain at the ankle [1, 2, 4, 8, 12, 22]. The objective of this article was to review the literature concerning the anatomy, pathogenesis, symptoms and treatment of the AITFL impingement and finally to formulate treatment recommendations. Anatomy of the anterior inferior tibiofibular ligament The distal tibiofibular joint was classically described as a syndesmosis between the rough, triangular surface on the medial and distal aspect of the fibular shaft and the fibular notch of the distal end of the tibia. The tibiofibular syndesmosis is established by three ligaments: the interosseous ligament, the posterior inferior tibiofibular ligament, and the AITFL [7, 10, 15, 24, 26]. The AITFL is a flat band becoming thicker from superior to inferior [24]. The ligament starts from the distal tibia, 5 mm in average above the articular surface, and descends obliquely between the adjacent margins of the tibia and fibula, anterior to the syndesmosis to the anterior aspect of the lateral malleolus [7, 22]. Its length ranges from 12 to 20 mm [4, 22]; its thickness, from 1 to 3 mm [4, 22]; and its width ranges from 7 to 12 mm at the fibular insertion and 9 to 22 mm at the tibial insertion [22]. This ligament is the weakest of the syndesmotic ligaments and is the first to yield to forces that promote an external rotation of the fibula around its longitudinal axis [15]. Resection of the accessory ligament did not disturb the stability of the syndesmosis, a finding that was in accordance with the results reported by Rasmussen et al. which suggests that mobility is minimally influenced by isolated cutting of the AITFL [23]. Nikolopoulos et al. studied 24 cadaveric ankles to describe the anatomy of the AITFL [22]. In five ankles the AITFL appeared to consist of two layers, one superficial and one deep, which were separated by a thin fibrofatty septum [22, 24]. However, the total thickness of these two layers did not exceed the average thickness of the AITFL in the rest of the specimens [22]. In five ankles, the articulated surfaces of the distal tibia and fibula that formed the syndesmosis were covered by articular cartilage and were surrounded by synovial bursa [22]. A completely separate accessory anteroinferior tibiofibular ligament was present in 22 ankles (Fig. 1). Nikolopoulos was the first to describe this accessory ligament [21]. However the results of his study were never published in English literature [26]. Nikolopoulos believed that this anatomical structure represents a separate ligament rather than a distal component of the AITFL [21]. The fact that there was clearly a fibrofatty septum that separates the AITFL from the accessory fascicle, similar to the one that covered the intermediate space between the interosseous and the anterior or the interosseous and the posterior tibiofibular ligaments, suggested that this fascicle could probably be considered an independent accessory ligament [22]. Some other authors described this accessory ligament as a complete separate distal fascicle of the AITFL [4, 15, 26]. Fig. 1The distal fascicle in relation to the anterior inferior tibiofibular ligament Ray and Kriz [24] founded in 10 of the 46 specimens an accessory ligament. This ligament was parallel and distal to the AITFL. The fibres of the accessory ligament descended obliquely from the lateral, anterior, and distal borders of the tibia to the anteromedial aspect of the lateral malleolus, approximating the fibular insertion of the anterior talofibular ligament (ATFL). Its length ranges from 17 to 22 mm, its thickness from 1 to 2 mm, and its width from 3 to 5 mm. The maximum, minimum, mean, and standard deviation for the length, width, gap, and angle of declination for the inferior fibre bundle are provided in Table 1. The ligament was not covered by synovial tissue, ran deeper than the AITFL and was described as being intra-articular, although how this was determined was not stated by the authors. The accessory ligament crossed the superior lateral margin of the ankle joint. The accessory ligament was separated from the AITFL by a distinct fibrofatty septum. The ligament could even be divided in two, three, or more fascicles, increasing in length from superior to inferior [24]. Table 1Characteristics of the inferior fascicle of the AITFL [24]MaximumMinimumMeanLigament length (cm)3.42.12.6 ± 0.3Ligament width (cm)1.30.20.4 ± 0.2Gap (cm)0.50.10.2 ± 0.1Declination angle (deg)155.0121.5136.3 ± 7.4 The incidence of the accessory fascicle differs very widely in the several studies (21–92%) [2, 4, 22, 24]. The discrepancies in the incidence between the several studies were probably due to the different definitions of a separate fascicle. Ray and Kriz did not consider it a separate fascicle if any digitations were present between the fascicle and the main body of the AITFL [24]. The incidence was up to 92%, which implied that the ligament may be considered as a normal finding. Vessels from the anterior peroneal artery penetrate through the interlaminar spaces [26]. It is not uncommon to have branches from the anterior tibial artery or peroneal artery or a branch arising from the anastomosis between these two arteries pass between these fascicles [11]. The most inferior fibres of the ligament merged at their fibular attachment with the origin of the ATFL [24]. Ray and Kriz [24] studied 23 cadaveric ankles. A classification scheme was designed to categorise the anatomical variations observed in the AITFL (Table 2). Table 2AITFL classification system [24]Type 1Multiple fascicles (more than three) with or without small gaps between adjacent fascicles  1AThe inferior fascicle is separated from the rest of the ligament by a gap and possesses its own distinct proximal and distal attachments  1BThe inferior fascicle is not completely separated from the rest of the ligament by a gap. Either its proximal or its distal attachment is continuous with the rest of the ligament  1CMultiple fascicles without gaps intervening between themType 2Three fascicles or less. A distinct inferior fascicle with both its proximal and distal attachments separate from the rest of the ligament. The inferior fascicle is separated completely from the main portion of the ligament by a gapType 3Three fascicles or less. A distinct inferior fascicle with either its proximal or its distal attachment continuous with the rest of the ligament. A gap does not completely separate the inferior fascicle from the rest of the ligamentType 4Three fascicles or less. The lower portion of the ligament possesses an inferior fascicle with both its proximal and distal attachments for the rest of the ligamentType 5Three fascicles or less. A ligament with no separations or gaps within its structure. It may or may not possess a fascicular arrangement Pathomechanism of the impingement syndrome It should be stressed that most studies were carried out in vitro and do not take muscular tonus and the effects of weightbearing into consideration, we performed a review of the pathomechanism of the impingement syndrome caused by the distal fascicle of the AITFL. Bassett et al. correlated the presence of this accessory ligament with pain from impingement in the anterolateral aspect of the ankle in patients with a history of inversion sprains [4]. They postulated that the post-traumatic anterolateral hyperlaxity, due to an injured ATFL, resulted in anterior extrusion of the talar dome with dorsiflexion, which now contacted the inferior fascicle of the AITFL with more pressure and friction [4]. This in turn might set up an environment favouring ligamentous impingement of the anterior aspect of the ankle [4]. Akseki et al. stated that the variations in width, length, and obliquity of the fascicle may be related to the pathological behaviour of the fascicle [2]. Mean width and length of the fascicle with bending during dorsiflexion and dorsiflexion-eversion were significantly higher than the fascicle without bending. Therefore this wider and longer fascicles had more potential to become pathological than thinner ones. They also conclude that if the fibular insertion point is far from the joint level, the fascicle has more potential to become pathological. These findings might be useful during arthroscopic procedures of the ankle. During dorsiflexion of the ankle the posterior shift of the anterior talus below the tibial plafond in ATFL intact cases was observed. Following incision of the ATFL this posterior shift of the talus was not observed; the talus remained in an anterior position and thus in contact with the fascicle [2]. Lesion of the ATFL led to bending of the fascicle with dorsiflexion and dorsiflexion-inversion in the specimens which the bending sign was negative. Lesion of the ATFL led also to contact between the talus and the fascicle in the ankles which showed no contact previously [2]. After sectioning the ATFL the anteroposterior laxity of the ankle increases especially in dorsiflexion [4, 13]. In 89.3% of the cases contact was observed between the ATFL and the anterolateral corner of the talus [2]. This contact may be normal in some of the cases. Contact was observed at an average of 12° of dorsiflexion of the ankle [4]. Bending of the accessory ligament, indicating further tensioning of its ligamentous bands caused by impingement of the talus, this was noted in an extent of dorsiflexion that varied from 5° to 10° (average 7°) [22]. Maximum dorsiflexion of the foot relieved the contact between the ligament and the superolateral corner of the talus [22]. In other studies the contact was also observed in neutral position [2, 24]. In one study the contact between the fascicle and talar dome was observed throughout the whole range of motion of the ankle [1]. They called this the “arthroscopic impingement test,” and it was positive in all of the cases with AITFL impingement they have studied [1]. Contact between the accessory fascicle and the talus was also observed during eversion of the foot, being more prominent when the foot was placed in a dorsiflexion-eversion position [22]. No interaction of the accessory ligament with the talus was noted during ankle plantar flexion [22]. In 82.9% of the cases with AITFL impingement Ray and Kriz [24] found an anterior abraded articular region of the talus. Akseki et al. [2] found this in 17% of the cases. In the study of Basset et al. [4] 71% of the patients, had an area of abraded articular cartilage on the anterolateral surface of the talar dome. There is no clarity if this abraded cartilage region is an anatomical variant of the talus or a pathological remodelling of the bone due to true ligamentous impingement [4]. On base of these studies we conclude that anterolateral hyperlaxity results in anterior extrusion of the talar dome with dorsiflexion, which contacts the inferior fascicle of the AITFL with more pressure and friction. This hyperlaxity was most likely due to an injured ATFL. Wider and longer fascicles and a fibular insertion point far from the joint level have more potential to become pathological. Often contact between the AITFL and the superolateral corner of the talus and an abraded region of the cartilage of the talus can be observed during arthroscopy. Diagnosis The diagnosis of ligamentous impingement in the anterior aspect of the ankle should be considered in patients who have chronic ankle pain in the anterolateral aspect of the ankle after an inversion sprain and who have a stable ankle, normal plain radiographs, and isolated point tenderness on the anterolateral aspect of the talar dome and in the anteroinferior tibiofibular ligament [4]. A popping sensation and aggravation of pain with dorsiflexion and eversion were noted [2, 4]. This audible popping is usually not seen in other causes of impingement of the anterior aspect of the ankle [4]. Pain in the beginning of plantarflexion-inversion may alert the physician for an impinging AITFL [2]. It is usually impossible to distinguish the source of pain in patients with both instability and impingement. So if the patient complains of giving way and frequent sprains, the surgeon may choose a reconstructive procedure [1]. But it should be noted that a painful lesion in the joint (synovitis, meniscoid lesion, or an impinging fascicle of the AITFL) may cause reflex sprains [1]. This process may confuse decision making in this type of combined pathology. The differential diagnosis for pain in the lateral aspect of the ankle after an inversion sprain includes osteochondral fracture, instability of the lateral aspect of the ankle, synovitis or rupture of the peroneal tendon, and ligamentous impingement in the anterior aspect of the ankle, with the latter being the least common [4]. Treatment Patients should be treated conservatively after an inversion sprain of the ankle for at least 6 months before operative intervention is considered [5]. This is with the exception of patients with an osteochondral fracture. Akseki et al. observed that conservative management with physical therapy, NSAIDs and bracing in patients with instability symptoms, for a period of 3 months, failed in all 21 patients [1]. Not every patient with chronic ankle instability needs a reconstructive procedure [1]. If the history, physical examination, and other diagnostic tests suggest impingement in addition to instability, arthroscopic debridement of the soft tissue lesion may be all that is needed [1. In cases with an area of abraded articular cartilage on the anterolateral surface of the talar dome, further debridement of this region was required [4]. At long-term follow-up arthroscopic excision of soft tissue overgrowth and osteophytes proved to be an effective way of treating anterior impingement of the ankle in patients who had no narrowing of the joint space [30]. Patients with less than 2 years of ankle pain before surgery for anterior ankle impingement showed significant better scores in pain, swelling, ability to work, and engagement in sports [31]. The decision to perform an arthroscopy of the ankle is typically based on the patient’s history and physical examination [9, 19, 28]. Arthroscopically resection of the distal fascicle of the AITFL should be considered when there (Fig. 2): (1) is contact between the AITFL and the talus was prominent also in the beginning of plantarflexion-inversion of the ankle, (2) is increased contact between the talus and the ligament and this continued until maximum dorsiflexion with abrasion of the articular cartilage (the surgeon should remember that this finding may be normal), (3) bending of the fascicle on the anterolateral edge of the talus with dorsiflexion and dorsiflexion-inversion, (4) is a distally inserting fascicle on the fibula, close to the origin of the ATFL on the fibula. This finding may be missed if the distraction is preserved throughout the procedure [2, 24]. For this reason, temporarily relieving distraction is advised, when a distal fascicle is seen during arthroscopy [2, 22]. Fig. 2There is contact between the distal fascicle of AITFL and the talus in dorsiflexion with bending of this fascicle Only three studies reported on the results of the treatment of AITFL impingement [1, 4, 12]. Akseki et al. [1] arthroscopically resected the distal fascicle of the AITFL in 21 patients with chronic ankle pain after an ankle sprain. During the procedure an impinging distal fascicle of the AITFL was found in all cases. Following anterolateral synovectomie, the fascicle was excised. At an average follow-up of 3 years good to excellent results were obtained in 17 patients. Nineteen patients were satisfied with the procedure and 17 patients returned to previous level of activity. Two patients who had mild laxity were graded as poor because neuromas of the terminal branches of the superficial peroneal nerve. These patients became asymptomatic after an injection of steroids. Bassett et al. [4] treated seven patients who had talar impingement by an AITFL and they were followed for a mean of 39 months. Two patients had arthroscopy and five had an arthrotomy. In all patients, a thickened distal fascicle of the AITFL was resected. Five patients also needed debridement of an area of abraded hyaline cartilage on the anterolateral aspect of the talar dome at the point where the distal fascicle made contact. Post-operative management consisted of 2–4 days of immobilisation, followed by progressive weightbearing. Four patients reported no pain in their ankle or limitation in activity, and the results were considered to be excellent. Two patients reported improvement, with only occasional pain related to overuse, and the result was considered good. The last patient had been followed for only 6 months with excellent results. Horner and Liu [12] treated nine athletes with anterolateral ankle pain during and following athletic activities without history of ankle injury. After 3 months conservative management each patient was explored arthroscopically. Patients’ outcomes were rated subjectively and objectively based on the UCLA ankle scoring system. Pathological characteristics were easily identifiable through arthroscopy. The structure may be resected during this procedure without sacrificing the stability of the ankle joint. They report that good to excellent results can be expected from this procedure including in the highest level of athleticism at an average follow-up of 18 months. The decision to perform an arthroscopy of the ankle is typically based on the patient’s history and physical examination. When an impinging distal fascicle of the AITFL and an abraded anterolateral region of the talus were observed during an ankle arthroscopy, the surgeon should look for the criteria described above to decide whether it is pathological and needs to be resected. Conclusion Because of the lack of evidence only preliminary conclusions can be drawn. The AITFL started from the distal tibia, 5 mm in average above the articular surface, and descended obliquely between the adjacent margins of the tibia and fibula, anterior to the syndesmosis to the anterior aspect of the lateral malleolus. The incidence of the accessory fascicle differs very widely in the several studies. The presence of the distal fascicle of the AITFL and also the contact with the anterolateral talus is probably a normal finding. It may become pathological, however, due to anatomical variations and/or anterolateral instability of the ankle resulting from an ATFL injury. When observed during an ankle arthroscopy, the surgeon should look for the criteria described to decide whether it is pathological and considering resection of the distal fascicle.

Purinergic_Signal-3-4-2072926

Adenosine A2A receptors modulate BDNF both in normal conditions and in experimental models of Huntington’s disease

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, enhances synaptic transmission and regulates neuronal proliferation and survival. Functional interactions between adenosine A2A receptors (A2ARs) and BDNF have been recently reported. In this article, we report some recent findings from our group showing that A2ARs regulate both BDNF functions and levels in the brain. Whereas BDNF (10 ng/ml) increased the slope of excitatory postsynaptic field potentials (fEPSPs) in hippocampal slices from wild-type (WT) mice, it was completely ineffective in slices taken from A2AR knock-out (KO) mice. Furthermore, enzyme immunoassay studies showed a significant reduction in hippocampal BDNF levels in A2AR KO vs. WT mice. Having found an even marked reduction in the striatum of A2AR KO mice, and as both BDNF and A2ARs have been implicated in the pathogenesis of Huntington’s disease (HD), an inherited striatal neurodegenerative disease, we then evaluated whether the pharmacological blockade of A2ARs could influence striatal levels of BDNF in an experimental model of HD-like striatal degeneration (quinolinic acid-lesioned rats) and in a transgenic mice model of HD (R6/2 mice). In both QA-lesioned rats and early symptomatic R6/2 mice (8 weeks), the systemic administration of the A2AR antagonist SCH58261 significantly reduced striatal BDNF levels. These results indicate that the presence and the tonic activation of A2ARs are necessary to allow BDNF-induced potentiation of synaptic transmission and to sustain a normal BDNF tone. The possible functional consequences of reducing striatal BDNF levels in HD models need further investigation. Introduction Brain-derived neurotrophic factor (BDNF) is an endogenous glycoprotein belonging to neurotrophins, a family of signalling molecules that play a key role in regulating neuronal proliferation, differentiation and survival [1–3]. Among the neurotrophins, BDNF has the widest distribution in the central nervous system [4, 5]. In the adult hippocampus, BDNF is critically involved in the regulation of synaptic plasticity [6] and facilitates long-term potentiation (LTP) [7–10; see 11 for review]. Most biological effects of BDNF are mediated by the tyrosine kinase receptor TrkB. Both BDNF and its receptors are highly expressed in the hippocampus, and the activation state of the complex BDNF/TrkB appears critical for modulating synaptic efficacy [12] and the response to excitotoxic injury [13, 14]. In hippocampal neurones, adenosine has been reported to transactivate TrkB receptors, an effect involving the A2A receptor subtype (A2ARs) [15]. Furthermore, Diogenes et al. [16] reported that A2ARs facilitate the excitatory action of BDNF on hippocampal synaptic transmission. Besides its involvement in hippocampal functions, BDNF is also very important for the survival of striatal neurons and the activity of corticostriatal synapses [17]. Impairment in BDNF function is though to play a major role in Huntington’s disease (HD), an inherited neurodegenerative disease caused by a mutation in the protein huntingtin and characterised by marked striatal degeneration (see [18] for review). Specifically, it has been shown that the activity of BDNF depends on the presence of normal huntingtin [19–21]. In vitro and in vivo data showed that wild-type huntingtin, but not the mutant protein, stimulates cortical BDNF production by acting at the level of Bdnf gene transcription [20, 21]. At the corticostriatal synapse, BDNF controls glutamate release and its exogenous administration allows striatal neurons to survive excitotoxin-induced neurodegeneration [22]. Mounting evidence also indicates an involvement of striatal A2A receptors in HD (see [23] and [24] for reviews). First, A2ARs are mainly localised on the neurons that degenerate early in HD [25]. Second, A2ARs and underlying signalling systems undergo profound changes in cellular and animal models of HD (see [24] for review). Thus, not only A2ARs seem to regulate BDNF functions in the brain, but both BDNF and A2ARs seem to be implicated in HD. In this article we report and critically discuss some recent findings on the following issues obtained by our group: (1) the regulatory role exerted by hippocampal A2ARs on BDNF functions and levels as revealed by studies performed in A2AR KO mice, and (2) the effects of A2AR blockade on striatal BDNF levels in experimental models of HD or HD-like striatal degeneration. In hippocampal slices from wild-type mice, the facilitatory effects exerted by BDNF on synaptic transmission require the endogenous activation of A2AR In hippocampal slices from wild-type (WT) mice (400-μm thick, see [26] for a detailed methodological description), BDNF (10 ng/ml over 30 min) induced a long-lasting increase in the slope of field excitatory postsynaptic potential (fEPSP) recorded in the CA1 area (Fig. 1). This effect was abolished not only by the inhibitor of Trk phosphorylation K-252a, but also by the selective adenosine A2A antagonists ZM241385 and SCH58261 (Tebano et al., manuscript in preparation). The effects of A2ARs seem to involve the cyclic adenosine monophosphate/protein kinase A (cAMP-PKA) pathway, the main transduction mechanism operated by A2ARs, as the selective PKA inhibitor KT 5720 abolished the excitatory action of BDNF (Tebano et al., manuscript in preparation). Fig. 1Brain-derived neurotrophic factor (BDNF) facilitates synaptic transmission in hippocampal slices from wild-type (WT) but not A2A receptor (R) knock-out (KO) mice. In hippocampal slices from WT mice, BDNF (10 ng/ml) induces an increase of the excitatory postsynaptic field potentials (fEPSP) slope, whereas a lower concentration (5 ng/ml) is ineffective. BDNF (5, 10 and 20 ng/ml) was totally ineffective in hippocampal slices from A2AR KO mice. *P < 0.05 vs. baseline (paired t test) The finding that BDNF enhances synaptic transmission in mice hippocampal slices confirms and strengthens previous data on the acute synaptic effects of BDNF at adult central synapses [17, 27]. Although the mechanisms responsible for such synaptic effects are not completely understood, they do not seem to be related to the neuroprotective ability of BDNF. That the above functions of BDNF recognise different molecular mechanisms is indicated by the fact that ligand-induced TrB translocation into lipid rafts is required for short-term synaptic modulation, but not neuronal survival, by BDNF [28]. Whatever the mechanisms, the blocking effect of ZM241385 and SCH58261 suggests that A2ARs have to be tonically activated by endogenous adenosine to manifest BDNF effects. To further explore the apparently “permissive” role of adenosine A2ARs on the synaptic effects of BDNF, A2AR KO mice were used. BDNF is no longer able to facilitate synaptic transmission in hippocampal slices from A2A KO mice A2AR KO mice (A2AR-/-) were generated, as previously described [29]. In hippocampal slices from A2AR KO mice, BDNF was no longer able to increase the fEPSP slope (Tebano et al., manuscript in preparation). As shown in Fig. 1, none of the tested concentrations of BDNF (5, 10 and 20 ng/ml) potentiated the synaptic response in slices from A2AR KO mice. Since the effectiveness of BDNF may depend on the proper expression of its receptors, we compared the levels of TrkB protein in WT and A2A KO mice by Western blotting. By using primary anti-TrkB antibody (Bioscience), we found that the levels of full-length TrkB isoforms were very similar in hippocampal extracts from WT and A2AR KO mice (Tebano et al., manuscript in preparation). Thus, the loss of BDNF activity observed in A2AR KO mice is not associated with a reduced density of TrkB receptors. BDNF protein levels are significantly reduced in the brain of A2AR KO vs. WT mice The BDNF protein levels were then measured on hippocampal extracts from A2AR KO and WT mice using the BDNF immunoassay system [enzyme-linked immunosorbent assay (ELISA) kit; Promega]. Interestingly, hippocampal BDNF levels were significantly lower in the A2AR KO mice compared with the WT littermates. As shown in Fig. 2, such an effect was not confined to the hippocampus, as a significant loss of BDNF levels was observed also in the striatum of A2A KO vs. WT mice. Fig. 2Brain-derived neurotrophic factor (BDNF) levels are significantly reduced in the hippocampus and striatum of A2A receptor (R) knock-out (KO) mice. BDNF concentration was significantly reduced in extracts of hippocampal and striatal tissues prepared from A2AR mice compared with wild-type WT mice. The amount of BDNF (total) is expressed as pg/mg of wet tissue [means ± standard error of the mean (SEM) from five mice/group]. *P < 0.05 vs. WT This result indicates that the presence of A2ARs is required to maintain normal BDNF levels in the brain. Although the mechanism by which A2ARs regulate BDNF is yet undetermined, it is worth mentioning that the cAMP-PKA pathway (the main transduction system operated by A2ARs) has been implicated not only in “gating” BDNF functions [30], but also in modulating BDNF gene transcription [31] and release [32]. Furthermore, Kolarow et al. [33] showed that the PKA inhibitor Rp-cAMP significantly inhibited and delayed BDNF secretion in hippocampal neurons. Interestingly, the endogenous state of activation of A2ARs seems to be adequate to sustain a normal BDNF tone in WT mice, as no increase in the hippocampal levels of BDNF were observed in WT mice treated with a single (0.5 mg/kg i.p sacrifice 24 h later) or with repeated (0.5 mg/kg per day over 5 days) doses of the A2A agonist CGS 21680. On their whole, the experiments performed in A2A KO mice indicate that A2ARs are essential for the tone and synaptic activity of BDNF. It should be noted, however, that the stimulation or the blockade of A2ARs can induce effects very different from those achieved after stimulation or blockade of the BDNF/TRkB system. For instance, whereas BDNF is thought to play an important role in memory processes [34, 35], adenosine A2ARs seem to negatively influence them. Indeed, an improvement in spatial memory has been reported in A2AR KO mice [36], whereas working memory was impaired in rats overexpressing A2ARs [37]. Another consideration that apparently weakens the importance of the A2AR/BDNF interaction is that A2ARs and TrkB Rs undergo opposite age-related changes in the hippocampus [38]. According to the authors’ interpretation, however, the relationship between age-related changes in the density of TrkB and A2AR receptors does play a role in the modulation of BDNF effects, thus even reinforcing the hypothesis of a close functional interplay between A2ARs and BDNF. A2ARs regulate striatal BDNF levels in experimental models of HD or HD-like striatal degeneration Two different models of HD were used: the quinolinic-acid (QA)-lesioned rats (pathogenetic model of HD-like striatal degeneration, [39]) and transgenic R6/2 mice (genetic HD model, [40]). For the method of QA-induced striatal lesions, see [41]. Briefly, QA (180 nmol/1 μl) was unilaterally injected in the striatum of anaesthetised rats, whereas vehicle [1 μl phosphate-buffered saline (PBS)] was injected in the other side. SCH58261 was administered at the doses of 0.01 and 1 mg/kg i.p. 20 min before QA lesion. The levels of BDNF were measured 24 h after surgery. As shown in Fig. 3, BDNF levels were significantly increased in the QA-lesioned vs. the control side. In animals pretreated with both doses of SCH58261 the rise in striatal BDNF was completely prevented. Worthy of note, SCH58261 markedly reduced BDNF levels in the control (vehicle-injected) side also (Fig. 3). As the early rise in BDNF levels most probably reflects a compensatory mechanism following QA injection, one can consider that SCH58261 may worsen QA effects by preventing BDNF increase. Previous studies have, however, demonstrated that this is not the case. We found, indeed, that whereas SCH58261 exerted very clear neuroprotective effects towards QA at 0.01 mg/kg i.p., it was no longer neuroprotective when administered at 1 mg/kg [39]. The finding that SCH58261 reduces BDNF levels irrespective of the dose (neuroprotective or non-neuroprotective) indicates that early changes in the striatal BDNF levels do not seem to correlate with the clinical and neuropathological outcome in the QA model. Fig. 3The A2A receptor (R)-antagonist SCH58261 reduces striatal brain-derived neurotrophic factor (BDNF) levels in control and quinolinic-acid (QA)-lesioned rats. QA (180 nmol/1 μl) was unilaterally injected in the striatum of anaesthetised rats, whereas vehicle [1 μl phosphate-buffered saline (PBS)] was injected in the other side. SCH58261 was administered at the doses of 0.01 and 1 mg/kg i.p., 20 min before QA lesion. The levels of free mature BDNF were measured 24 h after surgery. BDNF levels were significantly increased in the QA-lesioned vs. the control side. In animals pretreated with both doses of SCH58261, the rise in striatal BDNF was completely prevented. N 5/group. °P < 0.05 vs. PBS. *P < 0.05 vs. corresponding vehicle R6/2 HD mice may represent a more suitable model to evaluate whether A2AR antagonists have a worsening influence through the reduction of striatal BDNF levels. Indeed, the administration of BDNF has been reported to have beneficial effects in transgenic models of HD [42, 43]. Considering that in the transgenic mice the neurodegenerative process progressively takes place over several weeks, to ascertain whether A2AR blockade could influence striatal BDNF levels, we decided to treat R6/2 mice chronically with SCH58261. The drug was administered at the dose of 0.01 mg/kg per day i.p. over 1 or 3 weeks starting from the fifth week of age. Another group of R6/2 mice was treated with vehicle over 3 weeks. BDNF levels were then assayed at the beginning of the symptomatic phase (8 weeks of age). Three groups of age-matched WT littermates were used as controls and treated in the same way described as above. As shown in Fig. 4, BDNF protein levels were not reduced, and showed instead a nonsignificant tendency to increase, in the striatum of early symptomatic R6/2 vs. WT mice. This finding was unexpected, as a significant decrease in BDNF mRNA has been recently reported in the striatum of R6/2 mice of the same age [43]. Although we have not explored the mechanisms responsible for such a discrepancy, a dissociation between the levels of BDNF protein and mRNA has already been observed in the brains of transgenic R6/1 HD mice [44]. Another reason for which one would expect a reduction in BDNF levels in the striatum of R6/2 mice is that a marked reduction in the expression of A2ARs (which, as mentioned above, are very important to maintain normal BDNF levels) was reported in these mice [45]. More recently, however, the A2AR-stimulated adenylyl cyclase, the A2AR density, and their affinity for the agonist CGS21680 were found unchanged in the striatum of clearly symptomatic R6/2 mice [46]. Fig. 4The A2A receptor (R)-antagonist SCH58261 reduces striatal brain-derived neurotrophic factor (BDNF) levels in R6/2 HD mice SCH58261 was administered at the dose of 0.01 mg/kg per day i.p. over 1 or 3 weeks starting from the fifth week of age. Free mature BDNF levels were then assayed at the beginning of the symptomatic phase (8 weeks of age). Three groups of age-matched wild-type (WT) littermates were used as controls and treated in the same way as described above. Both schedules (1 and 3 weeks) of SCH treatment significantly reduced BDNF levels in the striatum of either R6/2 and WT mice. N  4–7/group. *P < 0.05 vs. corresponding control group (two-tailed unpaired t test) As for the influence of A2AR blockade, both schedules (1 and 3 weeks) of SCH treatment significantly reduced BDNF levels in the striatum of either R6/2 and WT mice (Fig. 4). Again, however, such an effect does not necessarily imply a negative influence of SCH58261 on HD mice. Indeed, in recent experiments, we found that SCH treatment (0.01 mg/kg per day i.p. between the 5th and the 6th weeks of age) exerted some beneficial effects, namely, normalisation of emotional behaviour and restoration of a normal sensitivity to NMDA in corticostriatal slices (Domenici et al., manuscript in preparation). Thus, although the pharmacological blockade of A2ARs significantly reduces striatal BDNF levels in experimental models of HD or HD-like neurodegeneration, this does not seem to have a negative impact on the course of the disease. Of course, it is still possible that reducing BDNF levels at other time points of the pathological process has a much marked influence on the disease. This issue, as well as the evaluation of BDNF protein changes at different stages of the disease, will be the object of future studies. Conclusions Over the last several years, BDNF has emerged not only as a potent neuromodulator but also as a substance exerting fast excitatory actions in neurons, controlling resting membrane potential, neuronal excitability and synaptic transmission and participating in the induction of long-term changes in synaptic transmission [47]. In agreement with the recent evidence that A2ARs play a major role in regulating BDNF functions [16, 48], data reported in this article indicate that the presence and tonic activation of A2ARs are necessary to allow BDNF-induced potentiation of synaptic transmission. As for the mechanisms responsible for the permissive effects of A2ARs, data indicate an involvement of the cAMP-PKA pathway, the main transduction mechanism operated by A2ARs. The finding of reduced BDNF levels in the brain of A2AR KO mice indicates that A2ARs are critical not only to allow the synaptic effects of BDNF but also to maintain normal BDNF levels. Findings also point out that A2ARs tonically regulate BDNF levels in different areas of the brain, irrespective of their levels of expression. The tonic effect exerted by A2ARs in the regulation of BDNF levels is also evident in experimental models of HD (R6/2 mice) and of HD-like striatal degeneration (QA lesion in rats). Given that BDNF delivery has beneficial effects in models of HD [42, 43], the possible functional consequences of reducing striatal BDNF levels in those models need to be clarified. In particular, further investigations on trophic factors and associated signal pathways are warranted before A2AR antagonists can be considered as a suitable therapeutic approach to HD [24].

Histochem_Cell_Biol-4-1-2386529

Intermediate filament cytoskeleton of the liver in health and disease

Intermediate filaments (IFs) represent the largest cytoskeletal gene family comprising ~70 genes expressed in tissue specific manner. In addition to scaffolding function, they form complex signaling platforms and interact with various kinases, adaptor, and apoptotic proteins. IFs are established cytoprotectants and IF variants are associated with >30 human diseases. Furthermore, IF-containing inclusion bodies are characteristic features of several neurodegenerative, muscular, and other disorders. Acidic (type I) and basic keratins (type II) build obligatory type I and type II heteropolymers and are expressed in epithelial cells. Adult hepatocytes contain K8 and K18 as their only cytoplasmic IF pair, whereas cholangiocytes express K7 and K19 in addition. K8/K18-deficient animals exhibit a marked susceptibility to various toxic agents and Fas-induced apoptosis. In humans, K8/K18 variants predispose to development of end-stage liver disease and acute liver failure (ALF). K8/K18 variants also associate with development of liver fibrosis in patients with chronic hepatitis C. Mallory-Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated K8/K18, chaperones and sequestosome1/p62 (p62) as their major constituents. MDBs are found in various liver diseases including alcoholic and non-alcoholic steatohepatitis and can be formed in mice by feeding hepatotoxic substances griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDBs also arise in cell culture after transfection with K8/K18, ubiquitin, and p62. Major factors that determine MDB formation in vivo are the type of stress (with oxidative stress as a major player), the extent of stress-induced protein misfolding and resulting chaperone, proteasome and autophagy overload, keratin 8 excess, transglutaminase activation with transamidation of keratin 8 and p62 upregulation. Intermediate filaments in health and disease Together with the actin microfilaments and the microtubules, intermediate filaments (IFs) are the components of the cytoskeleton of eukaryotic cells, which is involved in the maintenance of cell shape, locomotion, intracellular organization, and transport (Bershadsky and Vasiliev 1988; Ku et al. 1999). IF proteins comprise a large family, which includes ~70 different genes (Omary et al. 2004; Kim and Coulombe 2007; Herrmann et al. 2007; Goldman et al. 2008). They are further divided into six subtypes (Table 1), which are, at least in part, expressed in a cell type (and differentiation)-dependent manner (Omary et al. 2004; Kim and Coulombe 2007; Goldman et al. 2008). Accordingly, IFs serve as cell type markers and antibodies to IF proteins are widely used today in diagnostic pathology (Wick 2000; Barak et al. 2004). Individual IF proteins consist of a conserved central coiled-coil α-helical rod domain (interrupted by linkers) which is flanked by N-terminal (head) and C-terminal (tail) domains (Omary et al. 2006; Godsel et al. 2008; Kim and Coulombe 2007; Herrmann et al. 2007; Goldman et al. 2008). The N- and C-terminal domains contribute to the structural heterogeneity and are major sites of posttranslational modifications with phosphorylation being the best characterized one (Omary et al. 1998; 2006). This makes them important regulatory domains, since dynamic changes in phosphorylation status are responsible for alterations in IF dynamics, solubility, and organization (Omary et al. 2006).Table 1Intermediate filament proteinsTypeName/LocalizationDisease locationRemarksI (n = 28)K9-28 (epithelia)K10,14,16,17-skinAcidic keratinsK31-40 (hair/nail)K12-cornea(pI < 5.7)K13-stratified mucosaType I/II obligate 1:1 polymersK16,17-nailK18a-liverII (n = 26)K1-8, K71-80 (epithelia)K1,2e,5,9-skinBasic keratinsK81-86 (hair/nail)K3-cornea(pI ≥ 6.0)K4-stratified mucosaType I/II obligate 1:1K6a,6b-nailpolymersK8a-liverK75a,81,83,85,86-hairIIIDesmin (muscle)Muscle, heartDesmin, vimentin and GFAP are found in stellate cellsVimentin (mesenchymal)Peripherin (neurons)Braina, spinal cordaGFAP (astrocytes/glia)BrainIVNF-L (neurons)Braina, spinal cordα-internexin forms polymers with NFsNF-M (neurons)Braina, spinal cordaNF-H (neurons)Brain, spinal cordaSynemin β is also called desmuslin.α-internexin (CNS neurons)Nestin-stem cell markerSynemins (muscle)Syncoilin (muscle)NestinVLamin A/C (ubiquitous)Heart, muscle, fat, premature aging, complex defectsThe only nuclear IFs, longer rod domainLamin B1/2 (ubiquitous)OrphanPhakinin (lens)LensBeaded filaments in lens epitheliaFilensin (lens)Lensa Not a causative association, variants represent a risk factor. For an overview about the new keratin nomenclature, see Schweizer et al. (2006) In addition to the posttranslational modification, IF function is modified and complemented through interaction with a variety of IF-associated proteins (IFAP; Table 2; Green et al. 2005; Omary et al. 2006; Kim and Coulombe 2007). These proteins can be subdivided into several subgroups, which reflect multiple IF functions (Green et al. 2005). For example, IFs interact with a variety of anchoring proteins thereby forming transcellular networks which contribute to proper tissue architecture. IFAPs also include several cytolinker proteins, which provide the structural framework for coordinated cytoskeletal function (Table 2). By doing that, they are important mechanical stabilizers and accordingly, IF disruption results in increased mechanical fragility (Omary et al. 2004; Ku et al. 2007; Herrmann et al. 2007). The scaffolding function of IFs is best seen in IF-deficient animals, who exhibit disrupted cellular architecture, protein mistargeting as well as alterations in organelle localization and function (Toivola et al. 2005). In the case of lamin deficiencies, the impairment of nuclear composition has profound impact on many aspects of normal nuclear functions such as epigenetic changes, chromatin organization or DNA transcription, and repair (Dechat et al. 2008).Table 2Examples of IF-associated proteinsType of interactionExamplesFunctionAnchoringDesmoplakin, BPAG1, α-dystobrevinTissue architectureCytolinkerPlectin, Filaggrin,Cellular architectureChaperonesHsp27, α-B-crystallin, Hsp70Protein foldingKinasesPKC, Cdk5IF regulation, phosphate sink, cell cycleAdaptor proteins14-3-3 protein, AP-3Multiple effectsMembrane proteinsPolycystin-1UnknownApoptotic proteinsTRADD, TNFR2, C-Flip, Caspase3/9Apoptosis regulationMotor proteinsDynein, KinesinMovement of IF components IFs are not just simple cellular scaffolds, they rather build complex signaling platforms (Pallari and Eriksson 2006; Kim and Coulombe 2007). IFs interact with a variety of enzymatic and adaptor proteins, thereby affecting a multitude of cellular functions. For example, keratins associate with 14-3-3 proteins in a phosphorylation-dependent manner and this interaction regulates cell growth and cell cycle progression (Ku et al. 2007; Kim and Coulombe 2007). IF phosphorylation through associated kinases does not only regulate IF properties, but IFs also serve as phosphate “sponge” thereby preventing activation of other, potentially pro-apoptotic, substrates (Omary et al. 2006; Ku et al. 2007). In addition to that, IFs also directly participate in apoptosis regulation through binding of several apoptosis-related molecules (Marceau et al. 2007). In contrast to the actin and tubulin system, IFs emerged later in the evolution and are important supportive elements of the cell rather than their essential components. Therefore, IF variants are observed in various human diseases, which reflect their tissue specific distribution, whereas only few actin and tubulin variants have been described so far, likely due to their embryolethality (Ku et al. 1999; Omary et al. 2004). Currently, more than 30 diseases are caused by/associated with IF mutations (Omary et al. 2004). Among them, keratin-related- and lamin-related disorders are the best studied ones. For example, mutations in keratins 5/14 cause a blistering skin disease termed Epidermolysis bullosa simplex, whereas mutations in lamins A and C result, among others, in different diseases including premature aging, cardiomyopathy, and lipodystrophy (Omary et al. 2004; Dechat et al. 2008). The disease relevance of IFs is also highlighted by a variety of IF-containing inclusion bodies, which represent the pathological hallmarks of several neurodegenerative, muscular, and other disorders (Goebel 1998; Ross and Poirier 2004; Omary et al. 2004; Cairns et al. 2004). These aggregates share a variety of features such as presence of misfolded, ubiquitinated structural proteins together with variable amounts of chaperones and p62 (Kuusisto et al. 2001; Zatloukal et al. 2002; Ross and Poirier 2004). Among the IF-related inclusions, Mallory-Denk bodies (MDBs) are the most common and also the best studied ones due to the availability of animal MDB models (Denk et al. 1975; Yokoo et al. 1982; Jensen and Gluud 1994; Denk et al. 2000; Zatloukal et al. 2007). Therefore, one focus of our review will be to describe MDB as a prototype of IF-related inclusion body, which should offer useful insights into the formation of IF-related aggregates in multiple human diseases. Keratins as epithelially expressed IFs Keratins represent the largest subfamily of IFs consisting of >50 unique gene product members (Schweizer et al. 2006; Kim and Coulombe 2007; Godsel et al. 2008) which include 37 epithelial and 17 hair keratin members in humans (Table 1; Schweizer et al. 2006). Based on their pI, epithelial keratins can be subdivided in types I (acidic) and II (basic) corresponding to keratins 9–20 (K9−K28) and keratins 1–8 plus keratins 71–80 (K1–K8; K71–K80), respectively (Table 1; Coulombe and Omary 2002; Schweizer et al. 2006). Keratins are found as obligatory type I and type II heteropolymers (i.e., consisting of at least one type I and one type II keratin) and a homozygous disruption of a keratin results in degradation of its keratin partner at the protein level (Ku and Omary 2000; Omary et al. 2004). Similarly to IFs, keratins are expressed in a tissue-specific manner, with different pairs being the major cellular IFs in different cell populations (Moll et al. 1982; Ku et al. 1999; Coulombe and Omary 2002). For example, “simple” (i.e., single layered) epithelia, as found in digestive organs, express K8 together with variable levels of K7, K18, K19, and K20 depending on the tissue (Moll et al. 1982; Ku et al. 1999; Coulombe and Omary 2002; Ku et al. 2007). In contrast, stratified epithelia, like epidermis, express K5/K14 in the basal and K1/K10 in the suprabasal keratinocytes, respectively (Moll et al. 1982; Lane and McLean 2004; Coulombe and Omary 2002). Despite their similar molecular composition, “simple” and “stratified” keratins are not interchangeable, as shown in K14-null mice, whose phenotype was only partially restored by addition of K18 (Hutton et al. 1998). Furthermore, keratins have their preferential binding partners in vivo and the lack of such partner leads to their rapid degradation (Magin 1998). This contrasts with the in vitro situation, where IF assembly is more promiscuous (Hatzfeld and Franke 1985). K8/K18/K19 are promising serological markers based on their high abundance (approximately 0.3% of total liver protein) and intracellular localization under basal conditions with release into blood upon liver injury (Omary et al. 2002; Ku et al. 2007). However, one important caveat is the fact, that K8/K18/K19 are expressed in most simple epithelial cells and are therefore not liver-specific (Moll et al. 1982; Ku et al. 1999). The K8/K18/K19 epitopes used in serologic diagnosis can be divided into two classes, that is, non-specific and apoptosis-generated epitopes. The former class constitutes established tumor markers such as tissue polypeptide antigen (TPA, represents total K8/K18/K19), tissue polypeptide specific antigen (TPS, derived from K18), and CYtokeratin FRAgment 21-1 (CYFRA 21-1, derived from K19). Their original clinical use was to monitor treatment response and to detect recurring tumors (Barak et al. 2004). However, later studies showed that these epitope serum levels are also elevated in non-malignant diseases and might be a general marker of tissue injury (Gonzalez-Quintela et al. 2006a, b; Tarantino et al. 2007). The apoptosis-specific keratin antibodies are based on the finding, that type I keratins are cleaved at a conserved VEMD/VEVD residue during apoptosis. In addition to that, K18 posseses a second, K18 specific caspase-cleavage site at Asp396, which is an early event during apoptosis preceding the cleavage at the VEMD/VEVD motif (Oshima 2002; Marceau et al. 2007). The cleavage of human K18 at Asp396 can be monitored using the M30 antibody (Leers et al. 1999) and M30-Ab ELISA has become a useful serologic test for determining liver disease severity. For example, elevated serum M30 titers can distinguish simple steatosis from non-alcoholic steatohepatitis (Wieckowska et al. 2006) and predict several important prognostic parameters in patients with chronic hepatitis C infection (Bantel et al. 2004; Volkmann et al. 2006). As another tool for detecting apoptotic keratin fragments, an antibody specific to the conserved K18/K19 cleavage site at Asp237 was recently generated (Tao et al. 2008). It detects both mouse and human K18/K19 fragments and appears to be more sensitive than the established Asp396-Ab (Tao et al. 2008). Measuring the serum levels of apoptosis-specific keratin fragments should also improve our understanding of chronic liver disease, where apoptotic cell death is an important pathogenic feature (Malhi et al. 2006). In addition to that, monitoring the phosphorylation status of the circulating keratin fragments might be useful. Keratins undergo dynamic phosphorylation during mitosis, apoptosis and a variety of stress situations (Omary et al. 1998; Omary et al. 2006) and their in situ phosphorylation status is a marker of human liver disease progression (Toivola et al. 2004). However, it is currently unknown whether the phosphorylation status of the circulating keratin fragments correlates with the situation in situ. Hepatic phenotype in keratin-deficient transgenic animals The liver consists of different cell types with characteristic IF composition (Table 3; Omary et al. 2002). Adult hepatocytes are unique among simple epithelial cells in that they express exclusively K8 and K18, whereas other glandular epithelia exhibit a more complex keratin expression pattern (Omary et al. 2002; Ku et al. 2007). The hepatocytic keratin IF network is dense, particularly around bile canaliculi and at the cell periphery, and acts as cytoskeletal backbone to the functionally more dynamic and contractile actin microfilament system (Zatloukal et al. 2004). Biliary epithelial cells differ from hepatocytes by additional expression of keratin 7 and 19 (Omary et al. 2002; Zatloukal et al. 2004). Keratins in cholangiocytes, but not hepatocytes, exhibit polarized and compartment-specific expression pattern (Omary et al. 2002; Zatloukal et al. 2004). The biological significance of such an expression is enigmatic, but it may be related to polarity and secretory processes. Among non-epithelial cells, stellate cells express variable amounts of GFAP, desmin, vimentin, and nestin dependent on their activation status, localization, and other parameters (Table 3; Geerts 2001).Table 3IFs of liver cell populationsCell typeIF compositionHepatocyteK8/K18aOval cellsK7/K8/K18/K19CholangiocyteK7/K8/K18/K19Kupffer cellVimentinStellate cellGFAP, Vimentin, Desmin, NestinbEndothelial cellVimentinModified from Omary et al. (2002)a During embryogenesis, hepatocytes also express variable levels of K19 (Vassy et al. 1997)b Stellate cells represent a highly heterogeneous population with variable IF expression dependent on species, activation status of the cell, location within the hepatic lobe and many other parameters (Geerts 2001) Studies in keratin knock-out mice revealed that the regular liver development does not require the presence of K8, K18, or K19 (Ku et al. 2007). However, K8/K18 knockout mice exhibited mild chronic hepatitis, hepatocyte fragility and were markedly more sensitive to a variety of stress conditions (Omary et al. 2002; Zatloukal et al. 2004; Ku et al. 2007). Furthermore, K8/K18 transgenic mice were developed, which over-express different single-amino-acid variants (Ku et al. 2007). Among them, the K18 R89C variant resulted in disruption of hepatocyte IF network and exhibited a phenotype reminiscent of the situation in K8/K18-knockout mice (Ku et al. 1995, 1996). K18 R89C mice also predisposed to development of thioacetamide-induced liver fibrosis (Strnad et al. 2008). Ablations of different K8/K18 phosphorylation sites usually led to a somewhat milder phenotype which became apparent in stress situations, but not under basal conditions (Ku et al. 2007). For example, K18 S52A variant resulted in increased sensitivity to microcystin-LR-induced liver injury, whereas K8 S73A mice were predisposed to Fas-induced liver apoptosis (Ku et al. 1998; Ku and Omary 2006). The ablation of K18 S33 phosphorylation site, which regulates the binding to 14-3-3 proteins, caused limited mitotic arrest and accumulation of abnormal mitotic figures after partial hepatectomy (Ku et al. 2002a). In contrast, mice lacking K19 did not have an obvious liver phenotype (Tao et al. 2003), but surprisingly exhibited skeletal myopathy (Stone et al. 2007). GFAP/vimentin-knockouts displayed compromised astrocytic function with attenuated reactive gliosis, but no obvious alteration in the in vitro activation of hepatic stellate cells despite the lack of IF network (Geerts et al. 2001; Pekny and Pekna 2004). However, in vivo studies are needed to conclusively address the fibrogenic potency of these transgenic mice. Keratin variants in liver disease The large body of evidence from animal studies showing the importance of K8/K18 for liver homeostasis led to a search for keratin mutations in patients with liver diseases. Several K8/K18 variants were found to associate with the development of cryptogenic liver disease (Ku et al. 2001). In subsequent studies, K8/K18 were shown to represent susceptibility genes for development of end-stage liver disease of multiple etiologies (Ku et al. 2003a, 2005). In particular, biologically significant K8/K18 variants were found in 44 of 467 liver explants (12.4%), but only in 11 out of 349 analyzed blood bank donors, which were used as a control group (P < 0.0001, prevalence OR3.8; 95% CI = 2.1–7). Furthermore, K8/K18 variants associate with liver fibrosis progression in patients with chronic hepatitis C (Strnad et al. 2006). K8 R340H represents the most common amino acid altering K8/K18 variant and it is the only one, which was significantly associated with development of end-stage liver disease (Ku et al. 2005). Larger studies are needed to analyze the pathological significance of the other less common K8/K18 variants (Ku et al. 2007). In contrast to human association studies, experiments in K8/K18-deficient mice were shown to predispose mainly to acute liver injury (Ku et al. 2007). To address this issue, we recently analyzed a large cohort of patients with acute liver failure (ALF). K8/K18 variants were significantly more frequent in total ALF patient cohort (46/345; 13.3%) and in patients with acetaminophen-induced ALF (21/169; 12.4%) when compared to blood bank donors (11/349; 3.7%; P < 0.002 for both comparisons). Among the single polymorphisms, the K8 R340H variant was found at significantly higher frequencies in the whole ALF cohort as well as in the acetaminophen-induced ALF subgroup (frequency 6.6 and 7.1%, respectively vs. 3.1% in the control group; P < 0.03 for both comparisons). In addition, transgenic mice over-expressing K8 R340 variants displayed augmented acetaminophen-induced liver toxicity. In conclusion, K8/K18 are also susceptibility genes for development of ALF and K8/K18 variants may predispose to drug-induced liver injury (Strnad et al., unpublished data). Up to date, only one published study analyzed the polymorphisms in K19 gene and found no association between K19 variants and inflammatory bowel disease (Tao et al. 2007). Interestingly, we recently observed K19 G17S variant in three out of 190 patients with primary biliary cirrhosis, but none was found in control blood bank donors (200 samples; Zhong et al., unpublished data). However, larger studies are needed to address the importance of K19 in biliary diseases. The human K8/K18 variants described above do not cause a particular liver disease per se, they just pose a risk factor for its development. This is different from the situation in stratified epithelia, where keratin mutations result in several monogenic keratin diseases (Lane and McLean 2004; Omary et al. 2004). This discrepancy may be caused either by the intrinsic difference between keratins of simple and stratified epithelia (Hutton et al. 1998) or by the different localization of the variants within the protein backbone. To that end, disease-causing keratin mutations in stratified epithelia are clustered in the highly conserved helix initiation and termination motif, whereas K8/K18 disease-predisposing variants are observed in more variable domains (Owens and Lane 2004; Omary et al. 2004; Ku et al. 2007). The disturbance in cytoprotective function of keratins is the likely mechanisms by which K8 and K18 variants predispose to liver disorders. For example, K8/K18 are anti-apoptotic proteins and this ability is hampered in keratin-deficient animals (Oshima 2002; Ku and Omary 2006; Marceau et al. 2007; Ku et al. 2007). The ways of interaction between keratins and apoptosis are manifold. K8/K18 bind to several apoptotic proteins and type I keratins are established caspase substrates (Oshima 2002; Green et al. 2005; Marceau et al. 2007). In addition, K8/K18 serve as physiologic kinase substrates in vitro and in transgenic mice and an ablation of the K8 S73 phosphorylation site or introduction of the naturally occurring K8 G61C variant leads to increased apoptosis through increased phosphorylation of pro-apoptotic proteins (Ku et al. 2002b; Ku and Omary 2006). The keratin-mediated anti-apoptotic function may be highly relevant given the importance of apoptosis in liver disease and the pro-fibrogenic properties of elevated rate of apoptotic cell death (Friedman 2004; Malhi et al. 2006). Keratins exhibit anti-oxidative properties, as they sequester oxidatively damaged proteins, and similarly, the K18 R89C variant primes the liver towards oxidative injury (Tao et al. 2005; Zhou et al. 2005). This keratin property might be helpful in attenuating both liver fibrosis and acetaminophen-induced liver injury (Parola and Robino 2001; Jaeschke et al. 2003). Keratins are also established stress-inducible proteins, which are upregulated both in humans and mice under several, mainly cholestatic conditions (Fickert et al. 2002, 2003; Zatloukal et al. 2007; Ku et al. 2007; Strnad et al. 2008). Keratin variants may interfere with keratin upregulation or simply result in decreased keratin levels due to protein instability (as seen for K18 R89C variant in the liver; Ku et al. 1995). The naturally occurring K8/K18 variants interfere with basic IF properties such as K8/K18 filament assembly and keratin solubility (Ku et al. 2001; Owens et al. 2004; Ku et al. 2007). Due to altered protein conformation, some of them impair potentially cytoprotective keratin phosphorylation at adjacent residues (as seen in K8 G61C and K8 G434S variant; Ku et al. 2005; Ku and Omary 2006). K8/K18 variants may also affect organelle function, as seen in K8 knockout mice, which exhibit altered mitochondrial shape, localization, and alterations in several mitochondrial proteins (Toivola et al. 2005). Despite the various cytoprotective effects of keratins, their impact seems to be limited to certain conditions. For example, K18 R89C mice are predisposed to Fas but not TNF-induced apoptosis and the same mice develop more pronounced liver fibrosis after thioacetamide, but not after carbon tetrachloride injection (Ku et al. 2003b; Strnad et al. 2008). It is also unknown which one of the keratin-mediated effects is important in particular disease settings. The recently established transgenic mouse lines overexpressing the naturally occurring K8 G61C and R340H variants will likely offer valuable insights in this respect (Ku and Omary 2006; Zhou et al., unpublished data). Keratin network alteration in steatohepatitis of the alcoholic (ASH) and the non-alcoholic (NASH) type Steatohepatitis is characterized by hepatocyte “ballooning”, that is, swelling and rounding with clearing of the cytoplasm, which prevails in the perivenular zone and is often associated with pericellular fibrosis, predominantly granulocytic inflammation (“satellitosis”), steatosis (usually of macrovesicular type), and cholestasis (Brunt 2004; Lefkowitch 2005). Ballooned cells are often associated with granular, rope- or clump-like cytoplasmic inclusions, called Mallory-Denk bodies (MDBs) (originally also designated alcoholic hyalin, which is, however, a misnomer since they are not specific for alcoholic etiology) together with derangement or even disappearance of the keratin IF cytoskeleton (Mallory 1911; Denk et al. 2000; Zatloukal et al. 2007; Fig. 1a, b). The disappearance of the keratin immunostaining in ballooned hepatocytes is reasonably specific for ASH and NASH, since it is not seen in ballooned cells of viral hepatitis or toxic damage and can therefore be used as an objective morphologic parameter in grading of steatohepatitis (Lackner et al. 2008). However, ballooning (with concomitant cytoskeletal alterations) and MDB formation are not entirely interchangeable since not all ballooned hepatocytes contain MDBs. Fig. 1MDBs are readily detected by different methods. In clinical routine, MDBs are usually detected as eosinophilic aggregates in standard hematoxylin and eosin stained sections (a). After chromotrope aniline blue staining, MDBs appear as blue structures, often with red center (b). Immunofluorescence or immunohistochemical staining represents a more sensitive method for MDB detection than conventional histological stainings, but is strongly dependent on the antibody used as well as the staining protocol. MDBs can be reliably detected with antibodies against K8/K18 [green and redchannel in (c) and (d), respectively] or p62 [red channel in (c)], whereas only some MDBs stain with antibodies to phosphorylated keratins such as K8 pS431 antibody [green channel in (d)]. In both immunofluorescence pictures, MDBs are seen as yellow structure due to co-localization of both visualized epitopes MDBs are typical morphological features of ASH and NASH, although NASH usually exhibits slightly less prominent MDBs than the ones seen in ASH (Brunt 2004; Zatloukal et al. 2007). MDBs can also be detected after intestinal bypass surgery for morbid obesity, in chronic cholestasis, particularly in late stages of primary biliary cirrhosis, Wilson disease and other types of copper toxicosis, various metabolic disturbances, and hepatocellular neoplasms (Müller et al. 2004; Zatloukal et al. 2007; Fig. 2). In contrast, MDBs have not been observed in the context of acute cholestasis, acute viral hepatitis and a variety of acute toxic or drug-induced liver diseases (Jensen and Gluud 1994, Zatloukal et al. 2007; Ku et al. 2007). However, even in potentially MDB-forming liver diseases, MDBs are found only in a subset of patients, partially (but not completely) depending on the sensitivity of the detection method used. For example, when using immunohistochemistry for keratin or ubiquitin, MDBs were found in about 70% of ASH cases in contrast to 40% seen in hematoxilin-eosin-stained sections (Ray 1987).Fig. 2MDBs are seen in various human liver diseases. Immunohistochemical staining with p62 antibody visualizes the presence of multiple irregularly shaped aggregates in patients with alcoholic steatohepatitis (a), non-alcoholic steatohepatitis (b), Indian childhood cirrhosis and (c), idiopathic copper toxicosis (d) This suggests that MDBs require either a specific pathogenetic constellation or genetic predisposition for its formation, which is present only in a subset of patients. Apart from MDBs, additional features may be observed in some chronic cholestatic conditions. For example, a low percentage of hepatocytes express keratin 7 and to a lesser extent keratin 19 which indicate that these cells acquire features of precursor cells which normally express keratin 8, 18, 7, and 19 during regeneration (Van Eyken et al. 1988; Zatloukal et al. 2004). In idiopathic copper toxicosis and hepatocellular carcinoma, MDBs may coincide with another type of cytoplasmic inclusions, termed intracellular hyaline bodies (IHBs), which share several components with MDBs, but do not contain keratins (Stumptner et al. 1999; Denk et al. 2006). The ease of MDB detection makes them attractive morphologic markers. However, correlation between the clinical disease manifestation/progression on one side and hepatocyte ballooning with MDB formation on the other is imperfect. For example, patients with severe clinical symptoms of ASH sometimes show only moderate histopathological alterations with few or no MDBs, whereas patients with pronounced histological alterations do not necessarily exhibit significant clinical and laboratory abnormalities (Zatloukal et al. 2007). Despite that, controlled clinical-pathologic studies comparing NASH patients with ambulatory and hospitalized alcoholics revealed that hepatocellular damage, presence of MDBs, inflammation, and fibrosis collectively correlated with disease severity (Cortez-Pinto et al. 2003). Also in other studies, hepatocellular ballooning and MDB formation was positively correlated with disease progression, development of fibrosis, and cirrhosis and liver-related mortality (Orrego et al. 1987; Matteoni et al. 1999; Gramlich et al. 2004; Mendler et al. 2005). Morphology and composition of MDBs MDBs are irregularly shaped, usually dense cytoplasmic inclusions of different sizes (Mallory 1911). Small MDBs arise in association with IF bundles throughout the cytoplasm, whereas larger MDBs are often seen in the perinuclear region (Denk et al. 2000; Zatloukal et al. 2007; Ku et al. 2007). Ultrastructurally, they usually consist of haphazardly arranged filamentous rods, approximately 10–15 nm in diameter, covered by a fuzzy coat (designated as type II by Yokoo et al. 1972). In addition, MDBs with an electron dense granular to amorphous center (designated type 3) are seen predominantly in older inclusions, and filaments in parallel arrangement were also described (designated as type I) but seem to be exceedingly rare (Yokoo et al. 1972). Keratins 8 and 18, sequestosome 1/p62 (p62) and ubiquitin are major, and low and high molecular weight heat shock proteins (HSP 70, HSP 25), but also proteins of the protein degradation machinery, are minor constituents (Denk et al. 2000; Riley et al. 2002, 2003; Zatloukal et al. 2007; Ku et al. 2007; Figs. 1c, d; Fig. 2). Keratins 7, 19, and 20 have also occasionally been detected (Cadrin et al. 1990; Dinges et al. 1992). The K8/K18 within MDBs exhibit increased β-sheet structure (Cadrin et al. 1991; Kachi et al. 1993), are hyperphosphorylated, partially degraded and cross-linked (Hazan et al. 1986; Zatloukal et al. 1992; Cadrin et al. 1995; Stumptner et al. 2000; Fig. 1d). The list of MDB components is likely to grow since, in addition to intrinsic components, proteins non-specifically incorporated in the aggregate have to be expected. Pathogenesis of MDBs Studies on MDB formation and composition are greatly enhanced by the availability of animal models. MDBs can be induced under standardized conditions and their fate followed in mouse liver by chronic griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) administration (Denk et al. 1975; Yokoo et al. 1982; Denk et al. 2000; Zatloukal et al. 2004, 2007; Fig. 3c, d).Fig. 3MDBs formed in DDC-fed animals resemble inclusion bodies observed in human diseases. Liver sections were double labeled with antibodies to K8/K18 (green) and p62 (red). Samples from mouse fed DDC for 12 weeks (d) and from a patient with alcoholic steatohepatitis (b) exhibit multiple irregularly shaped inclusion bodies, which appear yellow due to presence of both epitopes. In contrast, control human (a) and mouse liver (c) display an unaffected keratin network with no apparent p62 staining. Note that DDC feeding leads to deposition of protoporphyrin, which can be seen as occasional blue pigment in (d) In patients, MDB formation is usually a chronic process requiring several years of alcohol intoxication or metabolic imbalance, that is, in association with the metabolic syndrome, Wilson disease, other metabolic disorders, and chronic cholestasis. An exception is idiopathic copper toxicosis of children in which end stage liver disease associated with MDB formation occurs at very early age (Müller et al. 2004; Zatloukal et al. 2007). Surprisingly, in alcoholics recovered from ASH, an alcohol excess may almost instantaneously lead to MDB recurrence, a situation which has been compared to an immunologic response and termed “toxic memory” (Jensen and Gluud 1994; Denk et al. 2000). The animal model of MDB formation does not only reproduce the structural and morphological features seen in humans, but also the phenomenon of “toxic memory” (Fig. 3; Denk et al. 2000; Zatloukal et al. 2004, 2007). To induce MDBs in mice, griseofulvin or DDC is usually fed for 2–3 months. MDB formation in mice is reversible, since MDBs disappear after recovery on standard diet for 1 month. However, after rechallenge of the recovered (“primed”) animals, MDBs reappear within a few days (Denk et al. 2000; Zatloukal et al. 2004, 2007). Rapid MDBs formation in primed mice is rather non-specific, since it can be triggered by a variety of stress conditions including colchicine (but not by lumicolchicine), bile acids, bile duct ligation, several other toxins, and proteasome inhibitors which were unable to induce MDBs in the naïve animal (Zatloukal et al. 2007, and references therein). The reasons for this phenomenon are as yet unclear but point to a final common pathway activated by the trigger. The availability of cellular and animal models of MDB formation led to valuable insights into the mechanism of MDB formation. Several pathogenic mechanisms were implicated in this process (Fig. 4; Dobson 2004):1.Enhanced oxidative stress2.Disproportional K8/K18 expression together with keratin modifications3.Chaperone dysfunction4.Elevated p62 levels5.Insufficient protein degradation Ad 1. The MDB-inducing conditions both in humans and mice cause elevated levels of oxidative stress (Tephly et al. 1981; Mehta et al. 2002; Dey and Cederbaum 2006; Farrell and Larter 2006) and MDBs themselves were shown to contain misfolded keratins with increased β-sheet formation (Cadrin et al. 1991; Kachi et al. 1993). Recent animal studies highlight the importance of altered methyl group metabolism and mitochondrial stress in this process (Li et al. 2008; Zatloukal et al., unpublished data). During DDC detoxification, N-methylprotoporphyrin is formed through transfer of a methyl group to heme moieties (Tephly et al. 1981). N-methylprotoporphyrin subsequently acts as a potent inhibitor of ferrochelatase, thereby causing porphyria (Tephly et al. 1980). Accordingly, MDB formation and DDC effects can be effectively attenuated by feeding S-adenosylmethionine, that is a compound involved in methyl group transfer (Li et al. 2008). This is reminiscent of the situation in ASH, which is also associated with disrupted methyl group metabolism (Schalinske and Nieman 2005).Fig. 4MDBs formation results from a complex interplay of several contributing factors. Since the cytoplasm represents a hydrophilic milieu, all exposed hydrophobic molecules (depicted by red stretches within the protein) are predisposed to aggregation. Properly folded proteins usually hide their hydrophobic stretches inside, but these get exposed in nascent protein chains or after proteins become misfolded as a consequence of oxidative stress. Chaperones bind to these hydrophobic residues and facilitate protein refolding. Alternatively, damaged proteins become polyubiquitinated and degraded either by the proteasomal or autophagic system. MDB-causing agents typically generate extensive amount of oxidative stress with increased protein misfolding. In addition, chaperone levels are downregulated and/or chaperone function is compromised. Dysbalanced K8/K18 expression precedes MDB formation, likely increases keratin misfolding and predisposes to posttranslational modifications, which may interfere with keratin refolding and/or repair. Accumulated misfolded proteins are sequestered as inclusions through the action of p62. p62 also shuttles polyubiquitinated proteins to degradative machineries. However, proteasomal degradation might be impaired by oxidative stress, may not be able to digest highly cross-linked protein species or might simply be overwhelmed by the excessive supply. On the other hand, autophagy is upregulated during MDB formation in mice and additional stimulation of autophagy attenuates MDB formation in certain conditions. Of note, supplementation of S-adenosylmethionine or mitochondrially targeted antioxidants effectively diminishes MDB formation, thereby pointing to a central role of oxidative stress in MDB generation DDC targets mitochondria, where it reacts with cytochrome P450 (Marks et al. 1985). An unbiased microarray analysis identified cytochrome P450 (Cyp) 2a5 as a major gene induced both after DDC exposure and particularly after DDC re-challenge. Moreover, Cyp2e5 overexpression spacially coincides with MDB formation (Zatloukal et al., unpublished data). Cyp2a5 is a “leaky” cytochrome which produces reactive oxygen species (Lewis et al. 1989). In that sense, it resembles human Cyp2E1, which was implicated as a source of oxidative stress in the pathogenesis of ASH and NASH (Villeneuve and Pichette 2004). To address the role of mitochondrial stress in MDB formation, we re-fed DDC-primed mice with DDC alone or in combination with the mitochondria-targeted antioxidant mito Q (Smith et al. 2003). Mito Q co-administration attenuated both MDB formation and DDC-induced liver damage. Therefore, mitochondrial oxidative stress seems to be involved in MDB formation in mice which is in good concordance to the mitochondrial dysfunction seen both in ASH and NASH patients (Pessayre 2007; Mantena et al. 2008; Zatloukal et al., unpublished data). Ad 2. Keratins are major constituents of MDBs and both altered K8/K18 expression and keratin modification seems to affect MDB formation (Zatloukal et al. 2007; Ku et al. 2007). Griseofulvin/DDC feeding leads to rapid induction of K8/K18 expression with disproportional K8 > K18 levels (Denk et al. 2000). The elevated K8/K18 ratio is crucial for MDB formation as shown in K18-knockout and K8 overexpressing animals, who are predisposed to MDB formation already upon short exposure to DDC and even form MDBs spontaneously during aging (Magin et al. 1998; Nakamichi et al. 2005). Accordingly, K8-null or K18 overexpressing mice are resistant to MDB formation and the protective function of K18 is not affected by its phosphorylation status or mutation (Zatloukal et al. 2000; Harada et al. 2007). However, the exclusive MDB inducing property of K8 in vivo cannot be reproduced in vitro, where aggregates resembling MDBs can also be produced by transfection of K18 (Nakamichi et al. 2002; Stumptner et al. 2007). Among posttranslational modifications, MDB formation is associated with K8 hyperphosphorylation and transamidation (Zatloukal et al. 1992; Stumptner et al. 2000). In a recent study, ablation of K8 S73 phosphorylation site in transgenic mice resulted in diminished MDB formation after DDC exposure (Harada et al., unpublished results). Several potential mechanisms might be responsible for this observation. K8 S73 is a well-known p38 kinase target site and p38 up-regulation induces keratin network reorganization with subsequent granule formation (Ku et al. 2002b; Wöll et al. 2007). P38 kinase-induced keratin network reorganization might be a necessary prerequisite for MDB formation, since p38 kinase inhibition prevented MDB formation in vitro (Nan et al. 2006). Alternatively, K8 hyperphosphorylation may induce MDB formation through inhibition of K8 degradation, which results in increased K8/K18 ratio (Ku and Omary 2000). MDBs contain highly cross-linked keratins and the ablation of tissue transglutaminase effectively inhibits DDC-induced MDB formation (Zatloukal et al. 1992; Strnad et al. 2007). Since K8 is a much better in vitro transglutaminase substrate than K18 and highly cross-linked proteins found in MDB-forming mice contain K8, but not K18, it was suggested that excessive K8 gets preferentially transamidated and acts as a nucleus for MDB formation. This is supported by studies in transgenic mice, where K8 overexpression accelerates and K18 excess inhibits not only MDB, but also cross-link formation (Strnad et al. 2007). Ad 3. MDBs were shown to contain misfolded keratins (Cadrin et al. 1991, Kachi et al. 1993). The protein misfolding is usually counteracted by the reparative function of chaperones (Ross and Poirier 2004; Macario and Conway de Macario 2005; Bukau et al. 2006), however, DDC feeding is associated with diminished chaperone expression, persistent chaperone modifications and impairment of chaperone function (Strnad et al. submitted). Similarly, chaperone function is impaired in a rat model of chronic alcoholic liver disease (Carbone et al. 2005). Ad 4. P62 is a stress-inducible protein with multiple functions which is a constituent of multiple inclusion bodies termed sequestosomes (Shin 1998; Kuusisto et al. 2001; Zatloukal et al. 2002; Moscat et al. 2007). It binds proteins polyubiquitinated at lysine 63 and shuttles them for proteasomal or autophagic degradation (Vadlamudi et al. 1996; Bjorkoy et al. 2005; Seibenhener et al. 2004; Wooten et al. 2008). p62 has been shown to enhance aggregate formation (Donaldson et al. 2003; Wang et al. 2005; Komatsu et al. 2007; Gal et al. 2007; Nezis et al. 2008). p62 action is beneficial under normal conditions, since it prevents accumulation of abnormal proteins (Bjorkoy et al. 2005; Ramesh Babu et al. 2008), but may become harmful when protein degradation is inhibited (Komatsu et al. 2007). There are several lines of evidence implicating p62 (containing the ubiquitin binding site) in MDB formation. In cell culture experiments, protein aggregates resembling MDBs formed only after p62 co-transfection, but not when K8, K18, and ubiquitin were transfected alone or in combination (Stumptner et al. 2007). Furthermore, p62 was rapidly induced in DDC-fed mice with p62-containing aggregates preceding the formation of MDBs (Stumptner et al. 2002). Furthermore, p62 inhibition attenuated, whereas p62 overexpression enhanced MDB formation in DDC-primed hepatocytes (Nan et al. 2006). Ad 5. Accumulation of protein aggregates, as seen during MDB formation, is counteracted by proteasomal or autophagic degradation (Glickman and Ciechanover 2002; Williams et al. 2006) and both degradation machineries are active in the liver. For example, conditional knock-out of the autophagy-related gene 7 (Atg7) leads to development of ubiquitin-positive inclusions in hepatocytes (Komatsu et al. 2005). Similarly, proteasomes seem to interact with MDBs and inhibition of proteasomal degradation leads to formation of MDB-like structures both in vitro and in DDC-primed mice (Harada et al. 2003; Bardag-Gorce et al. 2002; Riley et al. 2002; French et al. 2001). Furthermore, autophagic degradation is upregulated in DDC-fed mice and its further stimulation with rapamycin attenuates spontaneous MDB formation in K8 overexpressing mice (Harada et al. 2008). In contrast, MDB formation might be facilitated by proteasomal impairment, since MDBs are preferentially seen in proteasome-depleted hepatocytes (Bardag-Gorce et al. 2001). In addition, an aberrant form of ubiquitin, termed [UBB + 1] arising as a consequence of molecular misreading, is observed in MDBs and may inhibit proteasomal function (McPhaul et al. 2002; Lindsten et al. 2002). Similarly, alcohol and aging, both potential inducers of MDBs can be associated with decreased proteasome function (Hayashi and Goto 1998; Fataccioli et al. 1999). However, aggregate formation must not always be caused by a general inhibition in protein degradation. The degradative capacity may simply be overwhelmed by increased amounts of misfolded proteins. Alternatively, certain proteins may not be easily degradable, such as proteins with long polyglutamine stretches (Venkatraman et al. 2004). The latter scenario may apply to DDC-fed mice, which exhibit an accumulation of ubiquitinated protein only in the highly insoluble protein fraction (Strnad et al. 2007). This fraction is characterized by highly cross-linked protein species generated through extensive transamidation, which may make them resistant to proteolytic degradation (Strnad et al. 2007). Pathologic significance of MDBs In principle, cytoplasmic protein aggregates can be beneficial, detrimental or inert depending on the context. In many pathologic situations aggregation seems to be a protective response if the first lines of defense, that is, refolding and degradation, fail by sequestration of potentially harmful proteins (Arrasate et al. 2004; Bodner et al. 2006). On the other hand, large protein aggregates can be detrimental either by mechanical interference with cellular transport processes (e.g., shown with microtubule-dependent transport in neurons), deprivation of the cell of vital components by aggregation or coaggregation and by overwhelming/inhibiting the capacity of the chaperone system and/or the protein degradation machinery by indigestible material (Alonso et al. 1997; Stenoien et al. 1999; Suhr et al. 2001; Bence et al. 2001; Lee et al. 2004; Grune et al. 2004). It can be expected, therefore, that in different cell systems and pathologic conditions inclusion bodies differ regarding their cellular effects and consequences. MDBs per se do not seem to compromise the viability of transfected tissue culture cells (Stumptner et al. 2007) or hepatocytes in vivo, who even exhibit an activated phenotype (Denk et al. 2000). Further studies are needed to definitely characterize the molecular consequences of MDB formation.

Eur_J_Nucl_Med_Mol_Imaging-3-1-1914264

Clinical radionuclide therapy dosimetry: the quest for the “Holy Gray”

Introduction Radionuclide therapy has distinct similarities to, but also profound differences from external radiotherapy. Introduction Radionuclide therapy (or “targeted”, “metabolic-or “molecular-radiotherapy) may be defined as a radiation therapy that uses local, loco-regional or generally administered open (i.e. “unsealed”) radionuclides to achieve a transfer of radiation energy to a pathological target tissue and by this means to exert a destructive tissue effect. This “internal-radiation therapy has distinct similarities to, but also profound differences from the more commonly used external radiotherapy (EBRT). The tissue effect is expressed as the absorbed radiation dose in grays (Gy), i.e. the amount of transferred energy in joules per unit mass of target tissue. The fundamental use of this unit is similar to that in EBRT, and there is a similar relationship between radiation dose and response in terms of cell killing/survival. Therefore, calculation of the radiation absorbed dose to a targeted tissue makes sense at any point in treatment. In this respect, radionuclide therapy dosimetry may be considered an inherent part of radionuclide therapy in principle, as in EBRT. In the literature, there is considerable confusion over the proper use of the term “dose”, which actually refers to the “radiation dose-in the SI unit “grays”. However, dose in the context of radionuclide therapy is frequently used when actually the “administered activity-in GBq or mCi is meant. To avoid confusion, the terms “radiation dose-and “absorbed dose-are used when indicating Gy and the term “activity dose-when indicating GBq. Individual patient dosimetry has the following goals [1]: To establish individual minimum effective and maximum tolerated absorbed dosesTo establish a dose-response relation to predict tumour response and normal organ toxicity on the basis of pre-therapy dosimetryTo objectively compare the dose–response results of different radionuclide therapies, either between different patients or between different radiopharmaceuticals, as well as to perform comparisons with the results routinely obtained with external radiotherapyTo increase the knowledge of clinical radionuclide radiobiology, in part with the aim of developing new approaches and regimens In EBRT, the absorbed dose can be calculated relatively straightforwardly from the energy loss in the body from the point where the radiation enters the body to the target. Radiation in radionuclide therapy is directed to its target by a vehicle (hence “radiopharmaceutical”), which exhibits more or less specific binding to the target tissue. This is a highly dynamic metabolic process, both biochemically and physically, within the time interval of the decaying isotope, and entails a much more complex spatial and temporal radiation distribution than that in EBRT. Pharmacokinetics such as circulation, metabolisation, target expression heterogeneity and cellular uptake and release, as well as radiobiological phenomena such as biological or physical (“cross-fire”) bystander effects [2], play a determining role in the final radiation dose to the target. EBRT is typically a fractionated high dose rate radiotherapy in which episodes of radiation, aimed to cause as much lethal damage as possible, are interspersed with episodes of non-treatment, during which repair and repopulation occur. Radionuclide therapy is radiotherapy with a low and continuously decreasing dose rate, which requires a unique radiobiological approach [3, 4]. Integral activities can be determined numerically or by compartmental models [5] and presently rely heavily on the detection of the activity distribution by gamma cameras. Because the electron range is inferior to the spatial resolution of most molecular imaging devices, the “perfect-dosimetric study cannot be achieved, and calculations are always an approximation. Using the temporal and spatial radionuclide distribution data, radiation doses to target organs have generally been calculated using the MIRD formalism, formerly used for calculation of the biodistribution of diagnostic radiopharmaceuticals. Commercially available software such as MIRDOSE3 or the newer OLINDA/EXM [6, 7] is available for calculation of internal absorbed doses in organs and tumours. Although these models make important assumptions about anatomy (standard man and woman) and radiopharmaceutical distribution (uniformity of uptake in source and target) that are not necessarily valid in individual patients, they do provide a practical and standardised model for clinical end-users. Nevertheless, to date the need for dosimetry to individually optimise the therapeutic activity to be administered has been far from self-evident. Radionuclide therapy dosimetry has not gained wide acceptance as a clinical tool among the (nuclear) medical community because of an imbalance between the accuracy and the complexity of the procedure. A number of studies have even completely discarded dosimetry, instead using fixed activities for all patients or activities based on kg or m2 body dimensions. [8-10]. To underline that this is unjustified, this review will describe the significant progress that has been achieved over recent years, especially in the fields of instrumentation (with provision of clinically useful instruments), physical modelling and radiobiology. Furthermore, the EURATOM Council Directive 97/43 stipulated that in medical exposures for radiotherapeutic purposes, including nuclear medicine, “exposures of target volumes shall be individually planned”. In this context, the nuclear medicine physician is at present confronted with a “dosimetric dilemma”, because official guidelines and recommendations for most treatments do not include advanced dosimetric calculations. Therefore, this review primarily aims to provide the nuclear medicine practitioner with an up-to-date overview of clinically applied dosimetry techniques in radionuclide therapy. For those working at a more basic level of dosimetry, information is provided on the clinical application of methods and areas of further development. Given the large size of the field of radionuclide therapy, this review is limited to those oncology indications for which there is substantial literature concerning dose calculations, with in-depth discussion thereof. As it would be impossible to cover all areas of the complex subject of dosimetry, appropriate references will be provided where necessary, e.g. regarding basic physics, (radio)biology, radiopharmaceuticals and preclinical aspects. Radioiodine therapy in differentiated thyroid carcinoma Radioiodine therapy has proven to be a safe and effective method in the treatment of patients with differentiated thyroid carcinoma (DTC) after total or near-total thyroidectomy [11, 12]. It has been shown to be useful not only for ablation of benign remnant thyroid tissue, facilitating subsequent diagnostic testing, but also for treatment of any remaining cancerous cells either in the thyroid bed or at metastatic sites. Dosimetry plays an important role in answering questions concerning biodistribution since the biological half-time of radioactive iodine differs substantially between individual patients and even within distinct lesions of the same patient. It has proven exceptionally helpful in clinical trials of new drugs, for example retinoids, and in assessment of the use of recombinant TSH [13], as well as in the study of other questions such as the phenomenon of “stunning”. However, its possible role in extending therapy doses to the level of individual maximum effectiveness is currently less obvious. There is at present no consensus on the activity dose of 131I to be administered in various settings according to recent European and American guidelines [14, 15], largely because of the lack of prospective, randomised data. In the majority of cases, 1.1-3.7 GBq (30-100 mCi) is prescribed empirically for the first radioiodine therapy after thyroidectomy in newly diagnosed DTC, to ablate the remaining glandular tissue. Activity doses as low as 1 GBq are used when the size of the thyroid remnant is small, as measured by the postoperative radioiodine uptake in the neck, and/or to reduce local complications that could arise from radiation thyroiditis/oedema, which has been reported in 10-20% of cases, although usually mild. The effectiveness of this ablative approach based on standard activities is reported to be about 80%. For the treatment of residual tumour, relapses or lymph node metastases, generally higher activities of 3.7-7.4 GBq (100-200 mCi) are used. Approaches in which quantitative dosimetry is performed to estimate the activity dose needed to deliver an effective radiation dose are scarce in the literature. Maxon and co-workers, using sequential planar scintigraphy, established in the early 1980s that an effective mean radiation dose of at least 300 Gy is required for successful remnant ablation [16]. The validity of this recommendation remains unclear as it conflicts somewhat with clinical experience. It also neglects microdosimetry of radioiodine and dose heterogeneity, which may well determine the overall response. As far as metastases are concerned, these authors concluded in the same study that a radiation dose of at least 80 Gy is associated with a significant increase in response, while a dose of less than 35 Gy offers little chance of success. In a later study [17], they found that when metastatic disease was present only in lymph nodes, a target radiation dose of at least 140 Gy was successful in 86% of patients and 90% of involved nodes. When nodal metastases were associated with either residual thyroid tissue or other metastatic foci, a single treatment calculated to deliver at least 85 Gy to the metastases proved successful in 74% of patients and 81% of nodes. The aforementioned doses were mean absorbed doses for the whole remnant or tumour; no data were provided on the dose distribution in smaller tumour parts. It is also unclear what causes the large difference between radiation dose values for remnants and metastases. This method requires sequential measurements with a scintillation camera 24, 48 and 72 h after administration of 74 MBq (2 mCi) of 131I. De Keizer et al. [18] recently calculated thyroid tumour doses with a similar methodology but with the use of recombinant TSH. A tumour dose of >80 Gy was found in only 20% of metastases visible on post-therapy 131I scanning. In 55% of patients, progressive disease was evident after 3 months and none of these patients had radiation doses to the tumour in excess of 30 Gy, confirming the existence of a clinical dose-response relation. In cases of distant metastases, higher amounts of 131I are given in single doses and subsequent cumulative therapies. Most centres use a fixed dose of 7.4 GBq (200 mCi), but some use (much) higher activity doses. In order to avoid serious complications (bone marrow suppression, lung fibrosis), the commonly used “maximum safe-administered dose concept published by Benua et al. [19] restricts the blood absorbed dose to less than 2 Gy (200 rad) and the whole-body retention to 4.4 GBq (120 mCi) at 48 h in the absence of diffuse lung metastases. According to the protocol, activity concentration in the whole body as measured by 131I should not exceed 2,960 MBq (80 mCi) after 48 h when diffuse, functioning lung metastases are present. These measurements can be camera based without blood sampling. More recently, the blood dose formula derivation proposed by Hänscheid et al. [13] has provided a new tool for patient-specific blood dose assessment representing marrow dosimetry in DTC therapy. For more precise dosimetry, e.g. in dosimetric studies or for higher targeted blood absorbed doses, sequential blood sampling is recommended [20, 21]. The calculated radiation dose serves as a surrogate parameter for the organ at risk, the bone marrow, since to date direct determination of the bone marrow absorbed dose is not feasible. This method has been applied successfully in clinical practice. Dorn et al. [22] used a dosimetric study prior to therapy with 150-400 MBq 131I and obtained daily images up to 4-5 days. They maximised the administered activity to an absorbed dose of 3 Gy to the bone marrow or 30 Gy to the lungs, corresponding to the LD5/5 in EBRT, while aiming at >100 Gy to all known metastases. This resulted in a mean of 22.1 GBq (597 mCi) and a range of 7.4-38.5 GBq (200-1,040 mCi) activity administered per treatment. Many institutions will find it difficult to deliver these amounts owing to radioprotection restrictions, limiting the applicability of this method. To date, bone marrow depression has rarely been reported, while the rate of dose-related leukaemia has been estimated to be approximately 1% after 5-10 years. Lung fibrosis as a consequence of micronodular iodine-avid metastases has been reported in 1% of patients suffering from pulmonary metastases. The downside of these high-dose strategies may be an increase in the occurrence of these side-effects. On the other hand, assuming this “maximum safe dose-to be an optimal dosing strategy, extrapolation to a “fixed activity dose-strategy of 7.4 GBq (200 mCi), as is widely applied, would “under-treat-54% and “over-treat-3% of patients [23]. To date, there are no randomised and prospective trials that directly compare a low- and high-activity dose strategy in patients with metastases. The treatment of DTC in childhood varies substantially from the standard approach in adults mainly owing to the different tumour biology in this age group. Usually activities of 50-100 MBq/kg are given, treatment data on children being scarce and non-systematic. There are a number of potential drawbacks of dosimetry that may preclude its use in many centres. One problem is the uncertainty of volume determinations by neck ultrasound shortly after thyroid surgery since the differentiation between scar tissue, haematoma and thyroid remnant is often difficult. The same holds true for the use of computed tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET) in the evaluation of distant metastatic lesions, especially in the case of diffuse lung metastases. Also, it is difficult to predict radioiodine kinetics during therapy from prior diagnostic studies owing to the large difference in administered and measured activity and potential subsequent biological effects. PET studies using the cyclotron product 124I-NaI, if possible in combination with PET/CT tomography, may prove to be particularly valuable in improving treatment planning and patient-specific dosimetry. Compared with conventional methods, including CT and 131I scanning, this technique was found to be superior in terms of lesion detection and functional assessment [24]. Using three-dimensional 124I-PET voxel-based dosimetry, Sgouros et al. [25] showed a wide range of mean absorbed dose values for individual tumours, from 1.2 to 540 Gy. Unfortunately, no correlation with response was reported in that study. However, this wide variation certainly implies that the assessment of meaningful dose-effect relationships and clinical dosimetry should include further development of a quantitative approach. 131I-mIBG therapy in neuroblastoma and phaeochromocytoma For more than two decades, mIBG labelled with the γ/β emitter iodine-131 (131I-mIBG) has been used to treat neural crest-derived tumours, essentially neuroblastoma and phaeochromocytoma, both in relapsed and in newly diagnosed patients. In high-stage neuroblastoma, the treatment may represent (a) palliative therapy [26], (b) first-line therapy, as a single agent or combined with chemotherapy [27], (c) consolidation therapy after induction of a “good partial remission-[28] and, more recently, (d) second-line therapy after failed induction chemotherapy, combined with topotecan and stem cell rescue in children with metastatic neuroblastoma [29] or with myeloablative chemotherapy and autologous stem cell transplantation in refractory neuroblastoma [9]. In neuroblastoma, important efforts have been made to develop clinically useful dosimetry methods to predict the radiation dose to the red marrow as the critical organ for bone marrow toxicity, in order to apply optimal and individual dose maximisation in children with highly aggressive tumours. Three different methods of activity administration have evolved from the experience of treating such patients. The first approach, developed in Amsterdam, the Netherlands, is to give fixed activity dose “fractions-of 7.4-11.1 GBq (200-300 mCi), more recently combined with hyperbaric oxygen or topotecan. This approach has been proven to have a synergistic effect on cell killing in vitro when used in combination with 131I-mIBG [30]. Intervals between fractions are determined by blood count recovery and the treatment is continued to maximum response [31]. An advantage of this method is its simplicity, which may be viewed as important in very sick children and in those who require prompt treatment, especially when receiving first-line therapy. Another important advantage of lower activity doses is the shorter isolation and hospitalisation period required for reasons of radioprotection, which may be regarded as highly significant in these usually very sick children with a grave prognosis. As whole-body and tumour radiation doses are never calculated, any kind of dose-response assessment and optimisation on the basis of absorbed radiation dose is, however, impossible. Furthermore, the relatively low dose rate of radiation compared with (ultra-)high doses may be viewed as radiobiologically suboptimal. The clinical results of this approach need to be verified in a multicentre phase III trial that should include a dosimetric assessment of the whole-body dose. The second approach originates from the United Kingdom, where there has been a long tradition of using pre-therapy dosimetry. In a phase I/II study on advanced, chemo-refractory stage III/IV patients, Lashford et al. observed that 80% of patients developed grade 3 or 4 haematotoxicity at a whole-body absorbed dose of 2.5 Gy established from a pre-therapy 131I-mIBG scan [32]. Monsieurs et al. used 123I-mIBG prior to therapy to the same effect [33]. Previously, Tristam et al. had shown a highly skewed distribution of tumour radiation doses after a fixed, diagnostic activity [34]. Matthay et al. found a good correlation between whole-body and tumour dose [35]. As radiation dose is principally related to response, this approach may lead to a more standardised whole-body and tumour absorbed dose [36] and may offer more possibilities of dose escalation, especially in the presence of an altered biodistribution, e.g. due to partial kidney failure or high tumour load. Recently, an ESIOP experimental mIBG protocol has been proposed in which the aim is to deliver a total whole-body absorbed dose of 4.0 Gy in two fractions, in combination with topotecan, after failure of induction chemotherapy. Post-therapy dosimetry is performed after a first fixed fraction of 440 MBq/kg (12 mCi/kg), with calculation of the activity dose to be administered with the subsequent second fraction in order to arrive at the desired total whole-body absorbed dose of 4 Gy. The importance of this protocol is that whole-body and tumour absorbed dose can be estimated and the relation to both toxicity and response can be established, unlike with the non-dosimetry protocols. The schedule of intensification of mIBG therapy by dose escalation and radiosensitisation with topotecan with a haematopoietic autograft has been shown to be safe and practicable [29]. The next step will be to prove that this approach will also lead to a better outcome in neuroblastoma patients. The third approach can be derived from recent publications from the United States, in which patients received high activity doses of mIBG on the basis of an activity dose of 550-660 MBq per kilogram body weight (15-18 mCi/kg), with stem cell support if necessary. In an early study, Sisson and co-workers examined predictors of toxicity and found the best correlation to be between whole-body absorbed dose and platelet ratio; however, comparable and statistically significant correlations were found with GBq/kg body weight and GBq/m2 body surface [37]. This observation was supported by Matthay and colleagues [35], who found the amount of 131I (GBq/kg) and whole-body dose (Gy), but not administered activity dose (GBq), to be significantly correlated with platelet and neutrophil nadir. In a recent study by Dubois et al. [38], substantial haematotoxicity was seen at 660 MBq/kg (18 mCi/kg) in a group of patients with advanced, heavily pre-treated neuroblastoma, including myeloablative chemotherapy in 81%. Of these patients, 36% required autologous haematopoietic stem cell transplantation. The resulting median whole-body absorbed dose was 2.92 Gy with a median administered activity dose of 13.5 GBq (366 mCi, range 198-895 mCi). Interestingly, whole-body radiation dose was shown to correlate with failure to engraft platelets or red cells in those patients receiving autologous stem cell transplantation. An encouraging response rate of 27% was reported, however. The same authors recently established a maximum tolerated dose (MTD) of 440 MBq/kg (12 mCi/kg) with myeloablative chemotherapy and autologous stem cell transplantation [9]. The advantage is that there is no need for planning/simulation in this high-dose protocol, which may increase in importance if multiple centres participate. Malignant, metastatic phaeochromocytoma is a very rare disease but can be excellently treated by 131I-mIBG in both a palliative and a curative setting. Literature data on dosimetry are scarce and casuistic. In 1997, Loh et al. reviewed the literature concerning 116 previously treated cases [39]. They found a cumulative activity dose of 3.6-85.9 GBq (96-2,322 mCi, mean 490 α 350 mCi) in 1-11 (mean 3.3 α 2.2) therapies, with a mean single activity dose of 5.8 GBq (158 mCi). Dose prescriptions were as a fixed activity dose of 3.7-11.1 GBq (100-300 mCi or 3-9 mCi/kg). A recent study was performed in order to evaluate the performance of mIBG therapy with high activities after debulking surgery [40]. The median individual cumulative activity dose was 37.6 GBq (1,015 mCi), with a range of 14.3-62.5 GBq (386-1,690 mCi), in one to three consecutive therapies. Grade 3 platelet toxicity was observed after 79% of therapies, and grade 3 and 4 neutrophil toxicity after 53% and 19% of therapies, respectively. All patients had stem cells harvested before therapy, but 11 patients showed spontaneous reconstitution and only one required stem cell return. Response rate was 10/12 patients (83%). Radiopeptide therapy for neuro-endocrine tumours Several clinical trials have investigated the use of peptide receptor radionuclide therapy (PRRT) with a radiolabelled somatostatin analogue as one of the newly developed targeted tools for neuro-endocrine tumours [41-44]. Individual pre-therapeutic dosimetry is necessary for patient selection and therapy planning because there are huge inter-patient differences in radiopeptide uptake in normal organs and tumour tissues. This may be related to varying somatostatin receptor densities on tumour cells, as well as to factors such as tumour volume, interstitial pressure and viability. The dosimetric studies that have been performed in this treatment have been the most sophisticated in the field and exemplory for other types of treatment. Initial studies were performed with the radiopeptide used in diagnostics, [111In-DTPA0]-octreotide, given in high activities. Results were encouraging, with symptomatic and biochemical responses in a large percentage of patients, although objective responses were rare [42]. The analogue [90Y-DOTA0,Tyr3]-octreotide has been investigated in various phase I-II trials [41, 43]. 90Y-labelled lanreotide has also been investigated (in the MAURITIUS trial) [45]. For dosimetry of 90Y from post-therapeutic bremsstrahlung, images that substantially lack quality must be quantified. Therefore, two alternative approaches have been developed as a surrogate for the original radiopeptide, namely therapy simulation with either the 111In-labelled peptide or the 86Y-labelled peptide [46, 47]. These methods are intended to provide pre-therapy assessment of the optimal activity administration [48-51] (Table 1). The variation that can be appreciated among the various combinations in the table may be primarily technical/methodological in origin but also biological, based on differences in somatostatin receptors and isotope/chelator affinity. The reader is referred to the respective papers for more detailed information. Table 1Absorbed doses to principal organs and to tumour (Gy/GBq ±SD), deriving from different radiopeptidesStabin 1997 [109], Kwekkeboom 2001 [50]Kwekkeboom 2001 [50]Cremonesi 1999 [47]Forster 2001 [110]Helisch 2004 [111]Forrer 2004 [112]Therapy[111In-DTPA0, Tyr3]-octreotide[177Lu-DOTA0, Tyr3]-octreotate[90Y-DOTA0, Tyr3]-octreotide[90Y-DOTA0, Tyr3]-octreotide[90Y-DOTA0, Tyr3]-octreotide[90Y-DOTA0, Tyr3]-octreotideDosimetry[111In-DTPA0, Tyr3]-octreotide[177Lu-DOTA0, Tyr3]-octreotate[111In-DOTA0, Tyr3]-octreotide[86Y-DOTA0, Tyr3]-octreotide[86Y-DOTA0, Tyr3]-octreotide[111In-DOTA0, Tyr3]-octreotidePatients16530385Kidneys0.52 α 0.241.65 α 0.473.9 α 1.9b 2.73 α 1.412.84 α 0.64Kidneys + protection0.88 α 0.191.71 α 0.89Liver0.065 α 0.010.21 α 0.070.72 α 0.570.66 α 0.150.72 α 0.400.92 α 0.35Spleen0.34 α 0.162.15 α 0.397.62 α 6.302.32 α 1.972.19 α 1.116.57 α 5.25Red marrow0.03 α 0.010.07 α 0.0040.03 α 0.010.49 α 0.0020.06 α 0.020.17 α 0.02Tumour (range)0.72-6.8a 3.9-37.91.4-313.21-19.582.1-29.52.4-41.7aFrom reference [50]bSeries enlarged from the original one, as in Bodei et al. [44] Although [90Y-DOTA0,Tyr3]-octreotide (or -lanreotide) and its imageable counterpart [111In-DOTA0,Tyr3]-octreotide (or -lanreotide) are not chemically identical, the latter has been used for dosimetric simulation, based on the hypothesis that the similar physical and biological half-lives yield a comparable in vivo pharmacokinetics and biodistribution, especially concerning the renal uptake, which depends on aspecific phenomena. A drawback of this method is that the small structural modification may affect the somatostatin receptor binding affinity [52]. Regarding biodistribution, the organs receiving the highest predicted absorbed doses, in a first series of 18 patients, included the spleen (7.6 α 6.3 mGy/MBq), the kidneys (3.3 α 2.2 mGy/MBq) and the tumour (1.4-31 mGy/MBq, mean 10). When the series was enlarged to 30 patients, a slightly higher kidney absorbed dose (3.9 α 1.9 mGy/MBq) was observed [43]. Shortcut approaches involving use of the commercially available molecule [111In-DTPA0]-octreotide or OctreoScan® have been proposed [53]. Nonetheless, probably as a result of the quite different biokinetics and receptor affinity of [90Y-DOTA0,Tyr3]-octreotide and [111In-DTPA0]-octreotide, which are indeed chemically different, comparative data obtained appear not to be sufficiently overlapping for this tracer. A thorough dosimetric study using PET with [86Y-DOTA0,Tyr3]-octreotide, biochemically identical to the therapeutic molecule, was carried out in 24 patients [54]. This offers substantial advantages in terms of spatial resolution and quantification, but the short half-life of the radionuclide leaves later phases of the biokinetics to estimates based on extrapolation. The newest radiopeptide, [177Lu-DOTA0,Tyr3]-octreotate, offers further advantages. 177Lu has a lower energy (Emax 0.49 MeV) and penetration range (Rmax 2 mm) emission, but a longer half-life (6.7 days). The low abundance gamma emissions (113 and 208 keV) allow for dosimetry and imaging prior to as well as post therapy. Moreover, octreotate has a six- to ninefold higher affinity for somatostatin receptor 2, the somatostatin receptor most frequently expressed in neuro-endocrine tumours. Unfortunately, to date a thorough dosimetric analysis is lacking, but data deriving from a study comparing [177Lu-DOTA0,Tyr3]-octreotate with [111In-DTPA0]-octreotide indicate that, compared with [90Y-DOTA0,Tyr3]-octreotide, [177Lu-DOTA0,Tyr3]-octreotate delivers a lower burden to organs, with absorbed doses of 1.8-2.7 mGy/MBq to the spleen, 1.0-2.2 mGy/MBq to the kidneys (lowered to 0.7-1.1 mGy/MBq with protection) and 0.1-0.3 mGy/MBq to the liver. Red marrow absorbed dose, derived by the blood approach, ranged from 0.05 to 0.08 mGy/MBq [44]. Due to their marked radiosensitivity, the kidneys undoubtedly represent the critical organ, particularly after [90Y-DOTA0,Tyr3]-octreotide. Renal irradiation arises from the proximal tubular re-absorption of the radiopeptide and the resulting retention in the interstitium. According to EBRT studies, the renal maximum tolerated absorbed dose is conventionally considered to be in the range of 23-25 Gy. According to the National Council on Radiation Protection and Measurements (NCRPM) an absorbed dose of 23 Gy to the kidneys causes detrimental deterministic effects in 5% of patients within 5 years [55]. Sporadic cases of delayed renal failure, in some cases end-stage disease requiring dialysis, have been observed, especially in patients who have received an activity dose of more than 7.4 GBq/m2 [56]. Nephrotoxicity is accelerated by other risk factors, such as pre-existing hypertension or diabetes. Given the high kidney retention of radiopeptides, positively charged molecules, such as L-lysine and L-arginine, are used to competitively inhibit the proximal tubular re-absorption of the radiopeptide. This results in a reduction in the renal absorbed dose of between 9% and 53% [57]. Doses are further reduced by up to 39% by prolonging infusion over 10 h and by up to 65% by prolonging it over 2 days after radiopeptide administration, thus covering more extensively the elimination phase through the kidneys [43]. Despite kidney protection, renal function loss may become clinically evident years after PRRT. A median decline in creatinine clearance of 7.3% per year was reported in patients treated with [90Y-DOTA0,Tyr3]-octreotide and of 3.8% per year in patients treated with [177Lu-DOTA0,Tyr3]-octreotate. Cumulative and per cycle renal absorbed dose, age, hypertension and diabetes are considered factors contributing to the decline of renal function after PRRT [58]. Clinical experience and dosimetric studies clearly indicate that the renal absorbed dose estimated by conventional dosimetry does not accurately correlate with the renal toxicity observed in patients treated with [90Y-DOTA0,Tyr3]-octreotide. Consideration of additional parameters, such as patient-specific kidney volume and distribution of the radionuclide, appears to give a better correlation with the clinical effects [59]. Assessment of individual kidney volume by CT scan yields a wide variability when compared with the standardised phantom. Moreover, autoradiographic studies, performed on human kidney after in vivo injection of 111In-peptides, have demonstrated that the majority of radioactivity is deposited within the renal cortex, mainly in the juxtamedullary region. This leads to a higher deposition of energy per unit mass, compared with conventional dosimetry. Hence, calculation of the kidney absorbed dose assuming a homogeneous renal distribution of radioactivity is inadequate. New techniques accounting for the difference in radioactivity placement in the kidneys, on the basis of a CT-based volumetric analysis, appear more realistic [60]. Even if predicted absorbed doses are much lower than the threshold for toxicity, the other target organ that gives rise to concerns about acute and permanent toxicity after PRRT is the bone marrow, particularly in repeated administrations [41-43]. Acute haematological grade 3 or 4 toxicity is not uncommon, especially after [90Y-DOTA0,Tyr3]-octreotide, and sporadic cases of myelodysplastic syndromes or even overt acute myeloid leukaemia have been reported with all three therapeutic radio-compounds. Bone marrow dosimetry is usually modelled through a blood-based method, in which an equivalent distribution of the radioactivity from blood throughout the bone marrow is conservatively considered [61, 62]. Currently, the potential risk of kidney and red marrow limits the amount of radioactivity that may be administered. Indeed, when tumour masses are irradiated with suitable doses, volume reduction may be observed (Fig. 1) [63]. Tumour remission is positively correlated with high uptake during [111In-DTPA0] octreotide scintigraphy. Nevertheless, tumour radiation dose depends not only directly on the administered activity and the uptake versus time, but also on the tumour load. This is confirmed by clinical data regarding the response characteristics: patients with a limited number of liver metastases respond to PRRT, whilst patients with a high tumour load do not [45]. Mathematical models have shown that 177Lu is better in small tumours (optimal diameter 2 mm), whilst 90Y is better in larger ones (optimal diameter 34 mm): very small masses are likely not to absorb all the β-energy released in the tumour cells by 90Y, while larger tumours will suffer from lack of uniformity of activity distribution of 177Lu. Finally, differences in dose rate must be taken into account: the longer physical half-life of 177Lu means a longer period is needed to deliver the same radiation dose than when using 90Y. This may allow more time for tumour re-population [64]. Therefore, combination therapy with 90Y and 177Lu, either simultaneously or in distinct settings, has been suggested to overcome the difficulties of real clinical situations involving different sized lesions. Fig. 1Tumour dose-response relationship in 13 patients treated with 90Y-DOTATOC. Tumour volumes were assessed by CT before and after treatment. Tumour dose estimates were derived from CT scan volume measurements and quantitative 86Y-DOTATOC imaging performed before treatment. Data were further computed using the MIRDOSE spherical model. Reprinted by permission of the Society of Nuclear Medicine from [63] Treatment of solid tumours by radiolabelled antibodies Most clinical radioimmunotherapy (RIT) studies have been performed in colorectal cancer, using antibodies against CEA, TAG-72, A33 and KSA. Other tumours studied have included ovarian cancer (anti-MX35, anti-folate receptor Mov18, anti-HMFG1), prostate cancer (anti-PMSA), breast cancer (anti-mucin BrE3, NR-LU-10), glioma (anti-tenascin) and renal cancer (anti-G250) [65]. Although at later time points after injection of radiolabelled monoclonal antibodies, adequate to high uptake may serve to delineate deposits of solid tumours, the relatively unfavourable therapeutic window between the anti-tumour effect and toxicity hampers the introduction of these agents in the clinic. Solid tumours are generally more radioresistant than, for example, malignant lymphoma. The absorbed doses required to achieve a response of tumour deposits are higher than those needed to obtain a response in malignant lymphoma. Activity doses leading to adequate absorbed doses in tumour deposits therefore result in significant radiation-induced toxicity, primarily of the bone marrow, as the most radiation-sensitive organ; myeloablation may be the goal as well as the result of this. Most studies aim at administration of the MTD that results in acceptable toxicity to the bone marrow, as the organ responsible for dose-limiting toxicity. Several methods have been reported for calculation of the absorbed dose to the bone marrow, e.g. region of interest analysis of scintigraphic data or a model based on radioactivity in the blood. When radiolabelled antibodies bind to blood, bone and bone marrow components or when the radionuclide accumulates in bone or bone marrow upon metabolisation of the radionuclide-antibody complex, calculation of the red marrow dose is more complex than when using radiolabelled antibodies that lack these characteristics [66, 67]. The use of a model for bone marrow dosimetry using blood activity may result in better reproducibility of the bone marrow dosimetry. Wessels et al. [68] observed that historical variations as high as 200-700% between different institutions performing marrow absorbed dose calculations could be dramatically reduced to −29% to +20% by a central computing facility and the use of similar methodology, based on the standard American Association of Physicists in Medicine (AAPM)/Sgouros blood model [66]. In a study using 131I-labelled murine monoclonal antibody G250 in patients with metastatic renal cell cancer, haematological toxicity correlated with whole-body absorbed radiation dose [69]. In a retrospective analysis of 114 patients who underwent 131I-labelled antibody therapy, absorbed dose-based definitions of MTD and escalation variables proved to be better than activity-based methods [70]. In a study in which therapeutic doses of 131I-cG250 (the chimeric variant of G250) were administered, no correlation was found between haematological toxicity and either the radiation absorbed dose to the whole body or bone marrow or the administered activity [71]. Juweid et al. observed that besides red marrow dose, baseline blood counts, multiple bone and/or marrow metastases and recent chemotherapy are important factors related to haematological toxicity after radio-immunotherapy [72], making a dosimetric approach to identify the optimal radionuclide dose more complicated. One way to overcome the unfavourable therapeutic index of tumour response and normal organ toxicity is to focus on treatment of patients with minimal residual disease. For example, patients with ovarian cancer in complete remission after debulking and chemotherapy have an approximately 50% chance of relapse. Compared with results in matched controls, Nicholson et al. observed an increase in 5-year survival from 55% to 80% following intraperitoneal administration of 90Y-labelled HMFG1 in patients with ovarian cancer in complete remission after chemotherapy [73]. However, a more recent randomised clinical trial in patients with ovarian cancer in complete remission after debulking and chemotherapy did not reveal a benefit in disease-free and overall survival after treatment with 90Y-labelled murine HMFG1 [74]. Introduction of 90Y-muHMFG1 did not delay the time to relapse and did not result in prolonged survival as compared with the control group. One can argue that 90Y is far from ideal for treatment of minimal residual disease owing to its high β-energy, and that radionuclides with lower range emissions, such as 177Lu, have more favourable characteristics for this purpose. Furthermore, relatively large antibody doses were used, which may have resulted in saturation of the epitopes on cancer cells and subsequently in low tumour-to-non-tumour ratios of radioactivity uptake. Obviously, clinical dosimetry could play an important role in explaining these differences in therapy results. A very significant advance that promises to increase the radiation dose to the tumour while reducing that to the bone marrow is the development of pre-targeting strategies. In pre-targeting, the tumour is first targeted with a specific non-radioactive monoclonal antibody construct. The antibody construct is allowed to accumulate in the tumour and to clear from the blood and non-target tissues. Subsequently, a small radioactive hapten is injected which has high affinity for the antibody construct. The radioactive hapten targets the antibody construct, while demonstrating rapid renal excretion. Examples of these approaches are the use of bispecific monoclonal antibodies and the biotin-avidin system [75, 76]. As pre-targeting increases the tumour/normal organ uptake ratio [77], these approaches hold great promise for enhanced therapeutic efficacy. However, the use of multiple drugs poses the problem of fine-tuning dosing and timing of the interval between injections. Optimisation of the dosing and timing schedule is needed for optimal targeting of the tumour by the radioactive compound, while assuring low normal organ uptake. Robust dosimetric analysis of tumour and normal organ uptake of the radioactive small molecule is a requirement for the successful development and implementation of pre-targeting strategies [78, 79]. Radioimmunotherapy of B-cell lymphoma Research in RIT in lymphoma has resulted in two FDA-approved radiopharmaceuticals, 90Y-ibritumomab or Zevalin® (IDEC Pharmaceuticals and Schering AG) and 131I-tositumomab or Bexxar® (Glaxo Smith Kline), for the treatment of B-cell lymphoma [80, 81]. Both are directed against CD20, albeit not against the same epitope. They are both approved for the treatment of relapsed or refractory follicular/low-grade or transformed B-cell lymphoma including rituximab-refractory follicular B-cell lymphoma in the US, but only Zevalin is approved in the EU and only for follicular lymphoma. Pre-treatment with unlabelled monoclonal antibody (=preload) as part of the treatment with Zevalin and Bexxar is current practice, as it leads to a more favourable biodistribution. This has been studied in animal models, as well as in the setting of myeloablative RIT [82-84]. The preload may clear peripheral B cells from the circulation, improving tumour targeting of subsequently administered radiolabelled monoclonal antibodies. This optimisation has been accomplished by diagnostic and dosimetric 111In or 131I tracer studies. On the other hand, the intrinsic therapeutic efficacy of the antibody is a confounding factor, making evaluation of the relation between absorbed dose and treatment response more difficult than for other radiopharmaceuticals [85, 86]. Bexxar has been developed using the whole-body absorbed dose as a substitute for bone marrow dosimetry by administering a trace amount of 131I-labelled CD20 antibody and determining the total body clearance prior to therapy in order to calculate the patient-specific injected activity dose that should deliver the specified absorbed dose to the whole body. The method has been further simplified to an estimation based on three points [87]. In a dose escalation study with absorbed doses to the whole body ranging in 0.1-Gy increments from 0.25 to 0.85 Gy, the MTD was found to be 0.75 Gy in patients who had not received prior high-dose chemotherapy with stem cell support and who had platelet counts of ≥150 × 109/litre. A comparison between the therapeutic activity calculated on the basis of the diagnostic pre-therapy tracer study and the amount calculated per kilogram body weight showed that, using the latter method, 50% of the patients would have been either over- or underdosed by 10% or more, and 16% of the patients by 25% or more. Interestingly, a correlation between the duration of complete remission (CR) and the absorbed dose to the whole body was found. Patients receiving a whole-body absorbed dose between 0.65 and 0.85 Gy showed longer CR than patients receiving between 0.25 and 0.55 Gy [88]. In contrast, Zevalin has been developed with the notion that dosimetry may be dispensable. In a randomised study comparing Zevalin with rituximab alone, a secondary objective was to determine whether dosimetry was required [10]. A tracer dose of 185 MBq (5 mCi) 111In was administered for dosimetric purposes with the first infusion, followed by a therapeutic activity dose of 0.4 mCi/kg (15 MBq/kg) Zevalin 7 days later. The absorbed doses to normal organs and the marrow were found to be within the specified limits of 20 Gy for solid organs and 3 Gy for red marrow in 72 patients. The median estimated absorbed dose to the tumour was 15 Gy (range 0.6-24 Gy). There was no significant correlation between dosimetric and pharmacokinetic parameters and haematological toxicity, although the correlation between nadir of the neutrophil count and the whole blood half-life of 90Y exhibited borderline statistical significance. It was concluded that dosimetry may be excluded for populations of Zevalin patients who meet certain criteria for pre-treatment platelet count and percentage of marrow involvement by tumour. A possible explanation for this discrepancy may be found in the photon energy emitted by 131I; this may account for a large part of the cross-absorbed dose to the bone marrow, which is almost independent of the amount of bone marrow involvement. It has been reported that the self-absorbed dose accounts for 64% of the whole absorbed dose to the red marrow [89], leaving 36% as the cross-absorbed dose. Scintigraphic assessment of absorbed dose to the bone marrow has its shortcomings owing to over- and underlying tissues as well as to the local variability in bone marrow involvement by the disease targeted. It is especially difficult to use pharmacokinetic data for calculation of the absorbed bone marrow dose, since bone marrow involvement is a strong confounder. Other important issues are the bone to bone marrow ratio and variable values for the activity concentration in the blood and red marrow [90, 91]. A fixed ratio between blood and bone marrow has been used in bone marrow dosimetry based on blood radioactivity levels [10, 64]. However, it has been suggested that the red marrow to blood ratio is not fixed, but increases continuously up to 72 h post injection in both patients and rats [92]. Furthermore, haematological toxicity, like tumour response, is a deterministic effect of radiation, characterised by a sigmoid rather than by a linear dose relationship [93]. A sigmoid relationship may be discernible in a reasonably homogeneous population, but is likely to be more difficult to identify if the population is heterogeneous. The bone marrow reserve may be more relevant to the magnitude of toxicity than is the absorbed dose. Another particular challenge to haematology RIT dosimetry may be tumour regression during energy deposition, as is frequently the case in rapidly responding lymphoma. This may lead to underestimation of absorbed dose, since dose is per definition energy per unit mass [94]. The opposite will be the case if the tumour grows during energy deposition. To avoid this pitfall, repeated volume assessments during therapy would be necessary, or alternatively one could use voxel-based dosimetry, which is likely to be less sensitive to changes in mass. Myeloablative RIT at the MTD for normal organs is by definition dependent on dosimetry. Dose-limiting organs may be the lungs, kidneys or liver. In high-dose RIT using the 131I-labelled mouse antibody tositumomab, the lungs were found to be the dose-limiting organ in 28 of 29 patients and the kidneys in the remaining patient [95]. The opposite was found when the pharmacokinetics of the chimeric 131I-labelled rituximab were studied, i.e. the kidneys were found to be the critical organ [96]. These differences may be due to the significant difference in half-life between the antibodies, the chimeric and murine antibodies having half-lives of 88 h and 56 h, respectively. The MTD for myeloablative 131I-tositumomab was established to be 25 Gy to the lungs. Studies of myeloablative RIT using 90Y are ongoing and encouraging results have been reported [97]. In several experimental studies, Auger emitters have been therapeutically superior to β emitters when taken to the MTD [98, 99]. This is likely to be due primarily to lower bone marrow toxicity but is perhaps also attributable to the deposition of more energy in single cells or small tumour cell clusters. Auger emitters would thus be advantageous in an adjuvant setting, in leukaemia, but they may also be superior where there is tumour bulk. The challenge is how to estimate absorbed dose in single cells, but one may begin in patients with a significant amount of circulating tumour cells that can be studied ex vivo [100]. Interestingly, Kaminski reported that in a number of patients who relapsed following RIT the relapse occurred only at sites previously not known to be involved with tumour [101]; this indicates a possibility that small tumour manifestations receive lower absorbed doses than expected on the basis of antigen density and tumour diameter [102]. Discussion As stated by DeNardo [103], “claims for specific dosimetry have to demonstrate that the frequency of excess toxicity and/or tumour underdosing significantly decreases”. Dosimetry should provide a quantification procedure that is primarily of additional benefit over empirical, fixed dosing with or without visual scintigraphic assessment. In standard oncology practice, a new therapeutic agent undergoes phase I, II and III testing before becoming a standard treatment. In a phase I study, the maximum tolerated dose is established and side-effects recorded. Dosimetry should play an essential role in this phase and establish a threshold dose above which clinically significant side-effects occur. In phase II, the new radionuclide therapy is evaluated in terms of effect on tumour response and survival. Here, dosimetry should enable the determination of a clinical dose-response relationship. In phase III, the new therapy is compared with the standard one. At this stage, dosimetry helps to elucidate the clinical effects in a larger patient group, e.g. it may be observed that some subgroups have a better or worse result. Moreover, multicentre trials offer the opportunity to compare results in different institutions and countries, as well as provide the opportunity to standardise the dosimetry procedure in a larger context. Evidence-based medicine entails randomised and prospective trials. However, after 60 years of treating thyroid cancer patients, international guidelines still cannot provide a consensus on the amount of radioiodine that should be given. Undoubtedly, the need for randomised trials will increase in the coming years, as in other areas. This underlines the importance of stepping up scientific efforts to include optimal dosimetry not only as an inherent part of radionuclide therapy, but also as an inherent part of these studies. In this context, there was a remarkable recent editorial in the Journal of Clinical Oncology [104], discussing the limitations of the body surface principle that forms the basis for chemotherapy dosing. Indeed, the bioavailability of chemotherapy, and hence the dose to the target, suffers from a similar metabolic variability as is observed for radionuclides, and this variability seems to be more important than can be accounted for by the body surface in square metres as the sole parameter. Interesting parallels may be drawn for translational research, for example in pharmacokinetic modelling and molecular imaging. Many modern gamma cameras are optimised for photons below 200 keV and are less suitable for radionuclide therapy dosimetry if higher energy photons are involved. SPECT overcomes the problem of superimposition of target and other activity, but spatial resolution at depth is always worse than in planar imaging, so quantification has to rely on more or less representative phantom models [105]. PET has a 30-40 times higher sensitivity for a given spatial resolution that is typically 5 × 5 mm. This is a major advantage over SPECT in pharmacokinetic dynamic modelling. Correction for attenuation has become relatively easy with the new PET/CT and SPECT/CT cameras, but other correction factors, e.g. for scatter, linearity and calibration, are also critical. Partial volume effects are a major issue in the quantification of small tumours and occur at below about twice the spatial resolution. This is thus more of a problem for SPECT (spatial resolution for 131I, 25-30 mm) than for PET (spatial resolution 6-8 mm). Finally, as discussed in the radiopeptide section, the radionuclides used for dosimetry must show similar biochemical and physical behaviour to those used for therapy. As previously highlighted, the use of 124I PET and 86Y-DOTATOC PET dosimetry [25, 59] has been a landmark development. PET/CT and SPECT/CT and “molecular-radiopharmaceutical developments offer major opportunities for radionuclide therapy dosimetry [1]. Further improvements in the performance of these cameras, and hence dosimetry, can be foreseen. Also, the proliferation of microsystems such as micro-PET/SPECT/CT/MRI is allowing dynamic in vivo animal research, increasing our knowledge of radiopharmaceutical biodistribution, improving quantitation and permitting early selection of therapeutic radiopharmaceuticals. Table 2 lists some of the most important methodological issues involved in performing accurate dosimetry today. Table 2.Methodological issues in performing clinical dosimetry- Diagnostic tracer and/or therapeutic activity study- Planar and/or tomographic (SPECT and/or PET) quantification- Dynamic and/or multiple time point activity sampling- Linearity of detector response in low and/or high activity- Correction factors for attenuation, scatter and/or partial volume effects- Nuclear medicine and/or radiological volume and response- Standard (MIRD,...) and/or simulative (Monte Carlo,...) modelling- Tissue heterogeneity and/or spatial resolution limits- Treatment of minimal residual disease and/or partial volume effects- Disease-induced and/or therapy-induced changes in parameters- Macro- and/or microdosimetry techniques- Animal and/or human dosimetry data Radiobiology is a science in itself. Nevertheless, up to now very little consideration has been given to the effects of radionuclide therapy at the cellular and molecular level [106]. Rather, extrapolations have been made from EBRT, despite the fundamental differences in radiation kinetics. The majority of observations in EBRT have been made under the condition of a high dose rate, while clinical radionuclide therapy entails a decreasing, low dose rate. The effect of fractionated EBRT is primarily influenced by the 4 R’s of radiobiology: repair of DNA damage, repopulation of tissues, re-oxygenation of tumour and redistribution in the cell cycle. In radionuclide therapy, with its decreasing dose rate, tumour DNA repair takes place simultaneously with sublethal damage. Bystander effects, i.e. radiation-like effects in unhit cells, may be of significance in low-dose, low dose rate radiotherapy [2]. Furthermore, it is becoming increasingly apparent that the physical paradigm of direct cell killing by double-strand DNA breaks is insufficient. Non-DNA targets, such as cell membrane or RNA, may also be critical to target cell death or dysfunction. Moreover, new molecular-targeted oncology treatments may not produce direct cell death, but rather alter biochemical pathways or cellular homeostasis, that may interact with classical radiation targets or produce new radiation targets [107]. In this paradigm, the concept of radiation dose may need re-definition and the future radiation dose may be determined in terms of the biological and functional changes produced and observed, such as by blood markers and by PET, SPECT and/or MRI. Biologically effective dose (BED) is a concept which has been successfully applied to radionuclide therapy through the landmarking studies of Barone et al. [59] using 86Y-labelled DOTATOC and kidney toxicity. BED is the product of the total physical dose multiplied by the “relative effectiveness”, which takes into account radiobiological parameters such as dose rate, radionuclide decay and tumour cell repair time, and allows direct quantitative comparison with EBRT. It should be noted that late-responding normal tissues and slow-growing tumours allow easier modelling than early-responding normal tissues and fast-growing tumours because of repopulation during treatment in the latter. Nevertheless, this may be regarded as a major development, stimulating further research [108] aimed towards the creation of a firm fundamental basis for radionuclide radiotoxicity and radiodosimetry. In future, it looks as if we shall no longer be looking at a “Holy Gray”, but rather at the worldly “BEGray”. In conclusion, recent developments in molecular medicine, PET/CT and SPECT/CT cameras and radiobiology offer major scientific and clinical opportunities in radionuclide therapy dosimetry. However, only prospective, randomised trials with adequate methodology can provide the evidence that applied clinical dosimetry results in better patient outcome than is achieved with fixed activity dosing methods.

Purinergic_Signal-3-1-2096766

CD39 and control of cellular immune responses

CD39 is the cell surface-located prototypic member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family. Biological actions of CD39 are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides. This ecto-enzymatic cascade in tandem with CD73 (ecto-5–nucleotidase) also generates adenosine and has major effects on both P2 and adenosine receptor signalling. Despite the early recognition of CD39 as a B lymphocyte activation marker, little is known of the role of CD39 in humoral or cellular immune responses. There is preliminary evidence to suggest that CD39 may impact upon antibody affinity maturation. Pericellular nucleotide/nucleoside fluxes caused by dendritic cell expressed CD39 are also involved in the recruitment, activation and polarization of naïve T cells. We have recently explored the patterns of CD39 expression and the functional role of this ecto-nucleotidase within quiescent and activated T cell subsets. Our data indicate that CD39, together with CD73, efficiently distinguishes T regulatory cells (Treg) from other resting or activated T cells in mice (and humans). Furthermore, CD39 serves as an integral component of the suppressive machinery of Treg, acting, at least in part, through the modulation of pericellular levels of adenosine. We have also shown that the coordinated regulation of CD39/CD73 expression and of the adenosine receptor A2A activates an immunoinhibitory loop that differentially regulates Th1 and Th2 responses. The in vivo relevance of this network is manifest in the phenotype of Cd39-null mice that spontaneously develop features of autoimmune diseases associated with Th1 immune deviation. These data indicate the potential of CD39 and modulated purinergic signalling in the co-ordination of immunoregulatory functions of dendritic and Treg cells. Our findings also suggest novel therapeutic strategies for immune-mediated diseases. Introduction Our research interests have been in the purinergic modulation of vascular inflammatory and cellular immune responses in transplantation settings [1, 2]. It is generally accepted that extracellular nucleotides [e.g., ATP, uridine triphosphate (UTP), adenosine diphosphate (ADP)], and the derivative nucleosides (e.g., adenosine from ATP), are released in a regulated manner by most all cells to provide the primary components for purinergic responses [3]. High levels of ATP may be released by CD4+ and CD8+ T cells upon stimulation with Con A or anti-CD3 mAb and serve to activate cells [4]. Importantly, ATP [or uridine diphosphate (UDP)] stimulation of monocytes, lymphocytes and endothelium induces largely proinflammatory responses, such as the release of interleukin (IL)-1 (or IL-8) [4–6]. On dendritic cells (DC), exposure to extracellular ATP induces migration and differentiation to drive cellular immune responses [7]. Adenosine is also recognized as a bioactive agent in vascular inflammatory states, with effects mediated on both vascular cells and leukocytes [8]. In addition, adenosine has known anti-thrombotic effects, modulates the expression of anti-apoptotic genes and is immunosuppressive [9]. Adenosine is constitutively present in the extracellular space at low concentrations, but under metabolically stressful and hypoxic conditions, the levels rise dramatically [10]. Primary release of the mediator could occur ab initio, or this might follow conversion of released nucleotides to adenosine (see later). The nucleotide/nucleoside mediators alluded to above bind specific purinergic receptors that comprise the second requirement for this complicated signalling network. Almost all cells carry cell-surface type 2 purinergic (P2) receptors for nucleotides and adenosine or type 1 purinergic (P1) receptors [11]. There are seven ionotropic (P2X), at least eight metabotropic (P2Y) and four adenosine receptor subtypes that have been identified and characterized to date [12]. Multiple P2X and P2Y receptor subtypes are expressed by monocytes and dendritic cells, whereas lymphocytes express only P2Y receptors [11]. These various receptors operate in both auto- and paracrine loops and are considered to play a complex, important role in the regulation of vascular and immune cell-mediated responses. Depending on the P2 or adenosine receptor subtype, the cell types and signalling pathway involved, these receptors might preferentially trigger and mediate short-term (acute) processes that affect metabolism, adhesion, activation or migration. However, purinergic signalling also has profound impacts upon other more protracted reactions, including cell proliferation, differentiation and apoptosis, such as seen in several chronic inflammatory states [12]. These mechanisms could be also implicated in immune memory [9, 13]. The third, and final, component of purinergic signalling systems comprises ecto-nucleotidases [2, 14]. These ecto-enzymes hydrolyze extracellular nucleotides to generate nucleosides that in turn activate adenosine receptors, often with opposing effects to those seen with P2-mediated effects. Within the past decade, ecto-nucleotidases belonging to several enzyme families have been discovered, cloned and functionally characterized by pharmacological means. In this review, we will focus on CD39, the prototype of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family (EC 3.6.1.5) [15, 16]. These proteins comprise a group of ecto-enzymes that hydrolyze extracellular nucleoside tri- and diphosphates. The ecto-nucleotidase chain or cascade, as initiated by NTPDases, is terminated by ecto-5–nucleotidase (CD73; EC 3.1.3.5) [2, 17]. Together, ecto-5–nucleotidase and adenosine deaminase (ADA; EC 3.5.4.4), another ecto-enzyme that is involved in purine salvage pathways by converting adenosine to inosine, closely regulate local and pericellular extracellular concentrations of adenosine [2]. Most notably, however, in many tissues and cells, NTPDases also comprise dominant parts of a complex cell surface-located nucleotide hydrolyzing and interconverting machinery. Such ensembles also include the ecto-nucleotide pyrophosphatase phosphodiesterases (E-NPPs), NAD-glycohydrolases, CD38/NADase activity, alkaline phosphatases, diadenosine polyphosphate hydrolases, adenylate kinase, nucleoside diphosphate kinase, and potentially ecto-F1-Fo ATP synthases [2]. Many aspects of these ecto-nucleotidase families and detailed expositions of structure/function relationships of the E-NTPDases and their role in the vasculature and nervous systems have been reviewed recently. The interested reader is referred to the recent special issue of Purinergic Signalling entitled ‘Ecto-nucleotidases–(PUSI 2, 2: 2006). This review will address only the immunobiology of CD39, focusing on T lymphocytes and alluding briefly to co-expression of CD73 by immunosuppressive T cell subsets. CD39 in the immune system Recently, almost all of the published investigative focus on CD39 has been on the thromboregulatory properties of CD39 with respect to platelet and endothelial activation [1]. However, CD39 was first described as a B lymphocyte activation marker [18]. This ecto-nucleotidase is also expressed on natural killer (NK) cells, monocytes, DC and subsets of activated T cells [19]. The relevance of the expression of CD39 by these cells is not yet clear. Development of mutant mice either null for Cd39 or where human CD39 has been over-expressed have provided useful models to study the role of this ecto-nucleotidase in immune responses [20, 21]. In the Cd39-null mouse [20], B cell numbers [anti-CD45RA 14.8; anti-CD22.2 (Lyb-8.20; PharMingen, San Diego CA, USA)] in unstimulated blood and spleen have been noted to be normal (not shown). Somewhat surprisingly, elicited IgG xenoantibody responses to solubilized xenoantigens administered to Cd39-null mice were found to be markedly suppressed [14]. With Dr. M. Cascalho (Mayo Clinic, Rochester, MN, USA), we have investigated whether Cd39-null mice are capable of appropriate antibody responses. We have noted that Cd39-null mice exhibit impaired B cell memory responses to T-dependent antigens. Unexpectedly, however, these mutant mice show a significant increase in the frequency of somatic mutations post-immunization (not shown). These observations suggest that CD39 function may contribute to the affinity maturation of antibody responses and to facilitate post-germinal center terminal B cell differentiation (M. Cascalho et al., manuscript in preparation). Although CD39 has been shown to be the dominant ecto-nucleotidase expressed by NK and NK-T cells and to impact upon cytokine production ([19]; G. Beldi, unpublished), the relevance of this has not yet been fully elucidated. CD39 is also the major NTPDase expressed by monocyte-macrophages. Upregulation of tissue factor expression by these cells in vitro and alterations in splenic macrophage populations in vivo have been observed in Cd39-null mice. P2Y receptors that are impacted upon by CD39 are critically linked to monocyte and endothelial cell responsiveness [22, 23]. P2Y receptors on monocytes could be also subject to desensitization, comparable to what we have observed in platelet P2Y1 receptors in Cd39-null mice [20]. Therefore, we were interested to observe major defects in monocycle entry and migration into the substance of Matrigel plugs injected into the subcutaneous tissue of Cd39-null mice. In parallel, we also evaluated parameters of monocyte transendothelial migration influenced by ATP in vitro and noted failure of Cd39-null cells to migrate in response to exogenous nucleotides. This defect could be overcome by co-stimulation with serotonin, suggesting a degree of P2Y receptor desensitization in Cd39-null monocytes [14, 24]. Ecto-enzymes, specifically ecto-nucleotidases, are known to play an important role in leukocyte trafficking but this complex area will not be dealt with here (for an excellent review on this topic, see [25]). However, CD39 has been recognized as a critical control point in the regulation of leukocyte accumulation within hypoxic tissues and a sixfold increase in CD39/CD73 tandem functioning in the setting of hypoxia has been demonstrated [26, 27]. Langerhans cells (LC) are members of the DC family of antigen-presenting cells residing in the skin. NTPDase1 enzymatic function on antigen presenting DC is involved in the recruitment, activation and polarization of naïve T cells. Mizumoto et al. established that LC from the Cd39-null mice do not hydrolyze ATP and ADP, unlike wild-type cells [28]. Cd39-null DC are also markedly unresponsive to ATP and are susceptible to cell death, but only after prolonged exposure to nucleotides [28]. Mutant mice null for Cd39 have amplified inflammatory responses to irritant chemicals. In these Cd39-null mice, there are major defects in dendritic cell formation, antigen presentation and T cell responses to haptens. These result in markedly attenuated responses to contact allergens in type IV hypersensitivity cutaneous responses that are also seen in inflammatory bowel disease models following haptenic stimulation (not shown). These data suggest that Cd39 expression is required for optimal stimulation of hapten-reactive T cells in mice [28]. Somewhat paradoxically, CD39 also appears to function as an additional recognition structure on haptenated target immunocytes for HLA-A1-restricted, hapten-specific cytotoxic T cells [29]. Curiously, following deletion of Cd39, LC are fully functional with respect to homing and phenotypic maturation. However, the major defect is that these cells appear less able to stimulate T cells. These abnormalities are potentially also relevant to allograft rejection processes [14]. Sequelae of these putative immune abnormalities include the relative failure of Cd39-null mice to reject allografts under limited co-stimulation blockade [30]. These data indicate a previously unrecognized role of CD39 and the effects on nucleotide-mediated signalling in immunological responses [28]. Localization of CD39 within lipid rafts implies that this ecto-enzyme may be involved in cell-cell contacts and signalling [31]. Such membrane micro-domains serve as the ‘message center–in which numerous signalling molecules are concentrated and scaffolding developed [32]. The Cd39-null DC might exhibit defective functions because of putative defects in initiating and maintaining cell-cell contacts. This mechanism speculatively involves facilitating integrin associations by purinergic signals within the ‘immunological synapse–and would have parallels to the way chemokines have been implicated in this process [33–36]. CD39 on DC and/or T cells may be likewise translocated to the immunological synapse during antigen presentation to facilitate intercellular signalling. More typically, CD39 is considered to play a cellular immunoregulatory role by hydrolyzing ATP (and perhaps ADP) released by T cells during antigen presentation and thereby generating adenosine, a known immunosuppressive molecule. Recent work has also indicated that regulatory CD4+ve CD25+ve T cells (Treg cells) play important roles in the suppression of immunological reactivity and maintenance of tolerance [37, 38]. Patterns of expression of CD39 by Treg and the possibility that the balance of extracellular nucleotides/nucleosides influence(s) the function of these interesting cells have been a major focus of investigation in our laboratories of late. CD39 expression by immunosuppressive regulatory T cells Treg are central to the acquisition of immunological tolerance. Here the immune system does not mount cellular responses against specific antigens while reactivity towards other antigens is maintained. The precise mechanisms underlying the acquisition of tolerance are not fully understood. Transplanted graft outcomes in the absence of heavy immunosuppression depend on consistent balances between cytopathic effector cells and Treg [39]. Depletion of the cytopathic T cell clone has been proposed as one mechanism for the induction of tolerance. However, it is now clear that the long-term maintenance of tolerance is also dependent on self-perpetuating immunoregulatory mechanisms that limit or constrain alloresponses. The suppressive Treg populations are considered key to the development and maintenance of peripheral tolerance [33, 40]. Significant deficiencies are apparent in identifying Treg. Although traditionally defined by CD4+CD25+ expression, the latter marker along with other membrane proteins such as GITR and CTLA4 becomes non-specific and somewhat redundant following activation. Such markers are widely upregulated on other cells thereby losing specificity for the Treg population. The forkhead winged transcription factor FoxP3 is specific for Treg; however, its intracellular location limits its usefulness in the study of this population. Moreover, it is now apparent that CD25 does not encompass all Treg as defined by FoxP3+ expression [41]. Similarly, the mechanisms of Treg action are poorly defined. Putative mechanisms of suppression by Treg include cell-to-cell contact predominant in vitro and the release of soluble mediators that may predominate in vivo. As an example, IL-10 and transforming growth factor β have been identified as soluble factors that may mediate Treg suppression. Our recent studies and data from other groups have indicated that adenosine is an important mediator generated by Treg cells, and appears responsible for, at least in part, their functions [42]. Patterns of immune expression of CD39 by Treg were determined using standard techniques; using the Cd39-null mouse cells as negative controls. CD3+, CD8+, NK1.1+, B220+, CD11b+ and CD11c+ cells were positively selected from spleens and lymph nodes of 8- to 10-week-old C57BL6 mice through the use of MACS Sort magnetic beads in MACS LS Separation columns (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells enriched for CD4 were obtained from lymph node and spleen preparations using CD4 T cell columns (R&D Systems, Minneapolis, MN, USA). CD4+/CD25+ and CD4+/CD39+ were positively selected using the relevant antibody and MACS Sort magnetic beads in MACS MS Separation columns (Miltenyi Biotech, Bergisch Gladbach, Germany). Where indicated, the same subsets were also sorted after staining the purified CD4+ cells with CD25 or CD39, using the MoFlo cell sorter (BD Biosciences, San Jose, CA, USA). In selected experiments, CD4+/CD25+ cells were purified using the murine CD4+CD25+ T regulatory cell isolation kit from Miltenyi Biotech (Bergisch Gladbach, Germany). The purity of the different cell populations was verified by flow cytometry on FACSort (BD Biosciences, San Jose, CA, USA). Anti-mouse and anti-human CD4, CD8, CD19, B220, NK1.1 and CD25 were from eBiosciences (San Diego, CA, USA), as were anti-mouse CD5, CD62L, CD45RB, interferon (IFN)-γ and IL-4. Purified anti-mouse CD3 and CD28 were from PharMingen (BD Biosciences,San Diego CA, USA). Rabbit anti-mouse CD39 polyclonal antibody was used to stain cells purified from wild-type lymph nodes and from spleen, as described [43]. Using cells harvested from naïve C57BL6 mice, CD39 was found to be expressed by the majority of monocytes and by subsets of lymphocytes, also inferred by gating on forward scatter (FSC) and side scatter (SSC; not shown). Among node-derived lymphocytes, the majority of CD39+ cells are found in CD19+ (or B220+) B cells. The remaining CD39+ cells were shown to reside almost totally within the CD4+ subset (Fig. 1a). There, they consistently range from 8 to 12% of all CD4+ cells in lymph node (and spleen). Further characterization of resting C57BL6 lymphocytes revealed that CD39 is selectively expressed on CD4+/CD25+ T cells (Fig. 1a). CD39 is consistently and abundantly expressed in CD4+/CD25high T cells, while the CD4+/CD25dim populations show a dichotomic expression pattern of CD39, with ~50% of the cells positive. Less than 1% of CD39+ cells were found in the CD4+/CD25− compartment. The majority of peripheral CD4+/CD39+ cells are also CD45RBlow and mostly CD62Llow, with similar expression patterns observed in BALB/c mice (not shown). Fig. 1 a Expression of CD39 on mouse lymphoid cells. Lymph node cell suspensions were prepared from 8-week-old C57/BL6 mice and were gated based on FSC and SSC parameters (not shown). CD4+ cells were gated based on CD25 expression. CD39 expression on the different subsets is shown in the histograms (open profiles) plotted against an irrelevant control (gray profiles). Differential expression of CD39 on CD4+ cells can be shown to be closely associated with CD25. b RT-PCR analysis of foxp3 mRNA expression. This was done in sorted cells with the following markers: Foxp3+/CD39+, Foxp3+/CD39-, Foxp3-/CD39+ and Foxp3-/CD39-. CD4+ cells obtained from the Foxp3-GFP ‘knockin–animals that had been generated by Mohamed Oukka and Vijay K. Kuchroo with colleagues (Ref). B lymphocytes were used as CD39+ control and included for comparison (striped bars). c RT-PCR analysis of CD73 mRNA expression. This was done in sorted cells with the following markers: Foxp3+/CD39+, Foxp3+/CD39-, Foxp3-/CD39+ and Foxp3-/CD39-. B lymphocytes were used as CD39+ control and included for comparison (striped bars). CD73 is also a useful immunophenotypic marker for Treg cells (not shown); and when combined with CD39 provides near-concordance with foxp3 expression. The Foxp3-/CD39+ subset does not express CD73 and these cells resemble the memory phenotype (not shown) These results were confirmed by reverse transcription polymerase chain reaction (RT-PCR) analysis of selected cell populations. Both CD11b+ and CD11c+ cells expressed high levels of CD39 mRNA, indicating that macrophages and dendritic cells constitutively express CD39. Within the T cell compartment, the highest expression levels of CD39 transcripts were found in CD4+ cells. Gene expression profiling of CD4+ T cells, sorted on the basis of CD39 cell surface expression, indicated that the CD4+/CD39+, but not CD4+/CD39−, T cells robustly express markers of Treg cells [40], viz. Foxp3, CD25, GITR and CTLA-4. Further, CD4+/CD39+ cells are anergic in the absence of IL-2 (not shown) and suppress T effector (CD4+/CD25−) proliferation with an efficacy similar to that observed with classic CD4+/CD25+ Treg (S. Deaglio et al., submitted manuscript). We have also confirmed the utility of CD39 in defining human Treg populations as comparable patterns of CD39 antigen expression in human cells are observed. The majority of human CD19+ B cells express CD39; however, within the T cell compartment CD39+ cells are present in the CD4+ subset (not shown). As in the mouse, CD39 is highly expressed in the CD4+/CD25high T cells, with only ~50% of cells CD39+ within the CD4+/CD25dim population. Negligible CD39 expression is found in the CD4+/CD25− population (not shown). Gene expression profiling of human CD4+/CD39+ T cells also confirms that these cells express Foxp3, GITR and CD25 in a pattern analogous to that of traditional CD4+/CD25+ Treg (K. Dwyer et al., submitted). Our analysis demonstrates that CD39 expression in both murine and human T cells is restricted to a subpopulation of CD4+/CD25+ cells that expresses markers associated with T regulatory function. We have further examined the exact relationship between CD39 expression and the regulatory phenotype by using T cells from mutant mice with the green fluorescent protein (GFP) reporter gene introduced into the endogenous Foxp3 locus [designated as ‘Foxp3+(GFP+) knockin–cells; kindly provided for these experiments by M. Oukka and V. K. Kuchroo, Boston, MA, USA] [44]. Four populations could be defined by differential CD39 and Foxp3 expression: Foxp3+/CD39+, Foxp3+/CD39− (minor population grouping), Foxp3−/CD39+ and Foxp3−/CD39−. These populations were sorted and gene expression profiles determined. The GFP+/CD39+ fraction was shown to mirror the genetic profile of Treg, as defined by the presence of Foxp3 transcripts in this positive control (Fig. 1b). Interestingly, the next major subpopulation Foxp3–/CD39+ cells contain T lymphocytes that are not classic Treg and yet appear to be associated with the memory compartment (W. Gao, manuscript in preparation; Fig. 1b). Foxp3+(GFP+) Treg however were also found to co-express the ecto-nucleotidase CD73, a unique situation amongst T lymphocytes (Fig. 1c). Consistent with other phenotypic data (not shown), RT-PCR analysis confirms that Foxp3+/CD39+ cells have high levels of gene expression of both CD39 and CD73 (Fig. 1b). CD73, which converts AMP to adenosine downstream of CD39, has been independently identified on CD25+ (FoxP3+) Treg and CD25− uncommitted primed precursor Th cells [42]. This recent work further supports our observations that the expression of ecto-nucleotidases and consequent adenosine generation play a role in the mediation of some of the suppressive capabilities of Treg cells. Hence, CD73 is co-expressed with CD39 as a cell surface marker of murine Tregs (Fig. 1c). Adenosine as a Treg effector molecule Tregs from mutant mice deficient in CD39 have impaired regulatory function manifesting as a 50% decrease in the ability of Cd39-null Tregs to modulate effector T cell function in vitro and in vivo. These results indicate that CD39 expressed by Treg is the major and rate limiting ecto-nucleotidase responsible for the generation of adenosine and suggest that a putative CD39/CD73-adenosinergic axis (i.e., generating adenosine) may contribute to the immunoregulatory function of Treg (S. Deaglio et al., manuscript submitted; also Fig. 2). Fig. 2Schematic representation of Treg markers. The cellular phenotype of these suppressive T cells can be defined by FoxP3+/CD39+/CD73+ expression. Phosphohydrolysis of extracellular nucleotides by CD39 and CD73 generates adenosine, which exerts a component of the immunosuppressive effect Adenosine plays a central and direct role in the regulation of inflammatory responses and limiting inflammatory tissue destruction [9, 10, 45]. Potentially, close cell-cell contacts with pericellular generation of adenosine and regulated expression of adenosine receptors may be important modulatory factors directly suppressing T cell responses. Early in the immune response, adenosine favors recruitment of DC, which initiates specific immune responses [9, 10, 28]. The immunosuppressive effects of adenosine on T cells are thought to be mediated primarily through the A2A receptor. Adenosine inhibits the production of proinflammatory cytokines and superoxide anions. Adenosine activation of A2A receptor also induces heterologous desensitization of chemokine receptors, which are critical in leukocyte trafficking, through the activation of protein kinase A [46]. It is also feasible that the suppression mediated via Treg-generated adenosine could be exerted indirectly through downregulation of co-stimulatory molecules on DC or by competition for other non-characterized signal molecules [47]. Irrespective of the mechanism of action, we have shown that FoxP3+ Tregs are the only T cells that contain the full enzymatic machinery necessary to generate adenosine. This effect, in conjunction with the expression of the adenosine A2A receptor on effector (CD4+/CD25−) T cells, generates immunosuppressive loops limiting effector cell proliferation both in vitro and in vivo (S. Deaglio et al., submitted). Immune deviation and autoimmune diathesis in Cd39-null mice The pathophysiological relevance of the CD39-adenosinergic loop was further tested by examining effects of exogenous, pharmacological adenosine A2A receptor agonists on T cells, committed to either Th1 or Th2 lineages [48]. Adenosine might contribute to the resolution of inflammation by facilitating Th2 pathways by the inhibition of Th1 cell functions [49]; this property has been addressed further in the Cd39-null mice. Subsets of naïve CD4+ T cells were polarized to a Th1 or Th2 phenotype through the addition of IL-12 (10 ng/ml) and anti-IL-4 (10 μg/ml) or IL-4 (10 ng/ml) and anti-IFN-γ (10 μg/ml), respectively, following stimulation with plate-bound anti-CD3 (5 μg/ml) and soluble anti-CD28 (2.5 μg/ml). The polarization of cells was confirmed by intracellular cytokine staining and gene profiling via RT-PCR analysis. Heightened expression of the A2A receptor, as measured by RT-PCR, could be detected in CD4+/CD25− cells polarized in vitro to Th1, but not Th2, phenotypes at day 3 (not shown). In keeping with this observation, the addition of the adenosine A2A receptor agonist, ATL146e (kind gift from J. Linden, Adenosine Therapeutics), at day 3 of culture, when all cells are already committed to either a Th1 or Th2 phenotype, selectively inhibits Th1 proliferation. This suggests once more that the A2A receptor is the critical immunomodulatory adenosine receptor [9]. No inhibition of the Th2 proliferation response by ATL146e [50] could be noted (Fig. 3a). Fig. 3CD39 generation of adenosine preferentially regulates Th1 immune responses. a 3H-thymidine incorporation of CD4+ T cells purified from WT mice as polarized towards a Th1 or a Th2 phenotype for 5 days. The selective adenosine A2A agonist ATL146e was added after 3 days of culture. Data are expressed as % of inhibition and are the mean of duplicates; error bars represent the SEM of three independent experiments. b CD4+ T cells were purified from Cd39-null (filled histograms) or WT (open histograms) mice and polarized towards a Th1 phenotype. The mRNA was extracted at day 3 and assayed for IFN-g (left panel) by RT-PCR. Data are representative of more than four independent experiments. c CD4+ T cells were purified from Cd39-null (filled histograms) or WT (open histograms) mice and polarized towards a Th1 phenotype. The indicated adenosine receptor agonists and antagonists were added at the beginning of the experiment (left panel). d This panel shows the effects of apyrase on IFN-g production by Cd39-null CD4+ T cells polarized towards a Th1 phenotype. For these experiments, mRNA was extracted at day 3 and assayed for IFN-g (RT-PCR). Representative data are shown from three independent experiments for each. e Representative images of Cd39-null mouse on C57BL/6/129 SVJ (upper panel) or BALB/c backgrounds (lower panel) manifesting alopecia. Histology of skin samples obtained from Cd39-null mice affected by alopecia. Uninvolved skin areas from the same animals as well as samples from WT mice were used for comparison. Biopsies were fixed and stained for CD4 cells. Original magnifications: ×10 for the upper middle panel, ×40 for the upper right and ×20 for the lower panels. f Skin samples were obtained from Cd39-null mice affected by alopecia, the tissue homogenized and mRNA extracted. Cytokine profiling was performed by RT-PCR, using a preamplification technique. Uninvolved skin from the same animal or a matched WT mouse used as controls. Representative data are from three animals Consistent with these results, in vitro polarization of CD4+ T cells towards a Th1 phenotype results in the increased production of IFN-γ by Cd39-null T cells, with a Th1-deviated phenotype (Fig. 3b). Addition of ATL146e [50], or soluble NTPDases (apyrase), strongly inhibits the Th1 response in both the wild-type and Cd39-null polarized cells confirming that A2A receptors are present and functional in both groups. These data suggest lack of substrate in the Cd39-null cells to be responsible for the observed Th1 bias (Fig. 3c). The A2A antagonist 8-(3-chloro-styryl) caffeine was shown to augment IFN-γ production in wild-type cells polarized to a Th1 phenotype (Fig. 3c), further supporting this hypothesis. In addition, reconstitution of Cd39-null cells with apyrase restores the catalytic potential of the cells, producing adenosine and inhibiting IFN-γ production in a dose-dependent manner, effects similar to that of ATL146e (Fig. 3d). Additional evidence that the perturbation in adenosine generation causes Th1 deviation, is that Cd39-null mice spontaneously develop autoimmune alopecia. Fifteen per cent (9/59) of designated Cd39-null animals develop skin lesions characterized by extensive and well-demarcated hair loss (Fig. 3e). No lesions are observed in age-matched WT controls (0/28). Similar lesions were observed in Cd39-null mice on the BALB/c background suggesting that the phenotype is strain independent (Fig. 3e). The alopecia typically appears at ~20–0 weeks of age in the facial region and thereafter extends out to include the trunk. The skin lesions are characterized by the presence of a population of CD4+ and CD8+ lymphocytes within the damaged hair follicles (Fig. 3e). Moreover, sixfold increases in IFN-γ transcript levels are noted in areas of skin with active disease, when compared to areas of uninvolved skin from the same animal or from wild type controls (Fig. 3f). In addition, there is increased expression of IL-2, Foxp3 and CTLA-4, consistent with the cellular infiltration. IL-4 and IL-10 were not detected (Fig. 3f). Autoimmune manifestations impacting other organ systems and on renal function are under evaluation (D. Friedman, not shown). These data validate the importance of adenosine in directing T cell subset differentiation and support a role for CD39 in orchestrating Treg cell suppressive responses under both in vitro and in vivo conditions. Summation This review summarizes components of extracellular nucleotide-mediated signalling pathway in T cells that are impacted upon largely by CD39, the prototypic member of the E-NTPDase family of ecto-nucleotidases. Modulated, distinct NTPDase expression appears to regulate nucleotide- and nucleoside-mediated signalling in the immune system. As the vasculature uses similar mediators to regulate blood fluidity and hemostasis, expression of CD39 on either endothelial or immune cells might allow for full integration of vascular inflammatory and immune cell reactions at sites of injury. There is a wide field for future investigations of the role of nucleotides, nucleosides and ecto-nucleotidases in immune-mediated diseases. Increasing interest in this field may open up new avenues for investigation and the development of new treatment modalities for a large variety of illnesses, including atherosclerosis and the vascular or immune inflammation seen in transplant-related diseases.

Purinergic_Signal-4-2-2377323

Endogenous ATP release inhibits electrogenic Na+ absorption and stimulates Cl− secretion in MDCK cells

Our previous studies with a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) transfected with FLAG-labeled α, β, and γ subunits of epithelial Na+ channel (ENaC) showed that, although most of the short-circuit current (Isc) was amiloride sensitive (AS-Isc), there was also an amiloride-insensitive component (NS-Isc) due to Cl− secretion (Morris and Schafer, J Gen Physiol 120:71–85, 2002). In the present studies, we observed a progressive increase in NS-Isc and a corresponding decrease in AS-Isc during experiments. There was a significant negative correlation between AS-Isc and NS-Isc both in the presence and absence of treatment with cyclic adenosine monophosphate (cAMP). NS-Isc could be attributed to both cystic fibrosis transmembrane conductance regulator (CFTR) and a 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS)-sensitive Ca2+-activated Cl− channel (CaCC). Continuous perfusion of both sides of the Ussing chamber with fresh rather than recirculated bathing solutions, or addition of hexokinase (6 U/ml), prevented the time-dependent changes and increased AS-Isc by 40–60%, with a proportional decrease in NS-Isc. Addition of 100 μM adenosine triphosphate (ATP) in the presence of luminal amiloride produced a transient four-fold increase in NS-Isc that was followed by a sustained increase of 50–60% above the basal level. ATP release from the monolayers, measured by bioluminescence, was found to occur across the apical but not the basolateral membrane, and the apical release was tripled by cAMP treatment. These data show that constitutive apical ATP release, which occurs under both basal and cAMP-stimulated conditions, underlies the time-dependent rise in Cl− secretion and the proportional fall in ENaC-mediated Na+ absorption in FL-MDCK cells. Thus, endogenous ATP release can introduce a significant confounding variable in experiments with this and similar epithelial cells, and it may underlie at least some of the observed interaction between Cl− secretion and Na+ absorption. Introduction Madin-Darby canine kidney (MDCK, type I) cells grown as epithelial monolayers have been used extensively as a model of the mammalian collecting duct (CD). These monolayers exhibit many characteristics of the CD, including a high transepithelial resistance and amiloride-sensitive Na+ reabsorption that is mediated by the epithelial Na+ channel (ENaC) and stimulated by agonists that increase intracellular cyclic adenosine monophosphate (cAMP) [1, 2]. Similar cell lines that are derived from the CD, including mouse cortical CD (M-1) and inner medullary CD (mIMCD-K2) cells [3–5] and A6 cells from the Xenopus distal nephron [6, 7] share these characteristics. However, in contrast to the CD, all of these cell lines also have Cl− channels in their apical membranes, and, when studied under short-circuit conditions, exhibit Cl− secretion, which is observed as an amiloride-insensitive component of the short-circuit current (NS-Isc) that is also stimulated by cAMP [2, 3, 5–8]. We previously developed a line of MDCK cells (FL-MDCK) that had been retrovirally transfected with rat ENaC subunits containing the FLAG epitope in their extracellular loops [2]. Monolayers of these cells had higher rates of ENaC-mediated Na+ absorption, measured as the amiloride-sensitive short-circuit current (AS-Isc), than did the original MDCK cell line. Our previous studies with FL-MDCK monolayers in Dulbecco’s modified Eagle’s medium (DMEM) showed that cAMP treatment produced a rapid transient peak in the total short-circuit current (Isc) within 5 min, followed by a broad peak that decayed over 20 min [2]. This biphasic response to cAMP treatment has also been reported in A6 and M-1 cultures and has been attributed to rapid stimulation of Cl− secretion mediated by cystic fibrosis transmembrane conductance regulator (CFTR) followed by a slower activation of ENaC [6–9]. In FL-MDCK cells, the biphasic response to cAMP treatment was followed by a decay of Isc, which reflected a decrease in AS-Isc [2]. Morris and Schafer [2] found that this late fall in AS-Isc in FL-MDCK monolayer was prevented by the omission of Cl− from the bathing solution and attributed the effect to Cl− secretion via CFTR, which has subsequently been demonstrated to be present in this cell line [10]. A large body of evidence indicates that CFTR is a cAMP-regulated Cl− channel as well as a conductance regulator, which is colocalized with ENaC in airway, colonic, and other epithelial tissues [11, 12]. It has been proposed that when cAMP activates CFTR, ENaC is inhibited, and this inhibitory effect of CFTR has been hypothesized to explain the pathophysiology of cystic fibrosis in airway epithelia [13, 14]. In Xenopus oocytes expressing ENaC, coexpression of CFTR reduces the ENaC-mediated Na+ current [15], an effect that might be attributed to a direct effect of CFTR on ENaC activity. However, Kunzelman and his collaborators [16, 17] have shown that the inhibitory effect of CFTR can be mimicked by coexpression of other anion channels or treatment with amphotericin B and is explained by an increase in intracellular Cl− ([Cl−]i). We have shown that an increase in [Cl−]i also inhibits ENaC in FL-MDCK cells; however, this effect cannot explain the effect of Cl− secretion on Na+ absorption with cAMP treatment, because stimulation of Cl− secretion by cAMP results in a fall rather than a rise in [Cl−]i [10]. Moreover, in the Xenopus oocyte expression system, CFTR activation inhibits ENaC at a continuous holding potential at which CFTR mediates inward currents corresponding to Cl− efflux [18]. Under these conditions, activation of CFTR will not result in an increase in [Cl−]i. Therefore, a change in [Cl−]i is unlikely to fully explain the observed reciprocal regulation of ENaC and CFTR. In the present studies, we show that adenosine triphosphate (ATP) accumulation in the apical solution produces a progressive stimulation of NS-Isc and a corresponding decrease in AS-Isc when FL-MDCK cells are studied in Ussing chambers. The possibility of constitutive ATP release raises an important issue when interpreting changes in ion transport in experiments such as these. More important in the present context, ATP release can explain at least part of the inverse relationship between Cl− secretion and Na+ reabsorption, and, as suggested by Wilson et al. [19], it may be involved in the conversion of CD cells from absorptive to secretory in polycystic kidney disease. Materials and methods Cell culture The FL-MDCK cells used were a clone of the type-1 MDCK line that had been retrovirally transfected with flagged rat α-, β-, and γENaC by Morris and Schafer [2]. The cells used were from passages 5–35 and were cultured in T-75 flasks at 37°C in DMEM (Life Technologies) supplemented with 10% fetal bovine serum (FBS), 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.4), 1% Pen/strep-fungizone, and the selection antibiotics G418 (800 μg/ml), hygromycin (300 μg/ml), and puromycin (5 μg/ml). For transepithelial transport studies, cells were seeded onto 24-mm Transwell inserts (Costar; Catalog No. 3412) at a density of ∼105 cells/cm2. For bioluminescence assays, cells were seeded on 12-mm collagen-coated Millicell inserts (Millipore; Catalog No. PHIP 012 50) at the same density. After seeding, cells were fed daily with DMEM containing FBS and HEPES but without selection antibiotics for 5–7 days. Before use in the experiments, the cell monolayers were induced with 1 μM dexamethasone plus 2 mM Na+ butyrate in the culture medium overnight. Ussing chamber experiments FL-MDCK monolayers were carefully cut from the plastic insets and were mounted in Ussing-type chambers. In most experiments, both sides of the monolayers were bathed with Krebs-Ringer bicarbonate (KRB) solution, which contained (in mM): 113 NaCl, 1.2 Na2HPO4, 25 NaHCO3, 1.1 CaCl2, 1.2 MgCl2, 4.5 KCl, and 10 glucose. The KRB solutions were continuously gassed with a mixture of 95% O2-5% CO2 to give a pH of 7.40 at 37°C. As shown by the schematic diagram in Fig. 1a, transepithelial short-circuit currents (Isc, μA/cm2) and voltages (Vte) were measured and conductances were evaluated every 30 s by measuring the current deflections induced by 4-s symmetrical voltage pulses of ±2 mV, as described previously [2, 10]. In most experiments, KRB was continuously recirculated on both sides of the epithelium from 10-ml reservoirs (Fig. 1b); however, in the experiments involving “fresh perfusion,” as shown in Fig. 1c, both sides of the monolayers were continuously perfused with fresh rather than recirculated KRB. In those experiments involving cAMP treatment, 100 μM 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) plus 100 μM isobutylmethylxanthine (IBMX) were added to both the apical and basolateral solutions. AS-Isc was defined as the change in Isc produced by the addition of 10 μM amiloride to the apical solution, and the remaining Isc was defined as NS-Isc. Because we observed time-dependent changes in AS-Isc and NS-Isc in the voltage clamp experiments, we conducted additional experiments in which the monolayers were left under open-circuit conditions during the course of the experiment, and the open-circuit transepithelial voltage (Vte, mV) was measured continuously except for intermittent voltage clamping to 0 mV and ±2 mV for a total of 4 s every 30 s to obtain Isc and conductance measurements. Fig. 1a Schematic diagram of the electrophysiological circuitry. The Ussing chamber is constructed of two lucite half-chambers, which are clamped together with the Madin-Darby canine kidney (MDCK) cell monolayer (on a Transwell membrane) between them. Voltage-sensing electrodes are positioned close to the center and on either side of the monolayer. Current passing electrodes, made from silver disks with a central hole to accommodate the voltage electrodes, are placed at the back of each half chamber and parallel to the monolayer. The transepithelial voltage (Vte) is measured by the voltage clamp/analog to digital converter (called “the clamp”). When no current is being passed, the voltage recorded is called the “open circuit” Vte. To short-circuit the epithelial monolayer, the clamp passes a current (I) that is sufficient to drive Vte to zero. The current measured in this situation is called the short-circuit current (Isc). b In experiments in which the Krebs-Ringer bicarbonate solutions (KRB) in the Ussing chambers were recirculated, 10-ml syringes were used as reservoirs, and a roller pump was used to continuously move the KRB from each half-chamber to its respective reservoir. c In experiments in which fresh KRB solutions were continuously perfused, large beakers served as the solution reservoirs from which KRB was constantly infused into each half-chamber by a roller pump and constantly removed by suction to a waste reservoir. In both the recirculation and fresh perfusion methods, the solutions in the reservoirs (10-ml syringes or beakers, respectively) were constantly bubbled with a 95% O2–5% CO2 gas mixture Bioluminescence detection of ATP release ATP released from the MDCK monolayers was measured as described by Taylor et al. [20]. Briefly, culture medium was removed from the inserts, and the cell monolayers were washed three times with phosphate-buffered saline (PBS) solution to remove any FBS present in the culture medium. Opti-minimal essential medium (MEM) with 2 mg/ml luciferin-luciferase reagent (Sigma) was added to the apical or basolateral side of the monolayer. Each insert with cells contained 200 μl of solution, and the average light signal was measured for 10-s, nonintegrated photon collection periods with a TD-20/20 luminometer (Turner Designs; Promega). Treatment with cAMP was applied by adding 1 μl of 20 mM CPT-cAMP and 20 mM IBMX stock solution to the 200 μl apical solution of the inserts (final concentration 100 μM for both), and control experiments were done with addition of the same volume of vehicle alone. The light output was measured for at least 2 min, and the average was taken for the calculation of ATP release. Individual batches of luciferin-luciferase reagent were tested for relative sensitivity by measuring the light output response to serial dilutions of an ATP stock solution with 2 mg/ml luciferin-luciferase reagent in Opti-MEM. Data analysis and statistics StatView for Macintosh (SAS Institute Inc.) was used for statistical analyses. Statistical significance (P < 0.05) was determined by analysis of variance (ANOVA) with Bonferroni/Dunn post hoc testing for multiple comparisons and by paired or nonpaired t-tests, as appropriate, for single comparisons. Time dependence of transport was evaluated by linear regression analysis; the R value and significance of the slope are given. Correlation analysis was used to compare AS-Isc and NS-Isc and calculate a correlation coefficient, r. Materials All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted. Results Time-dependent changes in Isc The Isc in FL-MDCK cells was quite high and variable in the first 5 min after recording was begun using continuous short-circuiting. In six experiments, 10 μM amiloride added to the apical solution in the initial 5 min decreased Isc by 91 ± 2%, indicating that most of the current was due to ENaC-mediated Na+ absorption. The initial high Isc subsequently declined to a nadir at ∼15 min (Fig. 2a and b). Addition of amiloride at this nadir (Fig. 2a) almost completely inhibited Isc; however, when the experiment was continued beyond the nadir, amiloride was less effective in reducing Isc (Fig. 2b). In 35 experiments such as those in Fig. 2a and b, the time interval between the nadir and amiloride addition to the apical solution was varied from 0 to 45 min. In these experiments, the average Isc at the nadir was 15.2 ± 0.5 μA/cm2. Regression analysis showed that NS-Isc increased significantly (R = 0.76, P < .001) and AS-Isc decreased significantly (R = 0.53, P < .002) as a function of the time between the nadir and the addition of amiloride. The same high initial Isc and the time-dependent increase of NS-Isc were also observed in experiments using the intermittent short-circuit technique (see “Materials and methods”), which indicated that neither effect is produced by prolonged short-circuiting of the epithelium (data not shown). Fig. 2Time course of the short-circuit current (Isc) and response to amiloride. Isc was measured across monolayers of FL-Madin-Darby canine kidney (FL-MDCK) cells induced overnight with 2 mM Na butyrate and 1 μM dexamethasone. Amiloride (10 μM) was added to the apical solution either early (A) or late (B) at the point indicated by the arrow. The transepithelial conductance (Gte) is proportional to the height of the deflections in Isc produced by transiently clamping the voltage to ±2 mV, as described in “Materials and methods” As shown in Fig. 3, the effect of cAMP on Isc also depended on whether the treatment occurred early (at the nadir of Isc, Fig. 3a) or late (∼40 min after the nadir, Fig. 3b). With both early and late cAMP addition, Isc increased immediately, reaching a transient peak within 5 min; however, the magnitude of this initial peak was exaggerated with late cAMP treatment (Fig. 3b). After the initial response, Isc showed a secondary broader increase that decayed more rapidly after late (Fig. 3b) than after early (Fig. 3a) cAMP treatment. In 29 experiments such as those in Fig. 3, the time interval between the nadir in Isc and cAMP treatment was varied from 2 to 58 min, and 10 μM amiloride was added ∼30 min after cAMP treatment. Regression analysis showed a significant increase in NS-Isc with the time between the nadir and cAMP treatment (R = 0.60, P < .002). Fig. 3Time course of Isc response to cyclic adenosine monophosphate (cAMP) treatment. A mixture of 100 μM 8-(4-chlorophenylthio) (CPT)-cAMP plus 100 μM isobutylmethylxanthine (IBMX) was added as indicated by the lower bar, either early (a) or late (b) to both sides of the monolayers, followed by 10 μM amiloride addition to the apical solution only (upper bar) Further examination of the data from these experiments (see Fig. 4) revealed a significant negative correlation between AS-Isc and NS-Isc, both with and without cAMP treatment. In other words, the time-dependent increase in NS-Isc observed in both sets of experiments was correlated with a corresponding decrease in AS-Isc. As expected, the average AS-Isc was significantly greater in those experiments with cAMP treatment (20.2 ± 1.1 μA/cm2, n = 29) than in those without (12.8 ± 0.6, n = 35), but the correlation with NS-Isc was not significantly different between the two groups. Fig. 4Negative correlation between the amiloride-sensitive component of the short-circuit current ( Isc) (AS-Isc) and the nonamiloride-sensitive component (NS-Isc) in the absence and presence of cyclic adenosine monophosphate (cAMP). The 35 experiments in the first group (open circles, solid line) were conducted using the same protocol as the representative experiments in Fig. 1 with no cAMP addition. The 29 experiments in the second group (solid dots, dashed line) followed the protocol of the representative experiments in Fig. 2, with the addition of 100 μM 8-(4-chlorophenylthio) (CPT)-cAMP plus 100 μM isobutylmethylxanthine (IBMX) ∼30 min before the addition of amiloride. In both groups, there was a significant correlation between AS-Isc and NS-Isc: −cAMP group, r = 0.79, P < .001; +cAMP group, r = 0.65, P < .001 Effect of cAMP and intracellular Ca2+ on NS-Isc In the experiments shown in Fig. 5, NS-Isc was measured throughout the experiments in the continuous presence of 10 μM amiloride in the apical solution. Treatment with cAMP produced an initial rapid peak in NS-Isc, followed by a sustained plateau. We then tested the effects of two inhibitors of Cl− transport pathways: glibenclamide, which is a well-established inhibitor of CFTR; and 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS), which is a less selective inhibitor of Cl− channels including the Ca2+-activated chloride channel (CaCC) and the chloride channel (CLC) family of Cl− channels but not CFTR. (See the Discussion for more detail about these inhibitors and references to their specificity.) Addition of 200 μM glibenclamide followed by 300 μM DIDS to the apical solution ∼20 min after cAMP treatment significantly decreased NS-Isc to or below the magnitude prior to cAMP treatment (Fig. 5a & b). In other experiments, addition of apical DIDS before cAMP treatment reduced the late plateau but not the initial peak of NS-Isc (Fig. 5c and e), whereas apical glibenclamide significantly decreased the initial peak, with no effect on the late plateau (Fig. 5d & e). These data suggest that at least two types of Cl− channels are stimulated by cAMP. One is sensitive to glibenclamide and responds to cAMP quickly while the other is sensitive to DIDS and responds to cAMP slowly. Fig. 5Effects of glibenclamide and 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS) on the nonamiloride-sensitive component (NS-Isc). In these experiments, 10 μM amiloride was continuously present in the apical solution. A Effect of cyclic adenosine monophosphate (cAMP) treatment [100 μM cAMP plus 100 μM isobutylmethylxanthine (IBMX), lowest bar] on NS-Isc. The middle and upper bars indicate the time of addition of 300 μM DIDS and 200 μM glibenclamide (Gliben.) to the apical solution. B Summary of 14 experiments such as that in A. The mean NS-Isc values immediately before cAMP treatment (point 1 in A), 3–5 min (“initial”, point 2) and 15–20 min (“late”, point 3, just before DIDS or glibenclamide addition) after cAMP treatment, and after DIDS plus glibenclamide (point 4). * Significantly different from value before cAMP treatment, P < .001; † significantly different from the initial and late NS-Isc, P < .001. C Effect of 300 μM apical DIDS added before cAMP treatment. D Effect of 200 μM apical glibenclamide added before cAMP treatment. E Mean values of the change in NS-Isc (the cAMP-stimulated NS-Isc) produced by cAMP treatment in the initial (point 2 in A, C, and D) or late (point 3) response period in three sets of experiments: with no inhibitor (control, from summary of 15 experiments in B), DIDS before cAMP (as in C, n = 6) ,and glibenclamide before cAMP (as in D, n = 5). * Significant difference compared with the corresponding control value (calculated from the data in B), and between initial and late response to cAMP, P < .001 The Cl− channel that was quickly activated by cAMP and inhibited by glibenclamide has been attributed to CFTR, the expression and function of which has been identified by us in this same cell line [10]. Because cAMP increases intracellular Ca2+ ([Ca2+]i) in MDCK cells [21], we examined the hypothesis that CaCC contributes to the fraction of NS-Isc that is inhibited by DIDS by using thapsigargin to produce a modest but sustained elevation of [Ca2+]I [22, 23]. (Thapsigargin is known to elevate intracellular Ca2+ by releasing it from the endoplasmic reticulum [22].) In the experiments shown in Fig. 6, NS-Isc was measured in the continuous presence of luminal amiloride. The addition of 1 μM thapsigargin to both sides of the monolayer produced a transient spike followed by a sustained increase in NS-Isc, most of which was sensitive to DIDS (Fig. 6a). When 300 μM DIDS was added to apical solution before thapsigargin, it almost completely blocked the response of NS-Isc to thapsigargin (Fig. 6b). Fig. 6Effect of thapsigargin on the nonamiloride-sensitive component (NS-Isc) and prevention of that effect by 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS). All experiments were performed in the presence of 10 μM luminal amiloride. A Mean NS-Isc values for seven experiments at four time points: just before adding thapsigargin to the apical and basolateral solutions, 3–5 min after addition of 1 μM thapsigargin to the apical and basolateral solutions (TG-initial), 15–20 min after thapsigargin addition (TG-late), and 3–5 min after the addition of 300 μM DIDS to the apical solution. * Significantly different compared with NS-Isc before thapsigargin treatment, P < .001; † significantly different compared with both the initial and late NS-Isc after thapsigargin, P < .001. B Mean values of the change in NS-Isc produced by thapsigargin: the initial peak (gray bars) and the late response (open bars) for control experiments (as in A) and in three experiments in which 300 μM DIDS was added before thapsigargin. * Significantly different compared with the corresponding control values, P < .001 Effect of Ussing chamber perfusion on time-dependent changes in Isc In all of the experiments presented up to this point, the KRB solutions that bathed the MDCK monolayers were recirculated as shown in Fig. 1b. To examine whether the time-dependent changes in AS-Isc and NS-Isc might be caused by the accumulation of a secretagogue or metabolite in these recirculated solutions, we conducted paired experiments in which one Ussing chamber was perfused with recirculated KRB solutions and the other chamber was continuously perfused with fresh KRB solutions, as shown in Fig. 1c. The representative control experiment shown in Fig. 7a was conducted in the usual way: the apical and basolateral KRB solutions were recirculated through both sides of the Ussing chamber from 10-ml reservoirs. In the paired experiment in Fig. 7b, we continuously perfused fresh KRB on both sides of the Ussing chamber. In both the recirculation and fresh perfusion experiments, cAMP treatment produced an initial transient spike of Isc lasting less than 6 min, followed by a broader peak; however, Isc decayed more rapidly in the recirculation experiment than it did in the fresh perfusion experiment. In four sets of paired experiments summarized in Fig. 7c, AS-Isc was greater (39.8 ± 1.1 μA/cm2 vs. 25.2 ± 3.3 μA/cm2, P < 0.01) and NS-Isc was less (2.8 ± 0.2 μA/cm2 vs. 7.7 ± 1.6 μA/cm2, P < 0.01) with fresh perfusion than in the recirculation group, suggesting that some substance, which inhibited AS-Isc and stimulated NS-Isc, was accumulating in the recirculated bathing solution. Fig. 7Time course of the short-circuit current (Isc) in experiments with and without recirculation of the bathing solutions. The sequence of treatments was the same in these paired experiments: 100 μM cyclic adenosine monophosphate (cAMP) plus 100 μM isobutylmethylxanthine (IBMX) were added to both apical and basolateral solutions, followed by 10 μM amiloride to the apical solution. A Apical and basolateral Krebs-Ringer bicarbonate (KRB) solutions were recirculated through the two sides of the Ussing chamber from 10-ml reservoirs. 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS) (300 μM) was added to the apical solution near the end of the experiment. B In this paired experiment, fresh KRB solution was perfused through both sides of the Ussing chamber at the same rate (1 ml/min) as for the recirculated solutions in A. C Summary results for four sets of paired experiments such as those in A and B. *Significantly different from control (with recirculation as in A), P < .001 Effect of extracellular ATP on time-dependent changes in Isc We hypothesized that ATP was released by the FL-MDCK cells and accumulated in the recirculated solutions. Extracellular nucleotides are known to act as autocrine and paracrine agents that affect Na+ absorption and Cl− secretion, and ATP has been shown to increase intracellular [Ca2+] [24, 25], which might augment the Ca2+-activated component of NS-Isc as well as inhibit ENaC activity. As shown in Fig. 8a, in the presence of amiloride, the addition of 100 μM ATP to the apical solution increased NS-Isc, and, as shown in Fig. 8b, this effect was almost completely abolished when the ATP was added together with hexokinase as an ATP scavenger [26]. (These experiments were conducted using the recirculated KRB method.) In five experiments such as that in Fig. 8a, basal NS-Isc (just before ATP addition) averaged 4.1 ± 0.1 μA/cm2. With the addition of ATP, NS-Isc reached a peak value of 15.4 ± 2.2 μA/cm2 (P < 0.004) within 2–3 min, and then declined gradually to a plateau of 5.9 ± 0.3 μA/cm2, which was still significantly larger than the basal level (P < 0.01). Addition of 300 μM DIDS to the apical solution before the addition of ATP had no effect on the peak amplitude (14.6 ± 0.7 μA/cm2, n = 3), but decreased the plateau value to 4.5 ± 0.2 μA/cm2 (n = 3), which was not significantly different from the basal value. Fig. 8Effect of adenosine triphosphate (ATP) on the nonamiloride-sensitive component (NS-Isc) and prevention of that effect by hexokinase. All experiments were performed in the presence of 10 μM apical amiloride. A Effect of 100 μM ATP added to the apical solution on NS-Isc. Mean values of five such experiments are given in the text. B Effect of 100 μM ATP on NS-Isc. In these experiments, 6 U/ml hexokinase was continuously present in the bathing solution. The same results were obtained in three such experiments To test whether endogenous ATP production by the epithelium might be involved in the time-dependent increase of NS-Isc and decrease of AS-Isc, we examined the effect of hydrolyzing extracellular ATP with hexokinase on the time course of NS-Isc and AS-Isc. Paired experiments without or with hexokinase were conducted as shown, respectively, in Fig. 9a and b, both using the recirculated KRB method.. With hexokinase in the bathing solutions, the response of Isc to cAMP treatment was exaggerated, and the broader peak was more sustained (Fig. 9b). As shown by the summary of four such sets of paired experiments (Fig. 9c), hexokinase had no effect on the basal Isc measured just before cAMP addition; however, after cAMP, AS-Isc was significantly higher (40.4 ± 2.9 μA/cm2 vs. 29.7 ± 2.8 μA/cm2, P < 0.05) with hexokinase, whereas the DIDS-sensitive NS-Isc was not significantly different from zero and significantly less than in the control group (−0.3 ± 0.2 μA/cm2 vs. 3.6 ± 0.7 μA/cm2, P < 0.01). Thus, all of the above data were consistent with the hypothesis that the accumulation of extracellular ATP causes the time-dependent increase of NS-Isc and decrease of AS-Isc that were observed in those experiments in which the bathing solutions were recirculated. Fig. 9Time course of the short-circuit current (Isc) response to cyclic adenosine monophosphate (cAMP) treatment in the absence and presence of hexokinase. Both types of experiments followed the same general protocol: A mixture of 100 μM (4-chlorophenylthio) (CPT)-cAMP plus 100 μM isobutylmethylxanthine (IBMX) was added as indicated, followed by 10 μM apical amiloride and 300 μM apical 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS). A In this experiment, the Krebs-Ringer bicarbonate (KRB) solution had no hexokinase added. B A paired experiment in which 6 U/ml hexokinase was continuously present in apical and basolateral bathing solution. C Average data for four paired experiments: the mean values of Isc immediately before cAMP was added (Basal-Isc), amiloride-sensitive (AS-Isc) at 30 min after cAMP addition (AS-Isc), and DIDS-sensitive Isc (DS-Isc). * Significantly different compared with the corresponding control values Measurement of ATP released from MDCK cells ATP released from polarized MDCK cells was determined by a bioluminescence assay, in which a luminometer was used to measure light production from ATP consumption in the luciferin-luciferase reaction [20]. MDCK cells were grown to confluence in 12-mm permeable supports (Millicell), and the ATP was measured in the apical or basolateral solution by adding the reagents only to that chamber. As shown in Fig. 10, under basal conditions, apically directed ATP release produced a light emission of 16.4 ± 2.1 ALU (artificial light units), whereas basolaterally directed ATP release was nearly zero (0.7 ± 0.2 ALU). In ten paired experiments, addition of 100 μM cAMP and 100 μM IBMX to the apical solution significantly increased the apically directed ATP release from 16.0 ± 2.1 ALU (basal) to 51.1 ± 9.4 ALU (n = 10, P < 0.005), whereas addition of the same volume of solution without cAMP and IBMX (sham control for the possible effects of manipulating the monolayers) had no significant effect (17.4 ± 3.5 ALU basal vs. 15.1 ± 2.3 ALU sham; n = 4, P = 0.29). Treatment with cAMP had no significant effect on basolaterally directed ATP release (0.8 ± 0.3, n = 8, P > 0.5). Fig. 10Adenosine triphosphate (ATP) release across the apical and basolateral membranes of FL-Madin-Darby canine kidney (MDCK) monolayers. ATP release into the apical and basolateral solutions was measured for FL-MDCK monolayers in 12-mm culture inserts using the luciferin-luciferase assay. To measure apical ATP release, these reagents were present only in the apical solution (n = 14), and they were present only in the basolateral solution to measure basolateral release (n = 11). The monolayers were first incubated with 200 μl of standard Opti-minimal essential medium (MEM) solution on each side to measure basal ATP production. The monolayers were then removed from the instrument and either 1 μl of a stock solution of 8-(4-chlorophenylthio) (CPT)-cAMP + isobutylmethylxanthine (IBMX) stock solution (cAMP, n = 10; final concentrations: 100 μM cAMP and 100 μM IBMX) or 1 μl of plain Opti-MEM medium (Sham, n = 4) were added. Average values of the light emission in arbitrary light units (ALU) ± standard error of the mean (SEM) are given. * Significantly different from basal and sham, P < 0.005 Discussion Previous work [10] has shown that the triply transfected FL-MDCK cells used in these experiments (a subclone of αFβ FγF MDCK cells used in the experiments of Morris et al. [2]) expressed all three rat-ENaC subunits and CFTR, and that it was a very useful model epithelium for the study of ENaC regulation. In the current study, we examined in greater detail the relationship between Cl− secretion and Na+ absorption in this epithelium. Time-dependent changes in NS-Isc and AS-Isc FL-MDCK cells uniformly exhibited a high initial Isc, which was almost completely eliminated by 10 μM amiloride added to the apical solution. The initial Isc fell to a nadir within 15 min and then increased slowly (Figs. 2b and 3b) due to an increase in NS-Isc. This progressive rise of NS-Isc occurred consistently regardless of whether amiloride was present or not and whether the epithelium was continuously short-circuited or was left in the open-circuit condition with only intermittent short-circuiting to measure Isc. Moreover, the time-dependent increase of NS-Isc could not be inhibited by the further addition of amiloride but was partially blocked by apical DIDS, which is consistent with anion secretion (see below). The time course of the Isc response to cAMP using these FL-MDCK cells was like that observed in the previous studies of Morris and Schafer [2] and in other studies of similar epithelia [6, 9, 27]. There was an initial transient increase lasting less than 6 min, followed by a broader peak, which decayed over the next 30 min (Fig. 3). Morris and Schafer [2] have shown that when these experiments are conducted in a Cl−-free solution, the initial transient spike was absent and Isc was stable for at least 30 min. The initial transient and the broader peak of Isc were attributed to, respectively, stimulation of Cl− secretion and Na+ absorption by cAMP [2]. Interestingly, the effect of cAMP on Isc was also time dependent. With late-cAMP treatment (Fig. 3b), the decay of Isc was faster and the magnitude of NS-Isc was larger. As shown in Fig. 4, there was a significant inverse correlation between AS-Isc and NS-Isc both in the absence and in the presence of cAMP. This correlation does not indicate a causal relationship between the rise in NS-Isc and the fall in AS-Isc, but suggests that whatever process is responsible for the time dependence produces reciprocal changes in NS-Isc and AS-Isc. Cl− channels associated with NS-Isc As discussed previously [2], we attributed NS-Isc to Cl− secretion because in the presence of apical amiloride, Cl− was the only ion present in sufficient concentration to account for it. Furthermore, glibenclamide blocked the initial rise in NS-Isc in response to subsequent cAMP treatment, whereas DIDS blocked the broader late transport response but not the early peak (Fig. 5). Glibenclamide is a well-established inhibitor of CFTR at the concentration (200 μM) used in these experiments. Although glibenclamide also inhibits ATP-activated K+ channels [28, 29], previous studies have shown that the apical membrane of MDCK cells has no measurable K+ conductance [10, 30]. Although DIDS effectively inhibits Cl− channels such as CaCC as well as the outwardly rectifying Cl− channel (ORCC), and the CLC family of Cl− channels, it does not inhibit CFTR [31, 32]. These results indicate that at least two types of Cl− channels contribute to NS-Isc and that they respond differently to cAMP. Based on these data, we have attributed the early, glibenclamide-sensitive, increase in NS-Isc after cAMP treatment to CFTR, which we have demonstrated is expressed in the apical membrane of these FL-MDCK cells [10]. CaCC appears to be responsible for the later increase in NS-Isc after cAMP treatment. As shown in Fig. 6, we found that thapsigargin, which moderately increases [Ca2+]i [22], produced a transient spike and a sustained increase in NS-Isc that could be completely blocked by apical DIDS as expected for a CaCC [33]. Furthermore, cAMP increases [Ca2+]i in MDCK cells [21], and thus a CaCC may contribute to the DIDS-sensitive component of the NS-Isc response to cAMP. A CaCC has also been described in the M-1 and mIMCD-K2 cell lines [34, 35] and in primary cultures of rabbit proximal and distal tubule cells [36]. It should also be noted, however, that the molecular composition of CaCC is not as yet know. Thus, we were unable to provide unequivocal evidence for its existence in the FL-MDCK cell line. Endogenous ATP release inhibits amiloride-sensitive Na+ absorption When the MDCK monolayers were continuously perfused with fresh apical and basolateral KRB solutions rather than recirculated solutions, the time-dependent increase of NS-Isc and decrease of total Isc was prevented (Fig. 7). AS-Isc was also significantly increased during the continuous perfusion, suggesting that inhibition of ENaC was prevented by washing out accumulated metabolites (Fig. 7c). Furthermore, our experiments showed that hexokinase, a scavenger of extracellular ATP, produced the same effects on AS-Isc and NS-Isc as the perfusion of fresh KRB solutions (Fig. 9). In the presence of hexokinase, AS-Isc was significantly higher but the DIDS-sensitive NS-Isc was significantly lower than in control experiments, indicating that extracellular ATP caused the inhibition of ENaC and activation of NS-Isc, the later possibly via a CaCC. Extracellular ATP is a well-established agonist for purinergic receptors, leading to an increase of intracellular [Ca2+] and activation Ca2+-activated Cl- channels [24, 37, 38]. In parallel, ATP attenuates amiloride-sensitive Na+ absorption in a variety of tissues including airway [39–41] and renal epithelia [42–44]. Similarly, activation of CFTR also increases Cl− secretion and inhibits ENaC in native epithelial tissues [14, 45]. Because CFTR has been suggested to potentiate ATP release [46, 47] and it has been shown to be expressed in this MDCK cell line [10], we examined a possible contribution of ATP release to the time-dependent transport in FL-MDCK cells. As shown in Fig. 8a, addition of 100 μM ATP stimulated anion secretion, possibly via a Ca2+-activated Cl− channel, which was inhibited by DIDS, and this effect was prevented in the presence of hexokinase (Fig. 8b). Endogenous release of ATP by the FL-MDCK monolayers was demonstrated by the bioluminescence assay experiments shown in Fig. 10. Consistent with measurements in cortical CD (see [48]), ATP was shown to be released into the apical solution under basal conditions, and this release was augmented more than three-fold by cAMP treatment. In contrast, there was no significant release of ATP into the basolateral solution either without or with cAMP treatment. ATP has been shown to inhibit AS-Isc and stimulate NS-Isc in other epithelia of distal nephron origin, including M-1 [43], mIMCK-K2 [42], and A6 cells [49]. Release of endogenous ATP into the extracellular medium has been proposed as a general response to hypotonicity and mechanical stimulation in many types of cells, including MDCK and A6 cells [49, 50]. Ostrom et al. [51] demonstrated that mechanical stimulation increases ATP release from MDCK cells as well as COS-7 and HEK-293 cells, and they proposed that ATP acts to alter the set point for a variety of signal transduction pathways. Praetorius et al. [50] also observed that mechanical stimulation caused ATP release in MDCK cells and that this release was associated with a transient increase in [Ca2+]i. Because of the opposite actions of ATP in the extracellular apical membrane, it can serve to shift the balance between active NaCl absorption and active NaCl secretion in the MDCK cell, as shown by the hypothetical model in Fig. 11. It is also quite important to note that the evidence we have presented for ATP accumulation in the extracellular fluid with longer incubation periods in Ussing chambers raises a caution for all such experiments, and it suggests that recirculation of the bathing solution, which has been in common use, should be avoided. Fig. 11Model for the role of adenosine triphosphate (ATP) in regulating Na+ absorption and Cl− secretion in Madin-Darby canine kidney (MDCK) epithelia. The epithelial Na+ channel (ENaC) mediates active Na+ absorption and produces the amiloride-sensitive component of the short-circuit current (AS-Isc). Because of the increased Na+ conductance produced by ENaC, it also depolarizes the apical membrane, producing a negative transepithelial voltage (Vte) of −30 mV. The basolateral membrane contains the ubiquitous Na, K-ATPase and, putatively, an Na-K-2Cl cotransporter, which actively accumulates Cl− in the cell. Cl− can then move across the apical membrane down its electrochemical potential gradient through Cl− channels, which include the glibenclamide-sensitive cystic fibrosis transmembrane conductance regulator (CFTR) channel, and a 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS)-sensitive, calcium-activated Cl- channel (CaCC). The net secretory movement of Cl− gives rise to the amiloride-insensitive component of the short-circuit current (NS-Isc). Our results show that ATP is secreted across the apical membrane. The pathway for ATP secretion is not known, but it has been proposed that it may also occur via CFTR [46]. When ATP accumulates in the apical extracellular space (e.g., when the solution bathing this space is restricted in volume or is recirculated), ATP inhibits ENaC and stimulates CaCC (and possibly CFTR or other Cl− channels), resulting in a fall in AS-Isc and an increase in NS-Isc Implications for renal pathophysiology It must be recognized that the FL-MDCK cell line suffers from the limitations of any cell culture system when attempting to apply these results to an understanding of normal renal physiology or pathophysiology. Furthermore, even the type-1 MDCK cells used in these studies are heterologous, and their origin cannot be ascribed definitively to any nephron segment. Nevertheless, the ability of ATP to inhibit ENaC-mediated Na+ absorption and stimulate Cl− secretion, as shown in these experiments with FL-MDCK cells and in similar epithelia [42, 43, 49], suggests a role for such signaling in the nephron. Significant amounts of ATP are released from epithelial cells originating from all regions of the nephron, and multiple P2Y and P2X receptors have been identified in all segments of the nephron [48]. Thus, changes in the luminal ATP concentration in the cortical CD and other aldosterone-sensitive segments of the distal nephron, in which ENaC mediates Na+ transport, may be a physiologic signal for diminished Na+ reabsorption. Also, Wilson et al. [19] demonstrated significant accumulation of ATP in cyst fluid obtained from patients with autosomal dominant polycystic kidney disease. Thus, based on our observations in MDCK cells, it seems reasonable to speculate that ATP might favor cyst enlargement and consequent exacerbation of polycystic disease by inhibiting Na+ reabsorption and stimulating Cl− secretion [19, 52].

J_Mol_Biol-1-5-1950483

Experiments Suggest that Simulations May Overestimate Electrostatic Contributions to the Mechanical Stability of a Fibronectin Type III Domain

Steered molecular dynamics simulations have previously been used to investigate the mechanical properties of the extracellular matrix protein fibronectin. The simulations suggest that the mechanical stability of the tenth type III domain from fibronectin (FNfn10) is largely determined by a number of critical hydrogen bonds in the peripheral strands. Interestingly, the simulations predict that lowering the pH from 7 to ∼4.7 will increase the mechanical stability of FNfn10 significantly (by ∼33 %) due to the protonation of a few key acidic residues in the A and B strands. To test this simulation prediction, we used single-molecule atomic force microscopy (AFM) to investigate the mechanical stability of FNfn10 at neutral pH and at lower pH where these key residues have been shown to be protonated. Our AFM experimental results show no difference in the mechanical stability of FNfn10 at these different pH values. These results suggest that some simulations may overestimate the role played by electrostatic interactions in determining the mechanical stability of proteins. Fibronectin is an extracellular matrix protein composed of three types of repeating domains (type I, type II and type III). The type III (fnIII) domains (which are ubiquitous in many multidomain mechanical proteins) in particular have been found to play a pivotal role in regulating and mediating physiological functions of cells. This is achieved through the interaction of various domains with the integrin family of cell surface receptors.1 One of the critical interactions in fibronectin is the binding of the RGD (Arg-Gly-Asp) motif in the tenth fnIII domain of fibronectin (FNfn10) to cell-surface integrins. A linear RGD-peptide has been shown to bind (with reduced binding strength and selectivity) to different members of the integrin family.2 It may be the dynamic structure of these domains that determines the functional states of the protein. Indeed, it has been suggested that force-induced conformational change of these fnIII domains may be crucial in transmitting cellular signals.3 Thus knowledge of how fnIII domains respond to mechanical forces and their mechanical resistance to conformational change is of importance to understand the function of fibronectin at the molecular level. A number of studies have investigated the mechanical properties of the fnIII domains of fibronectin by both experiment4,5 and simulation.6–8 Using steered molecular dynamics (SMD) simulations, Vogel and co-workers6 predicted a “mechanical hierarchy” of a number of fnIII domains that is in reasonable qualitative agreement with the hierarchy obtained from the atomic force microscopy (AFM) forced unfolding experiments.4 One of the most mechanically weak fnIII domains is the tenth fnIII domain of human fibronectin, FNfn10, although, interestingly, this is thermodynamically the most stable fnIII domain to have been studied to date.9,10 The simulations suggested that the mechanical stability of FNfn10 is largely determined by the hydrogen bonds between the A and B strands around Arg6 and Asp23 (Figure 1). Recent solution studies by Koide and co-workers10 showed that FNfn10 becomes more thermodynamically stable at lower pH (<5.0) as a consequence of the protonation of three negatively charged residues: Asp7, Asp23 and Glu9, which have raised pKa values of 5.54, 5.40 and 5.25, respectively; they are essentially fully protonated below pH 4.7. Using their simulations, Vogel and co-workers6 showed that protonation of these side-chains allows them to move closer together to form side-chain–side-chain hydrogen bonds. They suggested that this stabilizes interactions between the A and B strands, resulting in a significant increase in the unfolding force in simulations at pH 4.7 even though it is well established that there is no correlation between mechanical stability and thermodynamic stability.11,12 This interesting result suggests that the mechanical stability of fibronectin might be modulated by a change in the pH of the tissues. We tested this prediction by monitoring the unfolding force of FNfn10 at different pH values: pH 4.5 (50 mM acetate), pH 5.0 (50 mM acetate) and pH 7.0 (50 mM phosphate). It is important to note that Asp7, Asp23 and Glu9 are fully protonated at pH 4.5 as shown by NMR experiments carried out by Koide and co-workers.10 Any increase in mechanical stability due to the protonation of these three residues, as shown in the SMD simulation, should be observed in the AFM experiments. Forced unfolding experiments were performed for a polyprotein containing eight repeats of the FNfn10 domain. Figure 2 shows a “typical” force-extension trace. As has previously been observed5 Fnfn10 unfolds either by a two-state unfolding mechanism (N → U, where N is the native state and U the unfolded state) or a three-state unfolding mechanism via an intermediate (I) (N → I → U). The unfolding forces for three transitions could thus be determined: FN→U, the force of unfolding of N directly to U; FN→I, the force of unfolding of N to I; and FI→U, the force of unfolding of I to U (Figure 2). The intermediate, I, has been shown, using site-directed mutagenesis, to be a species with the A-strand detached.5 Thus one might expect, if the simulations of the unfolding of FNfn10 are correct, that both the N → U and N → I transitions will be at higher force at lower pH due to the new stabilising interactions between the A and B strands. However, since the A strand is detached in I, any stabilizing interactions between the A and B strands should not affect the mechanical stability of the intermediate, and FI→U is expected to be unaffected by pH. Furthermore, the unfolding forces of N → U are higher than those of N → I. Li et al.5 suggested that this may be due to the “stochastic nature” of mechanical unfolding. They suggested that since N → I unfolding is observed at higher unfolding forces than the subsequent I → U unfolding, the I → U unfolding may sometimes occur at a force that is too low to be seen in the AFM force-extension traces.5 That is, the unfolding is likely always to occur via this intermediate but it may not be observed, particularly when N unfolds at high forces. Thus if the simulations are correct we might expect to see fewer I → U transitions at lower pH. To our surprise, the unfolding forces at all pH values are the same within error (Figure 3). This is true at all pulling speeds. The dependence of the unfolding force on the pulling speed remains unchanged, suggesting that there has been no change in the unfolding pathway. Note also that the proportion of I → U unfolding events remains about the same. Thus our results are in direct contradiction to the predictions from the steered molecular dynamics simulations. AFM combined with protein engineering Ф-value analysis and molecular dynamics simulations has been previously used to solve the mechanical unfolding pathway of TNfn3, a homologous fnIII domain from the extracellular matrix protein tenascin.13 The results suggest that the unfolding of fnIII domains is a complicated multi-step process. The major barrier to a forced unfolding event in TNfn3 is the conformational transition from a twisted to an aligned state, which involves the breaking of several key hydrogen bonds along the peripheral strands (A-B strands and some between the G and F-strands). But there is also significant loss of hydrophobic side-chain packing interactions of residues in the A, B and G-strands, and importantly there is significant core re-packing. Thus, hydrophobic contacts of the buried residues and the hydrogen bonding interactions along peripheral strands are both apparently critical to the mechanical stability of TNfn3. TNfn3 and FNfn10 fold into essentially identical tertiary structures encompassing seven beta-strands running in two antiparallel beta sheets. A structural alignment of TNfn3 and FNfn10 reveals that these two fnIII domains have essentially the same hydrogen bonding patterns14 and molecular dynamics simulations have suggested that they have similar forced unfolding pathways. It is therefore reasonable to anticipate that these two fnIII domains will have the same molecular determinants for mechanical stability. We have made a “core-swap” version of FNfn10, with the core of Tnfn3 which is significantly more stable than Fnfn10 itself.15 Considering these together, it seems that the mechanical stability of both FNfn10 and TNfn3 is likely to be associated with the complex interplay between key peripheral hydrogen bonds and hydrophobic effects from the packing of buried residues. Molecular dynamics simulations have previously proved to be of great value in predicting and understanding the behavior of proteins placed under mechanical stress.6,7,11,13,16,17 However, our results suggest that in the case of the simulations of forced unfolding of FNfn10 tested here,6 the relative strength and importance of electrostatic and hydrophobic components may not be adequately described. It is possible that this discrepancy lies in the different timescale of the simulations and the experiments. AFM experiments are typically performed at extension rates of ∼1 μm s−1, whereas the timescale of molecular dymamics simulations allows for full extension of the protein in 1 or 2 ns, equivalent to a pulling speed of several m s−1, many orders of magnitude faster. Many slow conformational changes in proteins will not be observed in simulations on the nanoseconds timescale. Furthermore, the pathway of forced unfolding may vary as a function of the loading rate.18,19 Our study serves to emphasize the point that simulations need constantly to be benchmarked against experiment. However, the fact that simulators are prepared to make ab initio predictions that can be tested experimentally can only serve to improve our understanding of molecular mechanisms underlying mechanical strength in proteins.

Eur_J_Health_Econ-3-1-1913175

Modelling the 5-year cost effectiveness of tiotropium, salmeterol and ipratropium for the treatment of chronic obstructive pulmonary disease in Spain

Our objective was to assess the 5-year cost effectiveness of bronchodilator therapy with tiotropium, salmeterol or ipratropium for chronic obstructive pulmonary disease (COPD) from the perspective of the Spanish National Health System (NHS). A probabilistic Markov model was designed wherein patients moved between moderate, severe or very severe COPD and had the risk of exacerbation and death. Probabilities were derived from clinical trials. Spanish healthcare utilisation, costs and utilities were estimated for each COPD and exacerbation state. Outcomes were exacerbations, exacerbation-free months, quality-adjusted life years (QALYs), and cost(-effectiveness). The mean (SE) 5-year number of exacerbations was 3.50 (0.14) for tiotropium, 4.16 (0.40) for salmeterol and 4.71 (0.54) for ipratropium. The mean (SE) number of QALYs was 3.15 (0.08), 3.02 (0.15) and 3.00 (0.20), respectively. Mean (SE) 5-year costs were €6,424 (€305) for tiotropium, €5,869 (€505) for salmeterol, and €5,181 (€682) for ipratropium (2005 values). Ipratropium and tiotropium formed the cost-effectiveness frontier, with tiotropium being preferred when willingness to pay (WTP) exceeded €639 per exacerbation-free month and €8,157 per QALY. In Spain, tiotropium demonstrated the highest expected net benefit for ratios of the willingness to pay per QALY, well within accepted limits. Introduction Owing to the chronic progressing nature and increasing prevalence of chronic obstructive pulmonary disease (COPD), its treatment will undoubtedly increase pressure on the drug budgets of developed countries in the future. Several new and competing treatments for COPD have recently been introduced, or may soon become available [1]. Healthcare authorities will be forced to decide upon reimbursement of these medications and will require high-quality information about the costs and effectiveness of these new drugs. Pharmaco-economic evaluations provide this information and may guide the positioning of new drugs in the treatment spectrum of COPD. To date, most economic evaluations of pharmacotherapy for COPD have been conducted alongside randomised controlled trials [2–7]. Consequently, their time horizon has been restricted to the duration of these trials, which is usually 6 months to 1 year, although economic evaluations of inhaled bronchodilators and corticosteroids piggybacked to clinical trials with durations of only 12–16 weeks have been reported [4, 5]. For reimbursement authorities and formulary decision makers, these short-term economic evaluations are of limited value, because COPD requires long-term maintenance treatment on a daily basis. Furthermore, these policy makers will need results that represent their national or regional setting. Because it is not feasible to conduct long-term empirical cost-effectiveness studies in all potential markets, some sort of modelling is required. This will allow expansion of the time horizon beyond that of a clinical trial, as well as the adapting and transferring of results from one setting to another. Decision analytical modelling has become an accepted approach to economic evaluations, and the use of modelling is supported by nearly all pharmaco-economic guidelines issued by health authorities involved in decision making about pricing and reimbursement of new pharmaceuticals, including Spain [8–11]. This study investigates the cost effectiveness of three bronchodilators for the maintenance treatment of COPD in Spain: tiotropium, salmeterol and ipratropium. Bronchodilators are central to the symptomatic management of COPD. Bronchodilators include three classes of medications: (1) the inhaled beta2-agonists, (2) the inhaled anticholinergics and (3) the oral methylxanthines. All three classes include drugs that are short acting or long acting. The most recently launched bronchodilator is tiotropium, an anticholinergic that provides 24 h bronchodilation with once-daily dosing. It was developed as a more effective and more convenient alternative to ipratropium, the most frequently used short-acting anticholinergic bronchodilator in COPD, which has to be used four times daily. At the time tiotropium became available, salmeterol, which is used twice daily, was the most effective and the most frequently used long-acting bronchodilator from the beta2-agonists class. Orally administered methylxanthines, such as theophylline, are not recommended for routine use due to their unfavourable risk/benefit ratios. In a series of multi-national trials tiotropium was directly compared with either placebo [12], ipratropium [13] or salmeterol [14]. The above-mentioned clinical trials have shown that tiotropium provides sustained bronchodilation, reduces exacerbation rate, and improves dyspnoea and health-related quality of life when compared with placebo or ipratropium. Improvements in lung function have been shown to be significantly better with tiotropium than with salmeterol [14]. Because information on the long-term cost effectiveness of these bronchodilators is lacking, a decision analytical model was developed to determine the 5-year cost effectiveness of tiotropium, salmeterol and ipratropium for the treatment of COPD. Efficacy data were derived from the above-mentioned clinical trials, and the potential cost effectiveness of inhaled bronchodilator therapy was assessed in scenario analyses. The model was populated with Spanish epidemiological data on COPD, Spanish healthcare utilisation, and unit costs and utilities based on Spanish population values. Spain was chosen because cost-effectiveness data on bronchodilator therapy from a Southern European country were lacking. Methods The model This 5-year model can be characterised as a Markov model [15]. It builds on our earlier work in which we constructed a fully probabilistic Markov model to assess the 1-year cost effectiveness of tiotropium, salmeterol and ipratropium [16, 17]. All mathematical and technical details of this 1-year model have been published elsewhere [17]. In brief, all COPD patients were classified into three disease states of increasing severity, based on their pre-bronchodilator forced expiratory volume in 1 s (FEV1): moderate COPD (50% ≤ the percentage of the predicted value (FEV1 % pred.) < 80%), severe COPD (30% ≤ FEV1 % pred. < 50%) and very severe COPD (FEV1 % pred. < 30%). In pre-specified time intervals (Markov cycles) patients move between disease states and are at risk of experiencing an exacerbation, either severe or non-severe. All probabilities to move between disease states (transition probabilities) and to experience exacerbations that went into the 1-year model were obtained directly from six clinical trials comparing tiotropium with salmeterol, ipratropium or placebo (study codes: 205.114/205.117, 205.115/205.128, 205.122A/205.126A, 205.122B/205.126B, 205.130 and 205.137) [12–14]. Transition probabilities and exacerbation probabilities for tiotropium were based on the pooled patient-level data from all six clinical trials. To obtain the transition and exacerbation probabilities for the comparator arms, we applied the relative difference between tiotropium and the comparators in the individual trials to the probabilities of tiotropium that were derived from the pooled data [17]. All trials used similar inclusion and exclusion criteria. They enrolled patients with stable moderate-to-severe COPD who had an FEV1 < 65% (salmeterol-controlled trials < 60%) of predicted normal and FEV1 ≤ 70% of forced vital capacity. The duration of these trials was 1 year for the those that compared tiotropium with placebo and ipratropium and 6 months for the trials that compared tiotropium with salmeterol. In total, 1,308 patients were randomly allocated to receive tiotropium, and 771 patients, 405 patients, and 179 patients were randomly selected to receive placebo, salmeterol and ipratropium. In all trials the same definitions of an exacerbation and its severity were used. An exacerbation was defined as a new onset or worsening of more than one symptom, such as cough, sputum, dyspnoea or wheeze, lasting for at least 3 days. Exacerbations were distinguished into severe and non-severe exacerbations. A severe exacerbation was defined as ‘incapacitating or inability to do work or usual activity’. All other exacerbations were non-severe. Severity was assessed by the clinical investigator. The risk of experiencing an exacerbation varied by disease state and treatment group. Given treatment group and disease state, exacerbation probabilities were assumed to be constant over time [17]. The current model expanded the time horizon from 1 year to 5 years and aimed to assess the cost effectiveness of tiotropium, salmeterol and ipratropium for treating COPD patients in Spain. To reflect the progressive nature of COPD in the long run and to incorporate mortality, an additional ‘death state’ was added to the model. The base-case analysis was performed from the perspective of the Spanish National Health Service (NHS) and included all costs covered by the NHS budget in euros at the 2005 value. A graphical presentation of the 5-year model is given in Fig. 1. In addition, an analysis from the societal perspective was performed. The difference between the societal perspective and the NHS perspective was that the first additionally included patients’ co-payments for medications and the costs of lost production from day 1 of sick leave onwards; however, it excluded value added tax (VAT), as this does not represent a cost to society.Fig. 1Graphical presentation of the Markov model. Backward transitions, i.e. from very severe to severe COPD and from severe to moderate COPD, were allowed during the first year, but not thereafter. The circles on the top left of each COPD severity state indicate that the patients can stay in the same state Scenarios The first year of the model incorporated the benefits of therapy as observed in the clinical studies for improvement in lung function, which delayed the progression to subsequent COPD severity states [17]. The first year of the model also incorporated the reduction in the number of exacerbations that were found in the clinical trials [17]. These first year probabilities are shown in the upper part of Table 1 and in Table 2. Because the clinical trials did not provide data on the probabilities to move between disease states and to experience exacerbations after the first year, the 5-year model was used to run three scenarios with different assumptions on transition and exacerbation probabilities for years 2 to 5: In the base-case scenario, the decline in FEV1 after the first year was assumed to be 52 ml/year [18] in all treatment groups, whereas the exacerbation probabilities remained as they were for the first year. The decline of 52 ml was the mean annual change in FEV1 among smokers and ex-smokers in the Lung Health Study [18]. The lower part of Table 1 shows the transition probabilities in the base-case scenario during years 2 to 5, whereas Table 2 shows the exacerbation probabilities.In the second scenario, both transition and exacerbation probabilities of the first year were assumed to remain constant during years 2 to 5. In other words, the first year probabilities as shown in Table 1 (under the heading “Subsequent cycle probabilities, year 1”) and 2 were applied to the later years.In the third scenario, it was assumed that disease progression and exacerbation risk no longer differed between treatment groups after the first year. Exacerbation probabilities for tiotropium, salmeterol and ipratropium were assumed to be equal to the first year probabilities of the ipratropium group (columns 4 and 7 of Table 2). The assumption on lung function decline was the same as in the base-case scenario. Note that the three scenarios do not differ with respect to the first year. During the first year, both forward transitions (transitions to worse disease states) and backward transitions (transitions to better disease states) are possible. In the base-case and third scenario, backward transitions during years 2 to 5 were not allowed, reflecting the progressive nature of COPD in general. Further note that no differences between treatments in terms of mortality risk were assumed.Table 1Mean (SE) probabilities to move between disease states in the base-case scenario. (These probabilities were also used in scenario 3. In scenario 2 the subsequent cycle probabilities for year 1 were applied to years 2 to 5. The cycle length was 8 days for the very first cycle and 1 month for of all subsequent cycles)To ⇒ From ⇓ TiotropiumSalmeterolIpratropiumModerateSevereVery severeDeathModerateSevereVery severeDeathModerateSevereVery severeDeathFirst cycle probabilities Moderate0.906 (0.018)0.092 (0.018)0.001 (0.002)0.001 (0.001)0.898 (0.033)0.100 (0.033)0.001 (0.003)0.001 (0.001)0.738 (0.092)0.257 (0.091)0.004 (0.013)0.001 (0.002) Severe0.259 (0.017)0.715 (0.018)0.025 (0.006)0.001 (0.001)0.201 (0.028)0.765 (0.030)0.033 (0.013)0.001 (0.002)0.102 (0.028)0.841 (0.034)0.056 (0.022)0.001 (0.002) Very severe0.010 (0.005)0.340 (0.024)0.648 (0.024)0.002 (0.002)0.001 (0.003)0.301 (0.042)0.696 (0.042)0.002 (0.004)0.005 (0.011)0.220 (0.066)0.773 (0.067)0.002 (0.007)Subsequent cycle probabilities, year 1 Moderate0.957 (0.010)0.040 (0.010)0.003 (0.003)0.001 (0.001)0.929 (0.025)0.066 (0.024)0.005 (0.007)0.001 (0.002)0.924 (0.050)0.073 (0.049)0.003 (0.010)0.001 (0.004) Severe0.023 (0.006)0.952 (0.009)0.023 (0.006)0.002 (0.002)0.023 (0.011)0.916 (0.020)0.059 (0.017)0.002 (0.003)0.013 (0.011)0.948 (0.021)0.037 (0.018)0.002 (0.004) Very severe0.001 (0.002)0.045 (0.012)0.947 (0.013)0.008 (0.005)0.006 (0.008)0.036 (0.019)0.951 (0.023)0.008 (0.009)0.003 (0.009)0.025 (0.026)0.964 (0.031)0.008 (0.015)Subsequent cycle probabilities, years 2 to 5 Moderate0.984 (0.007)0.015 (0.007)0.000 (0.000)0.001 (0.001)0.981 (0.017)0.019 (0.017)0.000 (0.000)0.001 (0.003)0.978 (0.033)0.022 (0.033)0.000 (0.000)0.001 (0.005) Severe0.000 (0.000)0.990 (0.004)0.008 (0.003)0.002 (0.002)0.000 (0.000)0.987 (0.008)0.011 (0.007)0.002 (0.003)0.000 (0.000)0.986 (0.012)0.011 (0.011)0.002 (0.005) Very severe0.000 (0.000)0.000 (0.000)0.992 (0.005)0.008 (0.005)0.000 (0.000)0.000 (0.000)0.992 (0.007)0.008 (0.007)0.000 (0.000)0.000 (0.000)0.992 (0.011)0.008 (0.011)Table 2Mean (SE) exacerbation probabilities in the base-case scenario. (These probabilities were also used in scenario 2. In scenario 3 the probabilities of ipratropium were also applied to tiotropium and salmeterol during years 2 to 5) Probability of experiencing an exacerbationProbability that the exacerbation will be severe, given that there is an exacerbationTiotropiumSalmeterolIpratropiumTiotropiumSalmeterolIpratropiumModerate0.051 (0.004)0.057 (0.013)0.080 (0.020)0.097 (0.024)0.030 (0.031)0.267 (0.114)Severe0.075 (0.003)0.089 (0.011)0.097 (0.013)0.136 (0.018)0.138 (0.033)0.188 (0.041)Very severe0.096 (0.005)0.104 (0.016)0.102 (0.022)0.192 (0.027)0.207 (0.048)0.186 (0.062) Input data: baseline distribution of patients among disease states The baseline distribution of patients among the disease states was based on a Spanish study by Miravitlles et al. [19] Re-analysis of the data according to the severity definition used in the model showed that, of 436 COPD patients, 55.2% had moderate disease, 34.9% had severe disease and 9.9% had very severe disease. A disease state for mild COPD was not included in the model, because mild COPD patients did not participate in the tiotropium trials. Input data: death state Probabilities of dying were based on all-cause mortality rates among Spanish patients with severe or very severe COPD as published by Miravitlles et al. [20]. The data from this study were re-analysed using the same cut-off values for the FEV1% predicted of severe and very severe COPD that were used in the model. The 1-year all-cause mortality rate was found to be 25 per 1,000 among patients with severe COPD [20], and the relative mortality risk in patients with very severe COPD compared with severe COPD was found to be 4.96 for a mean decline in FEV1% predicted of 19.4%. Under the assumption of constant proportional hazards, the relative mortality risk of patients in the trials was estimated to be 3.754 for very severe COPD and 0.248 for moderate COPD compared with severe COPD. We applied these relative risks to the all-cause mortality rate of 25 per 1,000 among patients with severe COPD to derive the mortality rates and, subsequently, the probabilities of dying, among patients with moderate and very severe COPD. These probabilities were the same for all treatment groups. Input data: utilities To reflect the impaired quality of life of patients with COPD, we attached utility weights to each disease severity state. These utilities were obtained from the EQ-5D scores at baseline in a subset of patients randomly accepted into the UPLIFT trial (n = 1,133) [21]. These scores were valued using the Spanish tariff [22]. A value of 1 represents perfect health, whereas 0 represents death. Mean (SE) utilities at baseline of the UPLIFT trial were calculated to be 0.809 (0.008) in patients with moderate disease, 0.762 (0.009) in patients with severe disease and 0.655 (0.024) in patients with very severe disease. During the months in which patients experienced an exacerbation, the utility value was reduced by 15% in the case of a non-severe exacerbation [23] and by 50% in the case of a severe exacerbation [24]. Each year, the number of quality-adjusted life-years (QALYs) was calculated as the sum of the multiplications of the number of months in a particular disease state and the utility of that disease state, divided by 12. Input data: costs The details and results of the cost estimates are presented in Table 3. Estimates of healthcare utilisation were primarily derived from two studies performed in Spain that had measured resource use and costs of the treatment of patients with COPD [25, 26]. Data from these studies were re-analysed to estimate resource use and costs by disease severity and exacerbation severity, using similar definitions as applied in the model. In the model, healthcare utilisation associated with COPD maintenance therapy varied by disease severity. Healthcare utilisation during exacerbations varied by the severity of the exacerbation (severe or non-severe). Calculations were conservative in assuming that costs per disease severity state and costs per severe or non-severe exacerbation were equal across treatment groups. Resources for maintenance therapy included visits to respiratory physicians inside and outside the hospital, visits to the general practitioner, pulmonary function tests, blood tests, imaging tests and respiratory medications. Resources associated with a non-severe exacerbation included general practitioner and respiratory physician visits and medications. Besides physician visits and outpatient medications, resources associated with severe exacerbations also included hospital admissions and visits to the emergency room (ER) department. Costs of pulmonary function and other tests, as well as costs of medications that were prescribed during inpatient stay or during ER visits, were not measured separately, because they were included in the overall costs per in-patient day and the costs per ER visit.Table 3Mean healthcare utilisation (HU), unit costs, and mean (SE) total costs of maintenance therapy and exacerbations from the NHS perspective for Spain in euros at the 2005 value (HU healthcare utilisation, RP respiratory physician, GP general practitioner, ICU intensive care unit, exa exacerbation)Maintenance therapy Unit costsModerate COPDSevere COPDVery severe COPDHU per patient per yearTotal costsHU per patient per yearTotal costsHU per patient per yearTotal costsVisit RP in hospital79.850.2722 (1)0.4435 (2)0.6350 (3)Visit RP outside hospital23.770.389 (1)0.5714 (1)0.6515 (1)Visit GP8.352.5922 (1)2.8824 (1)3.2727 (1)Thorax X-ray19.671.0320 (1)1.1823 (1)1.4929 (1)ECG22.530.8018 (1)0.8720 (1)1.1526 (1)Spirometry40.630.5522 (1)0.6627 (1)0.9237 (2)Blood analyses19.671.4128 (1)1.4328 (1)1.5831 (1)Blood gases27.250.339 (1)0.5615 (1)0.6718 (1)Influenza vaccination5.470.483 (1)0.644 (1)0.724 (1)Theophylline0.18122.0622 (8)161.7730 (4)159.0729 (5)Mucolytics0.3039.7411 (3)48.3114 (2)80.6024 (4)Oral corticosteroids0.2921.546 (3)23.737 (1)78.4823 (6)Inhaled corticosteroids0.61224.84138 (12)224.84138 (15)292.00179 (22)Oxygen3.7321.3279 (14)44.26165 (30)77.87290 (52)Other20 (3)44 (4)34 (5)Total costs per patient per year430 (24)587 (34)818 (58)ExacerbationsUnit costsNon-severe exacerbationSevere exacerbationStudy medication costs per dayHU per exaTotal costsHU per exaTotal costsICUa1,284––0.29374 (291)Tiotropium1.80Non-ICUa368––4.161,529 (307)Salmeterol1.20Emergency room115––0.94108 (11)Ipratropium0.19Visit GP8.351.6414 (1)1.008 (3)Visit RP in hospital79.85––0.5242 (10)Antibioticsb5.0011.0255 (7)7.5238 (7)Oral corticosteroidsb0.292.691 (1)4.981 (1)Inhaled corticosteroidsb0.617.014 (1)3.712 (1)Oxygenb3.732.058 (3)5.3220 (2)Other1 (1)1 (1)NHS sick leave benefitcs74.0913 (2)Total costs per exacerbation83 (7)2,136 (425)For medications and oxygen HU is expressed in number of days during which the medication or oxygen was usedaOf all severe exacerbations 52% required hospital admission. Of those, 14% were to the ICU. The length of stay on ICU is 4 days, and the length of stay on non-ICU is 8 days. Hence, 0.52 × 0.14 × 4 = 0.29 and 0.52 × 8 = 4.16bOnly medications prescribed in ambulatory settings. Costs of medications administered in hospital and at emergency rooms are included in costs of hospital stay/emergency room visitcAverage labour costs per day in Spain are €74.09. Of Spanish COPD patients, 33.2% have paid employment; 0.332 × €74.09 = €24.60. The NHS covers 60% of these labour costs during days 16–20 of the sick leave episode and 75% from day 21 onwards. Of severe exacerbations, 7% are associated with sick leave longer than 15 days. The duration of these absence spells is 26 days. So, (5 × 0.6 × €24.60 + 6 × 0.75 × €24.60) × 0.07 = €13.43 Unit costs of healthcare resources were obtained from the SOIKOS health database and have been described in detail elsewhere [25]. They were updated to 2005 using the Spanish General Consumer Price Index. Acquisition costs of pulmonary drugs from the NHS perspective were based on public prices and calculated as the ex-factory prices multiplied by a mark-up of 1.596 to convert these prices to public prices [27]. The co-payment of 10% of the public price for the people in the work force was excluded. It was estimated that 33.2% of Spanish COPD patients had to pay these co-payments because they were still in the work force [27]. From a societal perspective, the drug costs included co-payment but excluded the 4% VAT that is included in the public prices. Costs of tiotropium, salmeterol and ipratropium from the NHS perspective were €1.80, €1.20 and €0.19 per day, respectively. From the societal perspective these costs were €1.76 for tiotropium, €1.18 for salmeterol and €0.18 for ipratropium. The NHS covers the costs of absence from work due to illness from the 16th day of sick leave onwards. The NHS pays 60% of gross salary during days 16 to 20 and 75% from day 21 onwards. When we were calculating costs from the NHS perspective, these costs were added to the costs of all severe exacerbations with sick leave longer than 15 days (approximately 7% of all severe exacerbations). The costs from the societal perspective included the costs of lost production during all days of absence from paid work. The resulting mean (SE) annual costs of maintenance therapy from an NHS perspective were €430 (€24) for a patient with moderate COPD, €587 (€34) for a patient with severe COPD and €818 (€58) for a patient with very severe COPD. The corresponding values from the societal perspective were €429 (€21), €586 (€34) and €816 (€58). The mean (SE) costs of exacerbations from the NHS perspective were €83 (€7) for a non-severe exacerbation and €2,176 (€425) for a severe exacerbation. From a societal perspective these costs were €121 (€7) and €3,912 (€543), respectively. COPD severity To investigate the impact of COPD severity on the cost effectiveness of the bronchodilators we ran sensitivity analyses with the base-case scenario, assuming that, at the start of the model, 100% of the patients had either moderate, severe or very severe COPD. Discounting Because of time preference, costs and effects that will arise in the future are usually valued lower than costs and effects in the present. Discounting is used to calculate the present value of future costs and effects. In accordance with proposed Spanish guidelines, an annual discount rate of 6% has been adopted [28]. In the base-case analysis, costs and health outcomes were discounted at the same rate, whereas, in sensitivity analyses, discount rates of 0% for both costs and effects, and 6% for costs combined with 3% for effects, were applied. In scenarios 2 and 3 the same 6% discount rate was used as in the base-case scenario. Probabilistic sensitivity analysis To facilitate the interpretation of results of economic analyses, the reporting of uncertainty associated with costs and outcomes is equally important as reporting the point estimates of these parameters. Hence, the model was designed fully probabilistic, and uncertainty around the probabilities to move between disease states, the probabilities to experience exacerbations, utilities and healthcare utilisation was addressed by defining a probability distribution for each input parameter [17]. The uncertainty around these input parameters was propagated through the model simultaneously by conducting second-order Monte Carlo simulations. This means making random draws of the uncertain parameters from their probability distribution, running the model for each set of parameters that is drawn, and collecting the outputs from each run [29]. The current results were based on 5,000 iterations. The main outcome measures of the model were mean and standard error (SE, being the standard deviation of the 5,000 iterations) of the 5-year costs per patient, exacerbation-free months and quality-adjusted life years. The presentation of incremental cost effectiveness was based on the hierarchy of outcomes, i.e. each treatment option was compared with the next best treatment option in terms of effectiveness. The uncertainty around costs and effects was further explored by plotting of the 5,000 iterations on incremental cost-effectiveness planes (CE-planes) [30] and by presenting cost-effectiveness acceptability curves [30, 31] and frontiers [32]. The acceptability curve presents the probability that a treatment is the most cost effective of the three treatments at different threshold values for cost effectiveness (ceiling ratio), whereas the cost-effectiveness acceptability frontier demonstrates which of the three treatments should be adopted because it results in the highest expected net benefit, given the ceiling ratio. The net benefit is calculated as the total costs (C) minus the effects (E) multiplied by the ceiling ratio (C − (E × ceiling ratio)) [33]. The model was programmed in Microsoft EXCEL. Results Health outcomes The results of the Markov simulation for the different scenarios are presented in Table 4. In the base-case scenario, the mean (SE) number of exacerbations in 5 years was 3.50 (0.14) in the tiotropium group, 4.16 (0.40) in the salmeterol group and 4.71 (0.54) in the ipratropium group. The corresponding mean (SE) number of exacerbation-free months was 46.83 (1.11) in the tiotropium group, 45.29 (2.12) in the salmeterol group and 44.89 (2.86) in the ipratropium group. Estimates of the number (SE) of QALYs were 3.15 (0.08), 3.02 (0.15) and 3.00 (0.20), respectively. In all scenarios, differences in exacerbation-free months and QALYs between treatment groups were consistently in favour of the tiotropium group. Applying the first year probabilities to all subsequent years (scenario 2) increased the difference in exacerbation-free months between tiotropium and the other treatment groups to approximately 2.0 and the difference in QALYs to approximately 0.19. Assuming similar exacerbation probabilities across treatment groups in year 2 to 5 (scenario 3) reduced the differences between treatment groups considerably. In this analysis, the difference in exacerbation-free months between tiotropium and the other treatment groups was just above 1, and the difference in QALYs was about 0.13. There was almost no difference in health outcomes between ipratropium and salmeterol in this scenario.Table 4Results of the Markov simulation for the base-case analysis from the NHS perspective covering a time period of 5 years. The table gives mean (SE) or mean (95% confidence interval). QALY quality-adjusted life year, Tio tiotropium, Salm salmeterol, Ipra Ipratropium, exa exacerbations, CE-ratio cost-effectiveness ratio, exa-free exacerbation-free Costs In the base-case scenario from the NHS perspective, mean (SE) total costs over 5 years were lowest in the ipratropium group, with €5,181 (€682) per patient (Table 4). Costs were €5,869 (€505) in the salmeterol group and €6,424 (€305) in the tiotropium group. The higher costs in the tiotropium and salmeterol group were largely due to the higher costs of study medication. Compared with those of ipratropium, the 5-year costs of study medication were €2,477 (€67) higher in the tiotropium group and €1,521 (€85) higher in the salmeterol group. Savings in other categories of costs, mainly exacerbation-related costs, offset approximately half of these additional study medication costs. Because of a smaller difference in exacerbation rate between treatment groups in scenario 3, the difference in total costs between ipratropium on the one hand and tiotropium and salmeterol on the other hand increased by 64% and 75%, respectively. The impact of applying the first-year transition probabilities to years 2 to 5 (scenario 2) on the 5-year differences in costs between treatment groups was much less (Table 4). Cost effectiveness In the base-case scenario from the NHS perspective estimates of the incremental costs per exacerbation-free month were €360, when tiotropium was compared with salmeterol, and €1,711 when salmeterol was compared with ipratropium. The corresponding incremental costs per QALY were €4,118 and €38,931, respectively. The distribution of the results of the 5,000 model iterations on the CE-planes is also reported in Table 4. The comparisons between tiotropium and salmeterol show that the majority of simulations (approximately 65%) lie in the upper-right quadrants, signifying better health outcomes and higher costs for tiotropium. The CE-planes comparing salmeterol with ipratropium show that about 35% of the dots are found in the upper-left and about 45% in the upper-right quadrant, signifying similar health outcomes and higher costs for salmeterol. Figure 2 shows the cost-effectiveness acceptability curves in the base-case scenario from the NHS perspective. The curves show that, in terms of exacerbation-free months, ipratropium has the highest probability of being cost effective when the threshold value for cost per additional exacerbation-free month is below €1,050. In terms of QALYs, ipratropium has the highest probability of being the most cost effective for all threshold values for cost per QALY below €11,000. Above these values, tiotropium has the highest probability of being cost effective. However, when the distribution of the cost-effectiveness ratio is skewed, as is the case in the presented analyses, the treatment with the highest probability of being cost effective is not always the treatment with the highest expected net benefit [32]. In Fig. 2, the cost-effectiveness acceptability frontier is drawn in bold. The frontier follows the curve of the treatment with the highest expected net benefit for a given value of the cost-effectiveness threshold. Hence, it indicates which treatment should be chosen because it is optimal. The bold curve demonstrates that both ipratropium and tiotropium are on the frontier. For any ceiling ratio above €639 per exacerbation-free month and above €8,157 per QALY, tiotropium is the preferred treatment option. In scenario 2, these threshold values were lower: €551 and €6,226, respectively, whereas the corresponding values in scenario 3 were €1,918 and €15,635. The point on the frontier where the most optimal treatment switches from ipratropium to tiotropium in Fig. 2 corresponds to the base-case incremental cost-effectiveness ratio for the comparison between these two bronchodilators. Figure 2 also shows that, although tiotropium has the highest probability of being the most optimal treatment, this probability is 58% at maximum. This 58% is the value at which the acceptability curve of tiotropium using QALYs becomes asymptotic.Fig. 2Cost-effectiveness acceptability curves and frontier of the costs per exacerbation-free month and the cost per QALY. Ceiling ratio: threshold value for the cost-effectiveness ratio in euros. The curves in grey represent the probability that a treatment is cost effective for a given value of the ceiling ratio. In the case of skewed distributions of the ratio, the treatment with the highest probability of being cost effective is not always the treatment with the highest expected net benefit. The acceptability frontier (in black) shows which treatment is associated with the highest expected net benefit for each value of the ceiling ratio Societal perspective From the societal perspective, overall mean (SE) costs increased in all treatment groups to €6,574 (€321) for tiotropium, €6,125 (€541) for salmeterol and €5,545 (€720) for ipratropium. However, the savings in exacerbation costs generated by tiotropium and salmeterol compared with ipratropium were higher from a societal perspective than from the NHS perspective. Consequently, from the societal perspective, the difference in overall costs between the treatment groups was reduced. Also, the incremental costs per exacerbation-free month were reduced to €308, for the comparison tiotropium versus salmeterol, and €1,375 for the comparison salmeterol versus ipratropium. The incremental cost per QALY were reduced to €3,483, for the comparison tiotropium versus salmeterol, and €35,158 for the comparison salmeterol versus ipratropium. Tiotropium had the highest expected net benefit for any value of the cost-effectiveness threshold above €547 per exacerbation-free month and above €7,076 per QALY. Below these values ipratropium is preferred. Impact of COPD severity When the model was run separately for patients with either moderate, severe or very severe COPD, it showed that the threshold value above which tiotropium had the highest expected net benefit increased with the severity of COPD. The threshold values for the costs per QALY above which tiotropium became the preferred option were €7,600 for moderate COPD, €8,800 for severe COPD and €12,500 for very severe COPD. Below these values ipratropium was preferred. Tiotropium had the highest expected net benefit when the ceiling ratios for cost per exacerbation free month were €560 for moderate COPD, €700 for severe COPD and €1,200 for very severe COPD. Below these values ipratropium had the highest expected net benefit. Discount rates Because discounting affects both health outcomes and costs, the effect of discounting in this study was small. Analyses based on discount rates of 3% and 0% showed almost similar cost-effectiveness ratios. Discounting health outcomes at a lower rate than costs led to cost-effectiveness ratios that were slightly more in favour of tiotropium. Discussion Bronchodilators form the main therapy for the symptomatic relief of respiratory symptoms in COPD patients. In this study we have constructed a decision analytical model to synthesise clinical trial data on the effectiveness of bronchodilator treatment. We have shown how scenario analyses can be used to extend the time horizon of the cost-effectiveness study beyond that of the clinical trials. In addition, we have shown how a model can be populated with country-specific data to obtain estimates of the cost-effectiveness of bronchodilators in the Spanish setting. This comprehensive country adaptation and extension of the existing short-term Markov model [17] required much more than just the imputation of Spanish unit costs. We searched for Spanish sources for almost every type of input data, ranging from the distribution of COPD severity stages, mortality, the proportion of COPD patients in the work force, utilities, resource use and unit costs. We re-analysed patient-level data from previously published Spanish studies on prevalence, mortality, EQ-5D, and resource utilisation [19–21, 25, 26] to ensure that these Spanish data matched the model’s definitions of COPD severity and exacerbation severity. This included obtaining point estimates as well as distributions of these input data. The strength of the Spanish data is also that mainly patients from general practices were included, thus reflecting the routine care setting closely. The majority of Spanish patients are treated by general practitioners, and the proportion of patients referred to specialist care by pulmonologists is smaller than in many other west European countries [34]. This may be related to the gate-keeping function of the general practitioner in Spain that is not enforced in some other European countries. The base-case scenario showed that tiotropium was associated with an approximately 16% reduction in exacerbations when compared with salmeterol. Salmeterol was associated with 12% reduction in exacerbations, when compared with ipratropium. Differences between the three treatment groups in terms of QALYs were small. That was expected, given the 5-year time horizon and treatments that do not directly affect survival. The distribution of dots on the CE-planes showed that the higher effectiveness of salmeterol over ipratropium was associated with more uncertainty than the higher effectiveness of tiotropium over salmeterol, because, for the latter comparison, a larger proportion of the dots was found on the right side of the CE plane. Beside effectiveness, the choice between bronchodilators must involve many considerations, including economic considerations of costs and cost effectiveness. The final result of this cost-effectiveness analysis is an acceptability frontier that demonstrated that tiotropium has the highest expected net benefit for a threshold value of the costs per QALY that is well within the limits of other adopted therapies in Spain [35] and in other countries like the UK [36]. This threshold value increases with the severity of COPD, because the differences between treatments in the probabilities of developing a (severe) exacerbation decrease as COPD severity increases. Nevertheless, the cost-effectiveness threshold in the most severely affected patients is still relatively low. The threshold value for costs per additional exacerbation-free month of €639 above which tiotropium becomes the optimal choice in Spain is higher than the ceiling ratios above which tiotropium became most cost effective in the Netherlands (€0) and Canada (€10) in the 1-year model [17]. Similarly, when QALYs were used as the outcome measure, the ceiling ratio of €8,157 per QALY above which tiotropium is the preferred option in Spain is also higher than in The Netherlands and Canada, where tiotropium became the preferred option when decision makers could afford to pay more than €0 and €120 for a QALY, respectively. This difference between these countries is largely driven by the relatively low acquisition costs of ipratropium in Spain, which is related to the fact that there is no widespread use of the more expensive dry powder formulation of ipratropium. In addition, the savings due to the reduction in exacerbations by tiotropium and salmeterol are less, because the costs of treating a severe exacerbation in Spain are lower than in The Netherlands and Canada. This is caused by a smaller proportion of patients hospitalised, as well as a shorter length of stay. As a result, tiotropium generated very small net savings in the Netherlands and similar costs to the other two bronchodilators in Canada, whereas it increased total costs in Spain. Nevertheless, in the three countries investigated, the economic evaluations indicated that the health benefits gained with tiotropium are either at almost no additional costs or at costs that appear reasonable and acceptable, given other adopted treatments [37, 38]. Our base-case scenario was conservative with respect to lung function decline, as we assigned a similar decline of 52 ml/year to the patients in all treatment groups after the first year. We chose this to be the best assumption for the base-case scenario, because there is little evidence in the literature that bronchodilators alter the rate of decline of lung function [39]. However, frequent exacerbations seem to accelerate the decline in lung function [40, 41]. Therefore, our base-case scenario might have underestimated the long-term effect of a bronchodilator that reduces the number and severity of exacerbations. For that reason, we developed the second scenario, which is the least conservative, because we assumed that the differences in lung function decline and exacerbation rates between the three treatments that were observed during the first year would remain during the 4 years thereafter. This scenario is most favourable for tiotropium, and we see the threshold value above which tiotropium becomes most cost effective drop to €551 per exacerbation-free month and €6,226 per QALY. In addition to this optimistic scenario, we developed a very conservative scenario by completely eliminating the difference in exacerbation rate between the treatment groups after the first year and assuming that exacerbation rates would resemble the rate observed in the ipratropium group. This third scenario also assumed a similar decline in lung function across treatment groups of 52 ml/year. This third scenario is conservative because exacerbations were the main drivers of cost effectiveness. The threshold value above which tiotropium becomes most cost effective in this third scenario increases to €1,918 for an additional exacerbation-free month and €15,635 for a QALY, yet is still below acceptable limits reported for the Spanish setting. Below that, ipratropium was most cost effective. Altogether, these three scenarios give the range within which to expect the 5-year cost effectiveness of these bronchodilators. Note, that we had no scenarios assuming a differential effect of the treatments on, for example, utilities or costs of a single exacerbation, because the evidence of such effects is lacking. Furthermore, all scenarios had a time horizon of 5 years to meet the requirements of many European reimbursement authorities who prefer extensions to time horizons reflecting their budgeting process (typically for 3–5 years). Lifetime models that primarily aim to compare medications are soon outdated by the development of new therapeutic options. When the cost-effectiveness analysis was conducted from a societal perspective, the threshold value above which tiotropium is the preferred option is reduced considerably to €547 per exacerbation-free month and €7,076 per QALY. This improved cost effectiveness is primarily related to increasing exacerbation costs, because of the inclusion of the costs of production losses. Hence, a reduced exacerbation rate is associated with higher savings. The NHS perspective also included sick leave benefits paid for by the NHS from day 16 onwards. However, this applies only to severe exacerbations where it was less than 1% of its costs. In contrast, from a societal perspective, costs of production losses are 10% of the costs of a severe exacerbation and 30% of the costs of a non-severe exacerbation. We chose two outcome measures that are among the most relevant in the lives of patients with COPD: being free of exacerbations and having a good quality of life. The concept of an exacerbation-free month is comparable to the concept of a symptom-free day, which is a frequently used outcome measure in asthma [42]. As it is a positive outcome (i.e. more is better), its cost-effectiveness ratio is easier to interpret than the ratio ‘cost per exacerbation avoided’. In general, the number of exacerbations is not a good outcome measure to use in long-term models, especially when treatment improves survival. This outcome measure will bias the treatment which improves survival, because patients can experience exacerbations during the life years gained. In contrast, the number of exacerbation-free months takes account of the fact that the added years of life are partly lived free of exacerbations. The 1-year model, as well as the currently presented long-term model, has been used in reimbursement negotiations in several countries. The major strength of this model is its transparency. We have fully disclosed the model structure as well as all the input. Costs per exacerbation and costs per disease state do not differ between the three treatment groups, and mortality rates by disease severity were also set equally for all treatment groups. Hence, the difference in cost effectiveness is driven by the difference in acquisition costs of the study drugs and the difference in their effectiveness, i.e. the difference in the probabilities to move between disease states and the differences in exacerbation risks. For the first year of the model, estimates of effectiveness were directly obtained from patient-level data from clinical trials. Hence, estimates of probabilities were not based on expert opinion or literature, as is often the case in modelling studies. We acknowledge that differences may exist between trial results from which we derived the model input and the effectiveness of therapy in routine daily practice. A model can be used to adapt the trial data to better represent daily practice. Particularly, compliance is known to be worse in daily practice. However, not accounting for compliance was conservative for tiotropium and salmeterol, since once-daily and twice-daily dosing is more likely to be accompanied by long-term compliance than is four-times daily dosing in chronic diseases [43]. Owing to the double-blind, double-dummy design of the clinical trials, the dosing frequency was the same in the treatment groups, and any impact of dosing frequency on outcomes was eliminated. In conclusion, our model has demonstrated that tiotropium is the treatment with the highest expected net benefit, if decision makers can afford to spend additional budget to gain additional health benefits. The threshold value of the costs per QALY at which tiotropium becomes the preferred treatment is well within acceptable limits (i.e. €8,157 from the NHS perspective and €7,076 from the societal perspective).

Exp_Brain_Res-4-1-2373857

The haptic perception of spatial orientations

This review examines the isotropy of the perception of spatial orientations in the haptic system. It shows the existence of an oblique effect (i.e., a better perception of vertical and horizontal orientations than oblique orientations) in a spatial plane intrinsic to the haptic system, determined by the gravitational cues and the cognitive resources and defined in a subjective frame of reference. Similar results are observed from infancy to adulthood. In 3D space, the haptic processing of orientations is also anisotropic and seems to use both egocentric and allocentric cues. Taken together, these results revealed that the haptic oblique effect occurs when the sensory motor traces associated with exploratory movement are represented more abstractly at a cognitive level. Introduction Haptic perception (or active touch) results from the stimulation of the mechanoreceptors in skin, muscles, tendons and joints generated by the manual exploration of an object in space (Gibson 1962; Revesz 1934, 1950). Haptic perception allows us, for example, to identify an object, or one of its features like its size, shape or weight, the position of its handle or the material of which it is made. A fundamental characteristic of the haptic system is that it depends on contact. The “tactile perceptual field” (i.e., the portion of the skin that is in contact with the external stimulus) has a limited size (the surface of both hands at maximum) and a limited reach (the length of the arm). It results from these characteristics of the tactile perceptual field that the perception of the spatial properties of the objects almost always involves some displacements of the arm and the hand to explore the stimulus. In fact, it is also known that the nature of these exploratory movements often depends on the specific property of the touched object (Lederman and Klatzky 1987, 1993). For example, one might follow the contour of an object with one finger to perceive its shape or squeeze it with the whole hand to perceive its compliance. Moreover, the haptic system must integrate information about the parts of the body touching the object with information about the position of the body parts in space. It results from these properties of the haptic system that the haptic perception of space is far removed from the proximal stimulation that occurs during the manual exploration, and depends on spatio-temporal integration of the kinesthetics and tactile inputs to build a representation of the stimulus. Studies on the haptic perception of space have generally focused on the perception of the elementary spatial properties of objects such as their length, curvature or orientation in space (cf. Hatwell et al. 2003; Henriques and Soechting 2005; Millar 2008). The hallmark of these studies is that the dimension of interest is systematically manipulated and that exploratory movements, if present, are very precisely monitored. In general, the aim of these studies is to measure the sensitivity of the corresponding perceptual channel. In addition, evidence such as a systematic bias or some variation in the sensitivity of parameters is used to understand the underlying processes. Altogether, these studies have shown that the haptic perception of simple spatial properties such as orientation and length are systematically distorted with respect to the physical reality. For example, it is well known that the haptic perception of the length between two points is influenced by numerous factors such as movement direction (radial versus tangential) or velocity (cf. Walsh et al. 1979; Imanaka and Abernethy 2002; Gentaz and Hatwell 2004). Others have found that straight lines are often perceived as curved and vice versa (Sanders and Kappers 2007; see also Henriques and Soechting 2003). In the same vein, Kappers (1999, 2002) and Kappers and Koenderink (1999) have observed that, two parallel (in physical space) bars in different spatial locations can be felt as being almost perpendicular. This remarkable effect remains even after various types of feedback. Finally, it is noteworthy that judgments of different spatial properties can be inconsistent among themselves. For example, Fasse et al. (2000) found that the biases in the perception of the acute angles of a right triangle were inconsistent with the observed biases in the judgments of the lengths of the adjacent and opposed edges. This study suggests that diverse spatial properties of an object might be processed independently from each other and do not necessarily refer to a unique underlying representation of space. The present review is concerned by one very specific aspect of spatial perception: the haptic perception of spatial orientations. The perception of orientations is isotropic when the performances in orientation-adjustment tasks are similar regardless of the orientation values and anisotropic when performances differ according to these values. Historically, the orientation effect of a stimulus on the performance level was first studied in various detection, discrimination and reproduction tasks in the visual modality. A widespread observation, termed “oblique effect” by Appelle (1972), is that the performance level is in general better when the stimulus is aligned with the vertical or horizontal orientation. The original motivation for studying the oblique effect in the haptic modality was to test possible explanations of the visual oblique effect. The existence of an oblique effect in the haptic modality would suggest that orientations are handled by the same processes in both modalities and, thus, would reinforce the view that high-level, possibly amodal (i.e., independent of sensorial modalities), processes could be responsible for the oblique effect. In contrast, its absence would indicate the existence of distinct orientation-processing streams in the haptic and visual modalities (cf. Gentaz 2000; Gentaz and Hatwell 2004; Heller 2000; Millar 1994). In this respect, we will show that orientation processing is generally anisotropic in the haptic modality for adults, children and even in infants. However, unlike the visual oblique effect where such anisotropy is almost always present, we will show that this effect depends on the gravitational cues and memory constraints that are very specific to haptics. The second objective of this review is to identify the frame of reference associated with the haptic oblique effect. This question comes from the observation that the concept of orientation is by definition relative to a set of fixed axes, which define a coordinate system or frame of reference. For example, in the polar coordinate system, an orientation is defined by the angle between some features of the stimulus such as its main axis and an axis of the coordinate system. While the choice of this set of axes can be arbitrary, it is common to choose the vertical and its perpendicular (the horizontal) because of the permanent influence of the vertical gravity force on Earth. In fact, the concept of verticality is so dominant that even in a drawing put on a table that is on a horizontal plane, the verticality is projected as the line belonging to the sagittal plane and perpendicular to the axis of the body. However, the status of verticality must be questioned. In the visual modality, various studies have examined whether the oblique effect was tied to this geocentric reference frame or to a retinocentric frame of reference. In this respect, we will show that the haptic oblique effect observed in the frontal plane is defined in a subjective reference frame that combines ego and geocentric cues. We will also raise the question of which pair of angles is used to code the orientation of a stimulus in space, when the stimuli do not necessarily belong to a plane, where a single angle would suffice to code the orientation. As a matter of fact, it is important to note that different pairs of angles can be used to code an orientation in space in the same way as different coordinate systems can be used to code the position of a point in space. In conclusion, we will argue that the haptic oblique effect occurs at a relatively late stage of orientation processing, when sensory motor traces associated with exploratory movements are transformed into a more abstract representation of the orientation, because the experimental factors that modify the haptic oblique effect's strength either contribute to establishing such a representation (e.g. gravitational cues) or favor its use (e.g., memory constraints). Finally, we will discuss various hypotheses about how the processes involved in the recoding of the sensory-motor traces might yield an anisotropy in the perception or recollection of orientations. Hypotheses on the origins of the oblique effects Numerous studies have shown that vertically or horizontally oriented visual stimuli are generally perceived with greater precision than those oriented obliquely. While the existence of the visual oblique effect is well established, the explanations of this effect in vision are still debated today (e.g., Baowang et al. 2003; Essock 1980; Gentaz and Ballaz 2000; Gentaz and Tschopp 2002; Meng and Qian 2005; Westheimer 2003). Although orientation processing mainly involves the lower visual areas such as the Lateral Geniculate Nucleus (LGN) and the visual primary cortex (V1), it is completed at higher levels in the extra-striate and nonvisual areas (e.g., Goodale et al. 1991). The multitude of subcortical and cortical areas involved in orientation processing raises the question of where the processes responsible for the visual oblique effect specifically act in the anatomo-functional steps of visual processing. Two general hypotheses have been advanced to explain two types of oblique effect. The first hypothesis has been in general advanced to explain the so-called “Class 1 oblique effects,” a term coined by Essock (1980), which are observed in tasks measuring the basic functional properties of the visual system (e.g., acuity and contrast). The hypothesis is that the visual oblique effect is generated at the lowest levels of the visual system, such as LGN and/or V1 (for a discussion about the major role of V1, see Li et al. 2003). This hypothesis is supported by the very specific neurophysiological characteristics of orientation-selective neurons in these areas. In particular, it has been shown that neurons sensitive to the vertical and horizontal orientations are more numerous, sensitive and/or narrowly tuned than neurons sensitive to the oblique orientations (e.g., Furmanski and Engel 2000; Li et al. 2003; Saarinen and Levi 1995). According to this hypothesis, the processes generating the visual oblique effect would therefore be specific to this modality. In contrast, “Class 2 oblique effects” are observed in more cognitive tasks involving identifying, memorizing and categorizing orientations. Moreover, Class 2 oblique effects are influenced by factors, such as internal models (Morgan 1991), recognition (Heeley and Buchanan-Smith 1990) or attention (Shiu and Pashler 1992), that are not amenable to the characteristics of the early stages of visual processing. For example, the mere fact that an oblique effect is present in the reproduction of the orientation of two distant large dots is difficult to explain in terms of the properties of direction-sensitive neurons, since this stimulus in principle stimulates all orientations equally (Cecala and Garner 1986; Westheimer 2003). The second hypothesis is therefore that some oblique effects might occur not only at the low-level areas of the visual system such as LGN or V1 but also at higher level areas (from V2 to associative areas). This hypothesis suggests that the processes generating the visual oblique effect might not be specific to this sensory modality (Heeley et al. 1997). While some models of the Class 2 oblique effects make reference to neurophysiological mechanisms (e.g., McMahon and MacLeod 2003; Westheimer 2003), other explanations are formulated only in functional terms. For example, a widely shared idea is that orientations are represented in a reference frame having the vertical or horizontal orientations as principal axes at some later stage of visual processing. In this frame of reference, identification of these two orientations as principal axes can be carried out simply by identifying the stimulus with one of the two references axes, whereas the coding of oblique orientations would require the integration of information derived from both axes (Foster and Westland 1998; Gentaz and Hatwell 1996; Marendaz 1998; Regan and Price 1986). For example, the coding of oblique orientations might involve the computation of the ratio of the two values, which corresponds to the projections of the oblique on the references axes. According to this view, the oblique effect is caused by some additional processing needed to encode oblique orientations relatively to the reference axes. Alternatively, it has been suggested that all orientations are encoded with the same precision, but that participants have a remarkably accurate internal representation of the vertical and horizontal orientation, which can be used to produce the response when perceived orientation corresponds to these norms (Heeley et al. 1997). In other words, instead of assuming that some computation increases the error for oblique orientation, the higher level processes would bring additional information about the vertical and horizontal that decreases the variable error at the reference axes. Another possibility, adopted by Huttenlocher et al., is that orientations are recorded in categorical terms (Huttenlocher et al. 1991, 1994; see also the work on Categorical Perception, Harnard 2003). According to the Category-Adjustment (CA) model, each orientation is categorized in one of the categories that partition the orientation space. This model was developed to explain the biases toward the diagonals observed in various position- and orientation-recall tasks (see also Spencer and Hund 2003; Haun et al. 2005). According to the CA model, the recall of an oblique orientation would be biased toward the closest diagonal because it would combine a low-level (presumably unbiased) representation of the orientation with the center of the category including the orientation, typically one of the four quadrants. In other words, the gist of the model is that the mental partitioning or labeling of the orientations into a limited number of categories is at the origin of systematic bias in the encoding and recall of an orientation. Finally, Spencer et al. have recently developed an alternative theoretical framework, the Dynamic Field Theory (DFT) of spatial cognition, to explain the presence of systematic biases in orientation-recall tasks (Spencer et al. 2006, 2007). The DFT is based on the dynamic properties of several topologically organized and interconnected layers of units and can provide insights about developmental processes such as the change in direction of the bias in orientation recall tasks between childhood and adulthood, and about real-time processes such as the increase of the recall bias with time. More specifically, the DFT includes a perceptual map that interacts with the environment, a working memory map and a long-term memory map. According to the DFT, repeated exposure to a reference axis results in the storage of this piece of information in the long-term memory, which can be at the origin of a shift of the stimulus position in the working memory even in absence of explicit contextual information in the environment. It is important to note that both the CA and DFT models were originally conceived to explain the results of experiments dealing with visual stimuli showing the presence of systematic biases toward the diagonals in various orientation or position recall tasks, a type of spatial anisotropy that, as we shall see, has not yet been studied in detail in the haptic modality. In the following section, we start to review experimental studies of the haptic oblique effect. Isotropic or anisotropic haptic perception of spatial orientations? Existence of an haptic oblique effect Lechelt et al. (1976) were the first to show interest in the haptic oblique effect. The goal was to show the existence of an oblique effect in the haptic system analogous to the one observed in the visual system for the abovementioned reasons. To that end, they compared orientation perception in the visual and haptic systems by applying the same experimental paradigm to both systems. The visual and haptic stimuli were rods (30 cm × 0.6 cm), which could take one of six orientations: 0°, 45°, 90°, 135°, 225°, 315°. Adults were presented with two identical rods (a stimulus rod and a response rod) placed in the frontal plane, on either side and equidistant from their medial plane. In the visual condition, participants worked in a dark room with electroluminescent rods and gave verbal indications to adjust the response rod immediately after the stimulus rod had disappeared. In the haptic condition, participants were blindfolded and the response rod was adjusted with the contralateral hand immediately after the stimulus rod became unavailable. Globally, the analysis of constant errors did not show any systematic deviation in either modality for all orientations. The analysis of absolute errors revealed a classical effect of modality: errors were greater in the haptic condition (from M = 4.8° to M = 10.5°) than those in the visual condition (from M = 1.5° to M = 4.2°). The effect of spatial orientations was significant and revealed an oblique effect in both modalities. In vision, the errors were lower for vertical and horizontal (M = 1.5°) than for the oblique orientations (M = 3.5°). In haptics, the errors were lower for vertical and horizontal (M = 5°) than for the oblique orientations (M = 9°). Lechelt and Verenka (1980) replicated these results with a slightly modified procedure, where the rod exploration time was fixed at 5 s for both perceptual conditions, where the delay between exploration and reproduction was set at 10 s, and where the orientations were no longer indicated by the experimenters before the test. The authors concluded that there was an oblique effect in the haptic system, analogous to that observed in the visual system. Initial hypotheses on the origin of the haptic oblique effect: the role of prior knowledge and the mode of reproduction of the orientation Appelle and Countryman (1986) questioned the abovementioned conclusions of Lechelt et al. (1976), and Lechelt and Verenka (1980). They argued that the haptic oblique effect might be induced by the prior visual perceptual experience and/or the conditions of exploration–reproduction movements. To show the possible effect of the first factor, the authors proposed two “prior experience” conditions: an “informed” condition, in which standard orientations were verbally and visually presented to participants for as long as they wished; and an “uninformed” condition, in which participants did not know which standard orientations were being tested. On the basis of Appelle and Gravetter’s (1985) results, they predicted that prior verbal or visual knowledge of standard orientations would lead participants to produce responses using internal orientation models as a reference (mainly the vertical and horizontal orientations), instead of the immediate haptic percept resulting from the exploration of the stimulus, and induce the haptic oblique effect. With respect to the second factor (conditions of exploration–reproduction movements), these authors also argued that the haptic oblique effect observed in Lechelt et al.’s experiments could be due to the use of the contralateral hand to reproduce the orientation. Indeed, when one hand explores the orientation of a standard rod and the other hand reproduces the same orientation on a test rod, the symmetrical disposition of the shoulder–hand systems imposes nonhomologous movement patterns to explore and reproduce oblique orientations (agonist and antagonist muscles are mobilized differently). In contrast, exploration and reproduction movement patterns are homologous for vertical and horizontal orientations. Then, the authors proposed two exploration–reproduction conditions: contralateral condition (one hand explores and the other reproduces) and an ipsilateral condition (the same hand explores and reproduces). In the latter case, the exploration–reproduction movement patterns are homologous for all orientations and the oblique effect should not be present. However, Appelle and Countryman (1986) modified the experimental paradigm of Lechelt et al. (1976), and Lechelt and Verenka (1980). First, they used only four orientations (0°, 45°, 90° and 135°). Earlier results justified this choice, as they showed that performances did not differ between 45° and 225° or between 135° and 325°, for example. They introduced decoy orientations so that participants would not be aware that only four orientations were being tested. They presented the orientations in the horizontal plane (parallel to a table surface) instead of the frontal one. Finally, they fixed the delay between exploration and reproduction at 5 s. Absolute error analysis revealed that the performance on the oblique orientations, and therefore the amplitude of the haptic oblique effect, varied according to prior experience and exploration–reproduction conditions. Vertical and horizontal orientations were stable. The extent of the haptic oblique effect was maximal in the informed contralateral condition (vertical–horizontal: M = 5° and obliques: M = 9°) and diminished in the uninformed contralateral condition (vertical–horizontal: M = 5.5 and obliques: M = 7.5°). It was even smaller in the informed ipsilateral condition (vertical–horizontal: M = 3.5° and obliques: M = 5.5°). Finally, it was nil, and the oblique effect thus absent, in the uninformed ipsilateral condition (vertical–horizontal: M = 4° and obliques: M = 4.5°). In their study, Appelle and Countryman (1986) concluded that prior visual perceptual experience and exploration–reproduction movements were responsible for the haptic oblique effect, contrary to the propositions of Lechelt et al. (1976), and Lechelt and Verenka (1980). Role of gravitational cues in the haptic oblique effect Gentaz and Hatwell’s (1995) study provides a test of Appelle and Countryman’s (1986) hypothesis (second factor) regarding the existence of a difference between the one-handed exploratory movement patterns of an oblique orientation and the movement patterns needed to reproduce the oblique with the contralateral hand. As in Appelle and Countryman’s (1986) study, the participants worked in a nonvisual and uninformed condition with a rod oriented at 0°, 45°, 90° or 135° and they reproduced the orientation of the standard rod after a 5-s delay. The standard rod was presented in the horizontal (as in Appelle et al.’s experiment), frontal (parallel to the surface of a painting, as in Lechelt et al.’s experiments) or sagittal (the median plane, perpendicular to the horizontal and frontal planes) planes. The task was performed ipsilaterally (with the same hand) or contralaterally (with the other hand). Given that, in the sagittal plane, the oblique orientation exploratory movements of one hand become homologous with those needed to reproduce the oblique orientations contralaterally; the oblique effect should also be absent in this plane if Appelle and Countryman’s (1986) hypothesis (difference between exploration and reproduction movements) is determinant. Absolute error analysis revealed that results did not concord with these predictions, because similar oblique effects were obtained in the frontal and sagittal planes under both ipsilateral and contralateral conditions (vertical–horizontal: M = 5° and 45–135° obliques: M = 8°). However, results in the horizontal plane replicated those of Appelle and Countryman (1986), i.e., an oblique effect was present in the contralateral condition (vertical–horizontal: M = 8.9° and 45–135° obliques: M = 12.9°) and absent in the ipsilateral one (vertical–horizontal: M = 6.3° and 45–135°; obliques: M = 7.8°). Therefore, the presence or absence of an oblique effect does not appear to be due to the difference of movements between the two hands, but seems to be strongly linked to the type of spatial planes. To understand the results in the horizontal plane and, in particular, the lack of an oblique effect in the ipsilateral condition only, Gentaz and Hatwell (1995) asked blindfolded adults to symmetrically reproduce a previously presented orientation (a 45° right standard oblique was to be reproduced at 45° left and, similarly, a 45° left standard oblique was to be reproduced at 45° right) in the horizontal plane, either ipsilaterally or contralaterally. Given that the movements necessary for the exploration and the symmetrical reproduction of an oblique orientation become homologous in the contralateral condition and nonhomologous in the ipsilateral condition, the oblique effect should be present in the ipsilateral condition and absent in the contralateral condition if the Appelle and Contryman’s hypothesis was valid. Results did not concord with these predictions, since an oblique effect was present in both reproduction conditions in the horizontal plane (vertical–horizontal: M = 7° and 45–135° obliques: M = 9.3°). Taken together, these results partially (see below) invalidate Appelle and Countryman’s (1986) hypothesis about the role of exploration–reproduction movements. Gentaz and Hatwell (1995) account for these results by suggesting that the haptic oblique effect was linked to the gravitational cues produced by the shoulder–hand system. Indeed, the need to relatively move massive parts of the body such as the arm and forearm during the manual exploration of a stimulus brings antigravity forces into play. The role that gravity might play in the haptic system is clearly reduced in the visual system. The shoulder–hand system is much more directly submitted to gravitational constraints than the ocular system. When it explores a stimulus, it has to produce antigravity forces and these, in return, provide what are known as “gravitational cues.” These gravitational cues provided by the proprioceptive system result from the deformation of cutaneous, muscular and articular tissues, and depend on the specific muscular forces needed to maintain or displace the shoulder–hand system against gravity (Gentaz and Hatwell 1996). As such, these cues can provide a considerable amount of information on arm posture or motion, relative to some fixed “geocentric frame of reference” (Paillard 1991) that is linked to gravity and exterior to the body. This hypothesis was justified by an analysis of the task conditions proposed in the previous experiments. Indeed, the size of the stimulus rod (25 cm) and its position in relation to the participant (40 cm) required significant participation of the shoulder–hand system during exploration. To explain the “plane effect” in the ipsilateral reproduction conditions observed above (i.e., the presence of an oblique effect in the frontal and sagittal planes and its absence in the horizontal plane), Gentaz and Hatwell (1996) hypothesized that participants favored gravitational cues in orientation processing, because these cues specify the gravitational vertical, which is used as a reference axis. The characteristics of these gravitational cues would differ according to exploratory conditions. In a normal situation, their average amplitude is practically the same in all three planes, since the arm is always influenced by the same gravitational forces. On the other hand, the variability of these cues is not the same depending on the plane the experiment is carried out in. There is a high variability in the frontal and sagittal planes, as the arm deploys considerable antigravitational forces when moving from bottom to top, but falls from top to bottom with little effort. On the contrary, in the horizontal plane, gravitational cues hardly vary during exploration as the arm constantly operates in a direction perpendicular to gravity. Gentaz and Hatwell (1996) tested the hypothesis that the gravitational cues produced by the shoulder–hand system during exploration were involved in the oblique effect and in the “plane effect” in the ipsilateral reproduction condition. The amplitude of these cues was modified by manipulating the level of gravitational constraint, and variability was modified by changing the plane the task was effectuated in. The oblique effect and the plane effect should diminish in conditions where gravitational cues are weak, and increase in those where these cues are greater. In one experiment, only the horizontal plane was tested. Participants explored the orientation of a rod with one hand and reproduced it with the same hand, either keeping their forearm–wrist–hand in the air (“unsupported forearm” condition), or resting their forearm–wrist–hand on the rod supporting surface (“supported forearm” condition). In the first condition, antigravitational forces were necessarily produced, whereas these forces were very much reduced in the second condition. Absolute error analysis revealed that the oblique effect was absent in the supported forearm condition (vertical–horizontal: M = 4.7° and 45–135° obliques: M = 4.2°) and was present in the unsupported forearm condition (vertical–horizontal: M = 3° and 45–135° obliques: M = 7°). In another experiment, blindfolded adults were tested in one of the three planes, either in a “natural” condition, or in a “lightened” condition in which gravitational cues had been reduced by a pulley system. In the latter case, the participant’s forearm was attached to a display, which held it balanced in the air when the weight suspended on the pulley reached a certain value variable from one participant to another. The amplitude of the oblique effect was lower in the lightened condition (significant difference of 1.43°) than that in the natural condition (significant difference of 3.43°), and this is because that the accuracy of vertical and horizontal reproduction was deteriorated (thus increasing the difference with that of the oblique orientations, which remained stable). In addition, the exploratory plane had no effect. Taken together, these results revealed that gravitational cues are crucial when the haptic oblique effect is present in the three planes in blindfolded participants and in the ipsilateral reproduction condition. Role of previous visual experience and memory in the haptic oblique effect The previous results do not, however, exclude an influence of high-level factors, such as visual experience or cognitive resources or both. As a matter of fact, it is well known that the haptic perception of the sighted working in a nonvisual condition can be fed by visual representations (Hatwell 1978; Thinus-Blanc and Gaunet 1997). Gentaz and Hatwell (1998) examined whether the participant’s visual experience modified the action of the gravitational cues observed in sighted people. In this study, blindfolded sighted participants as well as early and late totally blind people were asked to explore a rod and to reproduce its orientation ipsilaterally. The magnitude of gravitational cues was modified by manipulating the level of gravitational constraints (natural and reduced) and their variability was modified by changing the task plane: horizontal (weak variability) and frontal (strong variability). By comparing the performances of the early and late totally blind, this study aimed at evaluating the role played by visual experience and visualization in the haptic oblique effect. If such is the case, the oblique effect should be present in the late blind and absent in the early blind. The comparison between blindfolded sighted and totally blind people, who are better-trained in the use of the haptic system, also allowed the authors to find out whether gravitational cues also played a role in the apparition of the haptic oblique effect in the blind as is the case with blindfolded persons. In this case, the oblique effect should be absent when cues are reduced and present when they are normal. Absolute error analysis showed that in the horizontal plane, the oblique effect was absent in both the early and late blind, regardless of gravitational constraints (vertical–horizontal: M = 7.9° and 45–135°; obliques: M = 10°). In the frontal plane, the oblique effect was present in both groups of blind people, regardless of gravitational constraints (vertical–horizontal: M = 5.8° and 45–135°; obliques: M = 9.9°). Furthermore, no difference was observed between the early and the late blind. In conclusion, these results confirmed that the variability of gravitational cues plays an important role in the presence or absence of the haptic oblique effect in the totally blind, although no visual experience effect was observed [first factor of Appelle and Countryman’s (1986) hypothesis]. Gentaz and Hatwell (1999) examined further the role of attentional and cognitive resources on the haptic perception of orientations and the oblique effect by increasing memorization constraints. We know that the characteristics of the haptic system differ in fundamental ways from those of the visual system. The limited size of the tactile perceptual field requires that it be moved to gain knowledge about, for example, the shape of an object. In contrast, eye movements play only a secondary role in the visual perception of the position or shape of an object, since both pieces of information can be extracted from static images. This difference was recently re-emphasized by Henriques and Soechting (2005) who argued in a recent review that “the processing of haptic information differs fundamentally from visual processing in that the former requires the integration of information that evolves in time as well as space.” It results therefore from this quality of proximal reception of the haptic system that haptic processing and haptic perception are consistently more sequential than visual ones. Moreover, haptic perception needs to rely more heavily on working memory to achieve the mental synthesis that is necessary to gain an unified representation of the object (Revesz 1950; cf. Hatwell et al. 2003). In all previous research on the haptic perception of orientations, the delay between the stimulus exploration phase and the reproduction phase was 5-s long and was unoccupied. In Gentaz and Hatwell (1999), memorization conditions were thus altered—the length and the nature of the gap was changed in the two exploratory conditions showing that the availability of gravitational cues affected orientation coding. Participants explored a rod in the horizontal plane with weak or natural gravitational cues and reproduced the rod’s orientation ipsilaterally. The orientations were reproduced according to one of four memorization conditions: after unoccupied 5-s gaps or 30-s gaps and 30-s gaps occupied by interfering verbal or motor activities such as reciting the alphabet forwards or backwards (verbal) or haptically exploring a raised sinuous trajectory (motor). Absolute error analysis showed that when the gap was unoccupied (regardless of how long it was), haptic orientation treatment and the oblique effect depended on the exploratory conditions: the oblique effect was absent when gravitational cues were weak (vertical–horizontal: M = 5.5° and 45–135° obliques: M = 5.5°) and present when they were natural (vertical–horizontal: M = 3.85° and 45–135° obliques: M = 6.8°), like in Gentaz and Hatwell (1995, 1996). On the other hand, when the 30-s gap was occupied by interfering verbal or haptic activities, the haptic oblique effect was present in both experiments and regardless of available gravitational cues (vertical–horizontal: M = 5.2° and 45–135°; obliques: M = 8.5°). Taken together, these results showed that high-level factors can indeed determine the presence of the haptic oblique effect, since the reduction of available attentional or cognitive resources by an interfering task caused an oblique effect in a condition in which it was not usually observed. In conclusion, the presence of a haptic oblique effect in blindfolded sighted participants, as well as in the totally early blind, with natural exploration and ipsilateral reproduction in the frontal plane (regardless of gravitational constraints) invalidates Appelle and Countryman’s (1986) hypothesis about the role of visual representations and shows the existence under very precise conditions of an oblique effect intrinsic to the haptic system (Table 1).Table 1Average absolute errors in the studies of the haptic oblique effect with blindfolded adultsExperimental conditionsOrientationsOblique effectReferencesTasksPlaneDelay (s)HandV/HObliqueDifferenceReproduction task Free-time exploration and haptically informed about standard orientationsF0contra594*Lechelt et al. (1976)Reproduction task Limited-time exploration (5 s) and haptically informed about standard orientationsF0contra48.14.1*Lechelt and Verenka (1980)F10contra47.53.5*Production task Free-time production and verbally informed about orientationsF0one3.28.35.1*Appelle and Gravetter (1985) Free-time and haptically informed about orientationsF0one3.58.14.6*Reproduction task InformedH5Contra594*Appelle and Countryman (1986) UninformedH5Contra5.37.52.2* InformedH5ipsi3.55.52* UninformedH5ipsi44.50.5Reproduction task Free exploration and uninformed about orientationsF5contra583*Gentaz and Hatwell (1995)F5ipsi583*S5contra583*S5ipsi583*H5contra8.912.94* Supported explorationH5ipsi6.37.81.5 Symmetric orientationH5contra79.32.3* Symmetric orientationH5ipsi79.32.3*Reproduction task Unsupported explorationH5ipsi374*Gentaz and Hatwell (1996) Supported explorationH5ipsi4.74.2−0.5 Normal gravity cuesF5ipsi3.58.24.7* Reduced gravity cuesF5ipsi6.17.21.1* NormalS5ipsi3.67.14.5* ReducedS5ipsi5.77.31.6* NormalH5ipsi5.47.31.9* ReducedH5ipsi7.691.4*Reproduction task Early and late blind Pooled NormalF5ipsi5.89.94.1*Gentaz and Hatwell (1998) ReducedF5ipsi5.89.94.1* NormalH5ipsi7.9102.1 ReducedH5ipsi7.9102.1Reproduction task Normal, unoccupied delayF5ipsi3.856.82.95*Gentaz and Hatwell (1999) Reduced, unoccupied delayF5ipsi5.55.50 Normal, unoccupied delayF30ipsi3.856.82.95* Reduced, unoccupied delayF30ipsi5.55.50 Normal, verbal taskF30ipsi5.28.53.3* Reduced, verbal taskF30ipsi5.28.53.3* Normal, motor taskF30ipsi5.28.53.3* Reduced, motor taskF30ipsi5.28.53.3*Reproduction task UprightF5ipsi2.55.53*Luyat et al. (2001) Body tiltedF5ipsi55.50.5Production task No context, smoothF5ipsi2.36.44.1*Luyat et al. (2005a, b) Congruent haptic cuesF5ipsi1.43.42* Incongruent haptic cuesF5ipsi1.84.83.05*The table reports the average absolute error for the vertical and horizontal orientations (V/H column), for the 45° and 135° orientations (Oblique column), as well as the existence of an oblique effect, that is, of statistically significant difference between the two sets of orientation (marked by * in the Difference column) as function of experimental conditions. The reported studies mainly used a reproduction paradigm where the subject explored the orientation of a rod with one hand before to be reproduced with the same (ipsilateral condition) or other hand (contralateral condition, see Hand column). The Plane and Delay column specify the plane in which the orientations were presented and reproduced or produced as well as the delay between the presentation and reproduction. The first column specifies additional experimental factors that were manipulated in the corresponding studies (see text) Haptic orientations defined in a subjective reference frame As previously indicated, the concept of orientation is by definition relative to one or more systems of coordinates with the cardinal vertical and horizontal orientations as norms of this system. However, on Earth, different verticals (gravitational, egocentric), which can constitute the norms for independent references, exist. Thus, the existence of an oblique effect raises the question of which kind of vertical is better perceived or in other words in which reference frame this effect is defined. A classical broad distinction is made between egocentric (referred to the participant’s body) and allocentric (referred to environmental cues) spatial frames (Howard 1982; Rock 1990). The allocentric reference frame can be divided into a gravitational frame defined by the direction of the gravity pull (geocentric frame) and in pattern-centric reference frames defined by (visual or haptic or both) contextual cues. In natural conditions, the different reference frames are in general congruent. Consequently, mapping of orientations could result from either egocentric or allocentric reference frames or most likely from both. In darkness, tilting the body uncouples the gravitational and the egocentric reference frames and allows us to specify in which kind of reference frame orientations are defined. Subjective reference frame In vision, certain studies have evidenced rather an egocentric (retinocentric) coding (Banks and Stolartz 1975; Chen and Levi 1996; Corwin et al. 1977), suggesting that the visual oblique effect could principally be accounted for by the properties of the orientation-selective neurons present in the primary visual cortex (see Introduction). Indeed, neurons tuned to vertical and horizontal retinal orientations are more numerous and feature particular response characteristics in relation to those tuned to oblique orientations. On the other hand, other studies have revealed the existence of gravitational orientation coding, implying that the origin of the oblique effect is probably more central, involving extraretinal information such as vestibular and somesthetic cues integrated at a higher level in the visual processing hierarchy (Attneave and Olson 1967; Buchanan-Smith and Heeley 1993; Ferrante et al. 1995; Lipshits and McIntyre 1999). However, very little research has been done concerning this topic in other perceptual modalities. In haptics, Luyat et al. (2001) tested the effect of tilting the body or the head on the haptic oblique effect. Blindfolded participants explored a standard rod in the frontal plane and reproduced its orientation with the same hand after a 5-s delay. Three conditions were examined: upright, inclined to the right (+45°) and to the left (+135°). Five orientations were tested: vertical, horizontal, 45° oblique, 135° oblique and the subjective vertical (SV). The SV is the individual’s perception of the direction of gravitational force (cf. Howard 1982; Luyat 1997). The most commonly used paradigm consists of adjusting a stimulus (a rod) to the physical gravitational vertical. The deviation from the gravitational direction constituted a measure of the subjective vertical. In upright posture, the SV is very close to the physical vertical in participants with no vestibular disease or parietal lesion. However, in darkness, lateral head or body tilt provokes systematic deviations of the haptic SV in the opposite direction to that of the head: the Müller effect. A first experimental phase estimated each participant’s SV in each postural condition and this SV was then tested in the exploration–reproduction task. What is more, as an insurance against a possible fluctuation of the SV in the haptic perception of orientation, the SV was measured again after the exploration–reproduction task. If the haptic oblique effect is defined in a gravitational reference frame, then the reproduction of the vertical and the horizontal should be more accurate than the reproduction of oblique orientations, regardless of postural conditions. On the other hand, if the haptic oblique effect is defined in an egocentric reference frame, then the reproduction of the two diagonals (45° and 135°), which are parallel and perpendicular to the body tilted to 45° or 135°, should be more accurate than the reproduction of the gravitational vertical and the horizontal, which become oblique in relation to the body. Finally, knowing the effect of tilting the body on the vertical, the SV could constitute a reference axis when the body is tilted. In this case, the reproduction of the SV should be more accurate than all the other orientations in the tilted body conditions. The results observed in the production task in which the participants had to manually orient the bar in the direction that they thought was vertical (phase 1, i.e., before the reproduction task and phase 2, i.e., after the reproduction task) showed systematic deviations of the haptic SV in the direction opposite to that of the tilted body (Müller effect). The comparison of phases 1 and 2 showed that SV perception was stable and faithful over time, thus justifying its study in the exploration–reproduction task. Absolute error analysis in the exploration–reproduction task showed that tilting the body affects the precision of reproduction, particularly of the gravitational vertical and the horizontal. Thus, an oblique effect was present in the upright condition (vertical–horizontal: M = 2.5° and 45–135°; obliques: M = 5.5°), but absent in tilted conditions (vertical–horizontal: M = 5° and 45–135° obliques: M = 5.5°). It should be noted that the same results were observed in a complementary experiment in which only the head was tilted. In conclusion, tilting affected vertical perception in the production task and also in orientation reproduction. This means that, in a tilted body condition, the gravitational vertical and horizontal orientations no longer appear to act as reference norms, as they were not better reproduced than the oblique orientations. Consequently, the hypothesis of a pure gravitational reference frame underlying the haptic oblique effect can be rejected. In the same way, the results did not confirm the hypothesis of a pure egocentric reference frame, as 45° (or 135°) oblique orientations (which are vertical or horizontal in relation to the body) were not better reproduced than the gravitational vertical and the horizontal. On the other hand, results showed that the SV was better reproduced than the other four orientations, particularly in the tilted body conditions (M = 2.7°). In these conditions, the SV could constitute a norm for a subjective vertical reference frame. Finally, these results questioned the nature of the processes responsible for the oblique effect across sensory modalities (Hatwell 1994; Thinus-Blanc and Gaunet 1997). Several studies in the spatial domain show that visual representations largely feed tactile representations and vice versa. Thus, Luyat and Gentaz (2002) examined whether the visual oblique effect is also coded in a subjective gravitational reference frame, analogous to that used in haptics. The effect of inclining the whole body on the visual oblique effect was studied with the same paradigm as that proposed in haptics. The classic oblique effect observed in the upright body condition disappeared in the inclined body conditions because of a reduction in the accuracy of the reproduction of the gravitational vertical and the horizontal. In inclined conditions, the subjective vertical appears to be the best perceived orientation. Thus, the visual oblique effect seems also to be defined within a subjective vertical reference frame. However, results in the production task showed that inclining the body produces systematic deviations to the visual subjective vertical (VSV) in the direction of the body incline axis, whereas deviation is in the direction opposite to body inclined in the haptic modality. In another study, Gentaz et al. (2001) examined the oblique effects in the visual, haptic and somato-vestibular systems by asking adults to reproduce (after a 10-s delay) an orientation presented in the frontal plane. In the visual modality, participants reproduced the orientation of a luminous rod presented in a dark room. In the haptic modality, blindfolded participants explored a rod with one hand and reproduced its orientation ipsilaterally. In the somato-vestibular modality, blindfolded participants reproduced the incline of their body. Results revealed similar oblique effects in the three tasks. However, no significant correlation between the magnitude of the visual, haptic and somato-vestibular oblique effects was observed. Taken together, these results suggest the existence of both processes specific to the haptic system (different subjective reference frames in haptics and in vision) and processes similar to the corresponding visual processes (access to a subjective reference frame) but not common across sensory modalities (absence of correlations between modalities). Let us now look at the effect of contextual cues on the haptic perception of orientation in adults by comparing it to the corresponding visual one. Haptic contextual reference frame In previous experiments, the stimulus consisted of a single rod without any contextual cues around it. In this case, it was not possible to determine whether the orientation perception in natural conditions could also be coded (at least partially) in a pattern-centric reference frame (defined by contextual cues). In vision, it is well known that spatial orientation is enhanced when vertical contextual cues are available, and as a consequence, it is strongly affected by roll or pitch of single tilted lines or by a more structured context such as a room (Groen et al. 2003; Luyat 1997). Recently, Luyat et al. (2005a) showed that the oblique effect decreased with visual contextual cues tilted 15°. Another characteristic of the haptic system is that the shape and size of the tactile perceptual field can vary according to the mode of exploration adopted by the observer (e.g., with one or several fingers). This observation stands in sharp contrast with the visual system where the shape and size of visual field are essentially invariant. The ability of the haptic system to perceive contextual cues might vary drastically in accordance with the number of contacts that are established with the environment. For example, two-handed exploration might enlarge the haptic perceptual field sufficiently to facilitate the perception of contextual cues (on the role of reference cues, cf. Millar 1994; Millar and Al-Attar 2002). The haptic perceptual field remains greatly reduced compared to the visual field even when the two hands are used with active exploratory movements. As a result, the perceptual cues, which are relevant in a task (or useful as a spatial exocentric reference frame), are less available in the haptic than in the visual modalities. Moreover, voluntary movements must be made to compensate for the smallness of the haptic perceptual field. As a result, haptic perception is highly sequential (cf. “Introduction”). However, this latter property permits contextual effects to be reduced in haptic perception compared to visual perception. Indeed, it is not the case in vision because of the presence of simultaneous peripheral (contextual cues) and central (the target) stimulation. The effect of contextual cues on the perception of orientations was investigated first by Walker (1972) in a short report in which the participant had to position a rod to the vertical while passing the right hand through a tilted grid. This author reported an effect, but unfortunately both precise data and important methodological aspects as the amount of tilt used were not provided. More recently, Luyat et al. (2005b) studied this question by asking blindfolded participants to position a rod with their right dominant hand to three different spatial orientations in their fronto-parallel plane: to the vertical and along two oblique orientations, respectively, at 45° to the left and to the right of the vertical one. This production task was carried out in four different backgrounds: (1) in the absence of contextual cues (smooth), (2) in presence of congruent contextual cues (stripes parallel to the orientation to be produced), (3) in presence of tilted stripes context to the left and (4) in presence of tilted stripes context to the right of the orientation to be produced. Using at the same time, the right and left hands with one hand exploring the context and the other (dominant hand) adjusting the stimulus-rod to the expected orientation allows a simultaneous stimulation by both the context and the stimulus. The analysis of the precision of adjustments (variable errors) revealed that the oblique effect obtained in the control condition (no context) (vertical: M = 2.3° and 45–135° obliques: M = 6.4°) was similar to that obtained previously by Gentaz et al. (2002) with a similar method of direct estimation of orientations (production task). Indeed, most experiments on the haptic oblique effect have been carried out with a reproduction task, which requires memorizing an unknown but previously scanned orientation. This experiment confirmed that a haptic oblique effect in the fronto-parallel plane can be evidenced with a production task, which involves a cognitive representation of orientations probably based on an internal model of gravity. More interesting is the fact that tactile contextual cues had an influence on the production of oblique and vertical orientations, particularly on the oblique effect. When the context was congruent with the standard orientations, the precision of adjustments was significantly improved (vertical: M = 1.8° and 45–135° obliques: M = 4.85°), an effect very similar to the effect exerted by visual vertical cues on spatial orientation. During noncongruent context scanning, the precision of orientations, particularly the obliques, also tended to be enhanced and, as a consequence, the oblique effect was significantly weakened. However, this improvement does not mean that the accuracy was enhanced, since analysis on constant error revealed that deviations erred in the direction of the stripes. Thus, the presence of a context, congruent or not, diminishes the variability of estimations and therefore enhances the precision of the production of orientations. Furthermore, the mismatch between the orientation to be produced and the context (noncongruent condition) revealed a tendency to adjust the rod in the direction of this context. In conclusion, these results suggest that the haptic oblique effect is defined in a subjective reference frame, which may integrate not only vestibular and somesthetic inputs (head or whole body tilt) but also haptic contextual cues according to task conditions. Moreover, it should be emphasized that tasks such as pointing to remembered visual targets have also been found to involve processing information in multiple reference frames (see Carrozzo et al. 2002; McIntyre et al. 1997a, b). We propose that the subjective vertical and the subjective horizontal (probably) constitute the norms of this subjective vertical reference frame. At the neurophysiological level, the questions of how and where vestibular or contextual information dedicated to spatial cognition (specifically to the subjective perception of “what is up”) is processed in the cortex is far from perfectly known. Research in humans with functional magnetic resonance imaging (fMRI) (Fasold et al. 2002) or electrical stimulation in patients with epilepsy (Kahane et al. 2003) have shown several vestibular cortex areas, in particular insulo-parietal (analogous to the Parieto-Insular Vestibular Cortex in monkeys) and temporal areas with a right hemispheric dominance. The integration of gravitational and contextual cues in the oblique effect suggests that these associative areas with multimodal neurons could be implicated. A last question raised in current haptic studies is the presence of the haptic oblique effect in 3D space in adults. Haptic orientation in 3D space In all previous experiments, we considered the haptic perception of orientations on a plane rather than in space. An orientation in space is defined by two parameters such as its azimuth and elevation. This observation raises the questions of whether the haptic oblique effect is still present in the absence of any spatial constraint when the participant needs to focus on at least two independent parameters to perform the task. More importantly, given the fact that there is a much larger set of alternative reference frames, which can be used to code an orientation in space than on a plane, the perception of orientations in 3D space raises the question of which reference frame is most adequate to describe the pattern of errors. To examine this question, Baud-Bovy and Gentaz (2006) used a haptic display to present the stimuli and record the participant’s hand movements to remove, if desired, the usual planar constraint imposed by the experimental apparatus used in most prior haptic studies. In a first experiment, the haptic reproduction of vertical, horizontal and diagonal orientations on a plane and in space was compared. Thus, participants were asked to explore the orientation of a “virtual rod” with to-and-fro movements on the frontal plane and then they were asked to respond in two different conditions. In the 2D reproduction condition, the finger movements were constrained to the frontal plane like in the exploration phase. In the 3D reproduction condition, the movements of the finger were unconstrained and could move freely in space. The first finding was that the vertical and horizontal orientations were better reproduced than the diagonal orientations in the 3D condition. Thus, removing the constraint during the reproduction phase did not prevent the occurrence of the haptic oblique effect. Decomposing the angular error in the 3D condition in an in-plane component and in an out-of-plane component revealed that the anisotropy in this condition concerned mostly the in-plane component. As a matter of fact, the error pattern in the 2D condition was very similar to the in-plane component of the 3D condition, while the analysis of out-plane errors showed that their distribution was much more uniform across orientations. This finding suggests first that an orientation in space is coded by two parameters (i.e., the angle inside the frontal plane and the angle between target orientation and the frontal plane) and second, that these two parameters are processed independently. To examine how orientations are coded in space in the absence of any sort of reference to a plane, Baud-Bovy and Gentaz (2006) tested thirteen target orientations, which belonged to different planes (three principal axes—vertical, sagittal and lateral axes, the two 45º and 135º diagonals in the frontal, sagittal and horizontal plane, respectively, and four “3D diagonals”). As previously, blindfolded participants were asked to explore the orientation of a “virtual rod” with to-and-fro movements and to respond in the 3D condition, i.e., without any constraint on the finger movements during the reproduction phase. The haptic processing of orientations was clearly anisotropic. The vertical orientation was reproduced most precisely. Once again the accuracy and consistency with which participants reproduced this orientation was striking. For the other orientations, the error pattern depended in a complex manner on participants, stimulus and error components, which makes its interpretation complex. On the one hand, the three principal axes were most accurately reproduced when all error components were pooled together. This observation has a straightforward consequence in a “plane-by-plane” analysis, as it implies that the horizontal and vertical axes in the frontal and sagittal planes, as well as the lateral and sagittal axes in the horizontal plane, are more accurately reproduced than the diagonal orientations. In other words, this observation is akin to a demonstration of the classic oblique effect in each one of these planes. Regarding the frontal plane, the results of this experiment were similar to those of the previous experiment. The analysis of the different error components showed a clear oblique effect in both experiments, with the vertical and horizontal orientations being most accurately reproduced, even in the absence of any planar structure in the design of the experiment and of any constraint during the reproduction phase. On the other hand, the presence of the classical oblique effect was much less obvious when we considered the various error components separately. The average angular error inside the horizontal and sagittal planes did not exhibit the expected pattern. Decomposing the in-plane errors in systematic, intersubject and intrasubject error components confirmed this negative finding. Further studies are needed to understand these complex patterns of results observed in 3D space. Let us now look at the ontogenetic development of the haptic oblique effect by comparing it to the corresponding visual one. Developmental aspect of the oblique effects The oblique effects in children and infancy In vision, Leehey et al. (1975) tested the oblique effect in infants aged from 6 to 50 weeks using contrast sensitivity. The stimulus was a circular grating consisting of light and dark bars uniformly alternated. Bars were of five different widths. When spatial frequency was above threshold discrimination, the pattern appeared contrasted, whereas it looked like a homogeneous gray field when spatial frequency was below threshold. Two gratings of the same spatial frequency differing in orientations were simultaneously proposed. Each trial consisted of an oblique grating (45° or 135°) and a vertical or a horizontal grating presented in a random fashion. As infants looked longer at the more contrasted of two patterns, a forced-choice judgment was used to determine which grating the young subject preferred. Given that the two stimuli have the same spatial frequency, an oblique effect would be present if, for a specific bar width, infants could discriminate the vertical or the horizontal lines but not the oblique lines. For this particular spatial frequency, infants should look preferentially at vertical or horizontal gratings rather than at oblique gratings. Results revealed an oblique effect. If the critical value of spatial frequency progressively decreased with age according to the development of visual acuity, an oblique effect was systematically observed at all ages. These findings were replicated by Gwiazda et al. (1978, 1984). According to Held and his colleagues, findings suggest that the visual oblique effect depends on endogenous maturation rather than exposure to a carpentered world, as assumed by other authors (Annis and Frost 1973; Ross 1992). Annis and Frost (1973) studied the oblique effect in Euro-Canadians raised in a carpentered environment characterized by the preponderance of cardinal contours, as well as in Cree Indians living in a traditional setting presenting more heterogeneous arrays of orientations. A classical matching task was used, subjects having to turn a test-rod until it matched the given standard-orientation. The four standard axes were tested: vertical, horizontal and the two diagonals. Prior knowledge of the tested orientations was given. The expected oblique effect was observed in Euro-Canadians, but it was absent in Cree Indians. In the same vein, Ross (1992) showed that its extent increases in an almost linear fashion between the ages of 7 and 12 years. In haptics, the oblique effect has been studied in school-age children and in infants. In children (from 6 years old to 10 years old), results showed that the oblique effect may be present under certain conditions (like in adults), and that its extent did not change (Gentaz and Hatwell 1995). In infancy, Gentaz and Streri (2002) initially examined whether the 5-month-old babies are capable of haptically discriminating (without visual control) between a vertical rod and a 45° oblique rod positioned in the fronto-parallel plane. The authors offered each baby a 90-s familiarization phase for one oriented rod. After this phase, a discrimination phase was proposed, in which the two orientations were alternately presented. By definition, haptic discrimination was said to occur when there was a significant difference between manual holding times in the discrimination phase for the familiar orientation (the one proposed in the familiarization phase) and for the novel orientation (for a discussion about the direction of preference, see Kerzerho et al. 2008). Results confirmed these predictions and thus showed that 5-month-old babies were capable of haptically discriminating between two orientations (vertical and 45° oblique). Then, Gentaz and Streri (2004) studied whether a haptic oblique effect is present from the age of 5 months. To answer this question, the authors used a method based on a critical angular value, as did Leehey et al.’s works in vision in babies. A haptic oblique effect is said to be present when the discrimination between two orientations does not only depend on their angular difference but also on the value of the tested orientation. In this case, the authors predicted that with the same angular difference (10°), discrimination should be observed between the vertical and a 10° oblique orientation, whereas no discrimination should be observed between a 55° oblique and a 45° oblique. Results confirmed these predictions. These results showed that an oblique effect may be present when the shoulder–arm–hand system of the infants from the age of 5 months could actively move freely in the air to hold and to slightly explore the rod positioned in the frontal plane with the finger movements. This effect could be explained partially by the presence of the gravitational cues provided by the arm–hand system. These cues could reinforce gravitational (vertical) direction as an important axis, which could be used by infants as a reference axis to define spatial orientations, as it is the case in children and in adults (Gentaz and Hatwell 1996). Subjective-reference frame in infancy As in adults, the effect of body tilt on the oblique effect was investigated in the visual and haptic systems in infants. In vision, Jouen (1985) showed a classical oblique effect in the upright body position of 5-month-old infants, with longer looking at the vertical and horizontal gratings than at the 45° and 135° gratings. In contrast, in the tilted body position, infants looked longer at oblique stimuli aligned with their body orientation. These results showed that the visual oblique effect in infants is not defined in a gravitational reference frame but rather in a retinotopic reference frame. Consequently, this suggests that this effect would be directly related to the properties of orientation-selective neurons of the low-levels of the visual system where the retinotopical mapping of most orientation-selective neurons is preserved (cf. “Introduction”). In the haptic modality, Kerzerho et al. (2005) investigated the effect of whole body tilt on the haptic oblique effect by using the same method as Gentaz and Streri (2004). Two postural conditions were tested: upright body and body tilted 20° to the left. Two orientations (vertical or +20°-left oblique), defined gravitationally, were proposed in the familiarization phase and four (vertical, 10°, 20° or 30°-left oblique) in the test phase. The results showed that the body tilt had an effect on the infant’s haptic discrimination of orientations. The understanding of this effect was complex, because it acted on both the spatial orientation discriminated and the direction preference of holding times. The pattern of results observed in the upright body position—the presence of a discrimination between vertical and 10°-left-oblique rods and a failure between 20°-left oblique and 30°-left oblique rods—confirmed Gentaz and Streri’s (2004) previous observations and were in line with those found in vision by Held and his colleagues. However, the pattern of results observed in the tilted conditions showed the presence of discrimination between vertical and 10°-left-oblique rods and between 20°-left oblique and 30°-left oblique rods. In other words, the haptic oblique effect found in the upright posture disappeared when the body was tilted. This effect of the body tilt on the patterns of results clearly showed that the hypothesis of a purely gravitational reference frame underlying the haptic oblique effect is not supported. Similarly, the results do not favor a purely egocentric reference frame, since a successful haptic discrimination was observed in the body tilted condition both (1) between the 20° oblique rod that was vertical by reference to the body axis in the 20° tilted position and the 30° oblique rod and (2) between the gravitational vertical rod and 10° oblique rod. In summary, the results suggest that spatial orientations are not defined only in a single reference frame. The results indicated that the haptic discrimination of the gravitational vertical (with the 10° oblique) occurred in the two body positions. This result suggests that in tilted position, gravitational vertical seemed still to play the role of a reference axis at least partially. In contrast, the haptic discrimination of the 20° oblique rod depended on the body position. It was absent in the upright position when the 20° oblique rod was oblique by reference to the body axis, whereas it was present in the 20° tilted body position when the 20° oblique rod was vertical by reference to the body axis. This result suggests that, in the tilted position, the egocentric vertical also seemed to have the role of a reference axis, at least partially. In conclusion, these results support the hypothesis of a reference frame, which integrates not only gravitational information but also egocentric information. As indicated before, this mixed-reference frame hypothesis underlying the orientation perception has already been proposed in the haptic (Kappers 2004) and visual (Lipshits et al. 2005; Lipshits and McIntyre 1999; Luyat et al. 2005a) systems in adults. General discussion and perspectives This review of experimental studies of the haptic oblique effect has showed that the perception of spatial orientations can be isotropic or anisotropic depending on various factors such as the presence of gravitational cues, the plane in which orientations are presented, the modality of response (ipsilateral or contralateral hand), etc. One of the most striking findings is the absence of an oblique effect in some conditions, which demonstrates that the haptic system can process all orientations isotropically. This observation stands in sharp contrast with vision where an oblique effect is observed much more systematically, probably because some form of directional anisotropy already emerges at the lowest levels of the visual system. Our general interpretation of these studies is that the haptic oblique effect is a classic Class 2 oblique effect, which occurs relatively late in the processing of sensory information. More precisely, we believe that these studies globally indicate that the oblique effect occurs when the sensory motor traces associated with the exploratory movements are transformed into a more abstract representation at the cognitive level, where presumably the perceived orientation is related to a frame in reference that favors the vertical and horizontal orientations. While the high-level representation would introduce some degrees of anisotropy, it would also be less taxing from a memory point of view and be more robust to motor interference tasks. In this framework, gravity would facilitate the transformation from the low-level to the high-level representation by adding information about the direction of the movement relative to the vertical in the sensory-motor traces. In absence of these cues, the oblique effect would be smaller or absent, because the axes of the reference frames would be known with less certainty. In addition, studies conducted in the haptic and visual modalities where the geo-centered and ego-centered reference frames was dissociated by tilting the body have shown that the axes of this reference frame were influenced by vestibular and somesthetic inputs. Thus, these findings suggest that orientations might be represented in a frame of reference based on the subjective vertical and horizontal, that mixes ego- and geo-centric cues. While it is tempting to postulate that this subjective reference frame is shared between the two sensory modalities, a detailed analysis of the results revealed a lack of correlation between the sizes of the oblique effect obtained in each modality (Luyat et al. 2001; see detailed review in “Haptic orientations defined in a subjective reference frame’’). Additional studies are necessary to determine if this lack of correlation reflects only a difference of degree of anisotropy present in the visual and haptic processing of orientations or if it is due to a different positioning of the subjective axes in the modalities. In either case, the absence of a purely gravitational or egocentric frame of reference is a strong argument in favor of a high-level haptic oblique effect. This review shows that gravity is a sufficient but not necessary condition for the occurrence of an oblique effect in the haptic modality. As a matter of fact, the haptic oblique effect can occur in absence of any gravitational cues. Moreover, the haptic oblique effect can also be strengthened by factors other than gravity. For example, some studies have shown the presence of an oblique effect with reduced gravitation cues when different hands were used in the exploration and reproduction phases (e.g., the contralateral condition in Appelle and Countryman 1986). To understand this result, it should be noted that the reproduction by the other hand of an oblique requires a complex transformation of the sensory-motor trace of the scanning movement given the symmetric arrangement of the two upper limbs. It is therefore plausible that the transformation from the low-level to the high-level representation is forced in this case, because the sensory-motor trace associated with the scanning arm is of little use for the reproduction phase and the haptic system must rely on the more abstract representation of the orientation (see Baud-Bovy and Viviani 1998 for a similar hypothesis in pointing context). Other studies have shown that an interference task during the delay between the presentation and the recall of the orientation (with the same hand) is responsible for an oblique effect in reduced gravity conditions or that it strengthens in normal conditions (Gentaz and Hatwell 1999). The idea is that higher attentional or cognitive demands decrease the amount of resources available to the haptic system. Such a situation would induce the transformation of sensory-motor trace or give more weight to a more abstract representation of the stimulus. Work in neighboring fields is consistent with this hypothesis. For example, Rossetti et al. (1996) showed that in proprioceptive pointing tasks, memorization leads participants to stop using sensory-motor representation to produce a movement and commits them to using a semantic spatial representation. In haptics, a similar shift in perception of parallelity was also observed by Zuidhoek et al. (2003). These authors tested the effect of a delay between the perception of a reference bar and the parallel setting of a test bar in the horizontal plane. They observed that a 10-s delay significantly improved performance. They suggest that a shift from the egocentric towards the allocentric reference frame during the delay period would lead to different spatial processing (for discussion, see also Faineteau et al. 2005; Gentaz and Gaunet 2006). The nature of the more abstract representation and underlying processes is still an open question. One possibility is that the orientations would be mapped to one or more topologically organized layers of processing units, and that the perceived orientation would correspond to the most active units in one of these layers. The DFT of spatial cognition developed by Spencer and colleagues assumes such representational scheme (Spencer et al. 2006, 2007). According to this theoretical framework, the stimulus would propagate from perceptual maps to the working-memory map, where the oblique effect would progressively occur under the influence of neighboring units and of the long-term memory map. While it is out of scope to enter into the details of this model here, it is important to note that the dynamic properties of these maps could explain the presence of a systematic bias toward or away from the reference axes as well as the increase of precision observed along the reference axes. Work on this model has so far focused on the visual modality and has not yet addressed issues specific to the haptic modality, such as the role of gravity or the coding of an orientation in space. In particular, the problem of extracting an initial representation of the orientation from the complex afferent and efferent signals that correspond to the sensory-motor trace of the scanning movement seems daunting, but this observation also holds true for all other explanations of the haptic oblique effect. Another possibility is that the orientation would be represented in categorical terms (Huttenlocher et al. 1991; see also the work on Categorical Perception; Harnard 2003). As described in “Hypotheses on the origins of the oblique effects”, the Category-Adjustment model assumes the existence of a double representation of the stimulus. According to the CA model, the oblique effect occurs at a late stage of processing, when both representations are combined, with possible different weights, to produce the response. From a theoretical point of view, such a double representation can be advantageous when the reliability of the low-level representation decreases, though it might introduce biases. In this theoretical framework, variations of the strength of the oblique effect could be understood in terms of a relative reliability of abstract representation of the sensory motor traces. The idea of a double representation could also be useful to explain the complex error patterns observed in the reproduction of 3D orientations showing that a participant responded using a heterogeneous set of cues that included the low-level sensory-motor trace as well as some more abstract-coding scheme based on the two angles between the orientation and the sagittal or frontal plane (Baud-Bovy and Gentaz 2006). However, a problem with the CA model is that it predicts an increase of the variability of response at the category boundaries (G. Baud-Bovy, 2008, A critical commentary of the category adjustment model and the oblique effect. Psychol Rev, unpublished). This observation is problematic insofar that it is usually assumed that the category boundaries correspond to the horizontal or vertical orientations. A possible remedy would be to assume the existence of narrow categories around the vertical and horizontal orientations in addition to the usual oblique categories centered on the diagonals. To conclude, it is important to note that both the DFT and the CA model were originally conceived to explain the presence of systematic biases toward the closest diagonal in various location-recall or position-recall tasks in the visual modality, which has not yet been observed in the haptic modality. As a matter of fact, the analysis of the constant errors in studies of the haptic oblique effect did not reveal systematic directional errors. This observation holds true whether the reproduction method (Appelle and Countryman 1986; Gentaz and Hatwell 1995, 1996, 1998, 1999; Lechelt et al. 1976; Lechelt and Verenka 1980) or the production method (Appelle and Gravetter 1985; Gentaz et al. 2002; Luyat et al. 2001) was used to assess the perceived position of the orientation in the haptic modality. This negative result does not, however, invalidate these models, since the aforementioned studies of the haptic oblique effect have used a limited set of stimuli (the vertical, horizontal and the two main diagonals) that precludes the observation of a bias toward the closest diagonal, since it can be observed only with oblique orientations that differ from the diagonals. The question of the accuracy of the perception, as opposed to its precision, has been so far neglected in studies of the haptic oblique effect and needs to be further investigated in future studies.

Virchows_Arch-4-1-2335297

Epithelial atypia in biopsies performed for microcalcifications. Practical considerations about 2,833 serially sectioned surgical biopsies with a long follow-up

This study analyzes the occurrence of epithelial atypia in 2,833 serially sectioned surgical breast biopsies (SB) performed for microcalcifications (median number of blocks per SB:26) and the occurrence of subsequent cancer after an initial diagnosis of epithelial atypia (median follow-up 160 months). Epithelial atypia (flat epithelial atypia, atypical ductal hyperplasia, and lobular neoplasia) were found in 971 SB, with and without a concomitant cancer in 301 (31%) and 670 (69%) SB, respectively. Thus, isolated epithelial atypia were found in 670 out of the 2,833 SB (23%). Concomitant cancers corresponded to ductal carcinomas in situ and micro-invasive (77%), invasive ductal carcinomas not otherwise specified (15%), invasive lobular carcinomas (4%), and tubular carcinomas (4%). Fifteen out of the 443 patients with isolated epithelial atypia developed a subsequent ipsilateral (n = 14) and contralateral (n = 1) invasive cancer. The high slide rating might explain the high percentages of epithelial atypia and concomitant cancers and the low percentage of subsequent cancer after a diagnosis of epithelial atypia as a single lesion. Epithelial atypia could be more a risk marker of concomitant than subsequent cancer. Introduction Breast biopsies for infraclinical lesions are more frequent with mammographic screening programs, but the distribution of the corresponding histological lesions and their associations are still imprecise. Difficulties encountered in following up patients without cancer account for the fact that the clinical significance of certain non-malignant lesions and the management of patients are still debated. Moreover, the problem of surgical biopsy sampling has never been fully investigated and has added additional confusion in appreciating the distribution and clinical significance of such lesions. In 1981, breast epithelial atypia were hardly mentioned and not clearly defined in the World Health Organization/International Union Against Cancer (WHO/UICC) histologic classification of breast tumors [73]. The histologic classification of noncancerous lesions has been mainly based on studies analyzing for each lesion the associated risk of subsequent cancer. These studies were initiated by the survival studies of Dupont and Page [15, 46] based on lesions discovered by palpation before the era of mammography. Thereafter, further studies [6, 9, 17, 24, 31, 51, 62, 67] substantiated these results, which were ratified in 2003 by the new American Joint Committee on Cancer (AJCC)/UICC classification of breast tumors [70]. Schematically, this classification differentiates benign epithelial lesions (usual ductal hyperplasia and other lesions) from atypical lesions of ductal or lobular type. Although this historical classification is challenged by a new classification [68], it remains the most widely used in practice. Interestingly, the occurrence of epithelial atypia was low in Page’s study [46] and has increased with mammographic screening programs [60, 66] and with the development of percutaneous large core needle biopsy (CNB) methods using stereotactic mammography or ultrasound guidance. At present, CNB is frequently used for the initial evaluation of clinically occult breast lesions, thus generating dilemma for the subsequent management of certain noncancerous lesions. At our institution, surgical biopsies (SB) have always been managed in the same way, and most patients with atypical and malignant lesions have been followed. The objectives of our work were to analyze the occurrence of epithelial atypia and their association with a concomitant cancer in a large series of SB performed for microcalcifications without a palpable tumor and to assess the subsequent cancer probability in the group of patients with an initial diagnosis of epithelial atypia. Finally, we provide some practical considerations for the management of patients with epithelial atypia in this era of mammographic screening and CNB. Materials and methods Selection of patients At Institut Bergonié, from January 1975 to December 2002, 3,166 breast biopsies for diagnostic purposes, 2,833 SB and 333 CNB, were performed for microcalcifications without any palpable mass in 2,708 patients (mean age 51.8 years, range 19.7–81 years). Among them, 248 (9%) had several biopsies in the same or contralateral breast. Since 1998, microcalcifications have been classified according to the classifications of the American College of Radiology [2]. SB for diagnostic purposes were defined before 1998 by the absence of a preoperative diagnosis based on the clinical–mammographic–cytologic triplet and by the absence of a positive frozen section and, since 1998, by the presence of epithelial atypia on CNB. Excluded from this study were 132 cancers and 139 non-atypical benign lesions diagnosed on CNB as well as 49 re-excisions performed elsewhere than in our center. Thus, 2,833 SB in 2,375 patients were available for analysis, among which 13 corresponded to re-excision after a CNB with epithelial atypia. Since 1989, needle localization, intraoperative specimen radiography, and post-excisional biopsy mammography have been performed in most cases. Surgical biopsies and tissue sampling: serial macroscopic sectioning SB was removed in one fragment and measured more than 3 cm in 94% of the cases (mean size 60 mm, 5–250 mm). For SB margin assessment, either the surface of the specimen was inked or the surgeon during the same operation removed additional tissue in the remaining cavity after excision of the specimen (surgical margins). After fixation in Holland Bouin, SB and margin specimens were serially sectioned in their entirety into numbered slices every 2 mm [12]. In most cases (89%), careful macroscopic examination of the specimen failed to reveal any lesion. Each numbered slice was put in as many numbered separate cassettes as necessary and paraffin-embedded in sequence. The median number of blocks per SB was 26 (from 2 to 180) and 8 (from 1 to 44) for surgical margins. Each block was examined on one hematoxylin–eosin–safran stained slide. Classification of lesions and review of slides Since 1975, all patients have been prospectively included in our clinical, histologic, and biologic database by senior pathologists (IM, GMG, IS, JMC). For each SB and each lesion, we prospectively entered in our pathologic database morphological descriptive criteria by using 65 pathological items for noncancerous lesions and 181 for cancers. Definitions and terminologies given in the literature were used to report columnar cell lesions (CCL), non-atypical ductal hyperplasia, atypical ductal hyperplasia, ductal carcinoma in situ (DCIS), and lobular carcinoma in situ (LCIS) [1, 4, 7, 10, 17, 19, 21–28, 38, 43–45, 47, 49, 50, 56, 59, 61, 69, 72, 74, 75]. The interest of our database was to collect morphological descriptive criteria of nearly all the breast lesions without labeling them. In fact, labels and definitions of breast lesions have varied throughout the past 30 years, while neither lesions nor their corresponding descriptive criteria (i.e., size, type, architecture, cellular and nuclear features, etc...) have changed. The only changes during this period were the definitions and/or the names given to these lesions. As we have listed for each lesion all the corresponding descriptive criteria among the 236 available items, we have been able to reclassify each lesion according to the “new” criteria recommended by referent authorities for a new definition, by selecting in our database the “new” correspondent descriptive criteria corresponding to this new definition. Consequently, this provided a homogeneous approach to the pathological lesions at the time of our study. For example, low-grade DCIS ≤ 2 mm have been reclassified as atypical ductal hyperplasia/ductal intraepithelial neoplasia (ADH/DIN) 1B (n = 30) according to the new AJCC/UICC classification of breast tumors [70], and lesions that we used to term before 1997 [71] as clinging carcinoma of the monomorphic type [4] have been reclassified as flat epithelial atypia (FEA)/DIN 1A or columnar cell change (CCC) with atypia (n = 84) [61]. About half of these 114 cases have been systematically reviewed by one (IM) or two senior pathologists (IM and GMG or IS), and there was a complete concordance between the second review and the initial descriptive criteria listed in the database. Similarly, lesions that we used to name LCIS before 1997 have been renamed lobular neoplasia (LN) since 1997, corresponding either to atypical lobular hyperplasia (ALH) or to LCIS. On the contrary, all the cases with micropapillary lesions were reviewed (n = 155) because there was no item corresponding to precise descriptive criteria of micropapillations (number, topography around the duct, type). Atypical ductal hyperplasia: definition and sizing Among the group of ADH/DIN 1B, we individualized two morphologic types of ADH. Neither had any high-grade cytological atypia or necrosis. ADH “mimicking DCIS” (Fig. 1). In this type, architectural atypia were qualitatively insufficient to allow a diagnosis of DCIS, therefore this “mimicking” DCIS lesion was classified as ADH whatever its size. Tufts and short micropapillations formed by cells had a broad base and were cohesive. There was no polarization of cells, i.e., no true cribriform spaces. Pseudo-cribriform patterns comprised irregular or relatively round microlumina with incomplete polarization of surrounding epithelial cells. Cellular bridges were wavy without any cellular polarization. Cells corresponded either to columnar cells with uniform ovoid to elongated nuclei or to cells with a slight increase in the nuclear/cytoplasmic ratio with more or less distinct cell borders and round or ovoid nuclei. These cells were sometimes admixed in the same lesion displaying a morphological gradient, but there was no regular arrangeent. Nuclear chromatin was evenly dispersed, homogeneous, or slightly marginated, and nucleoli were inconspicuous. Apical snouts, intraluminal secretions, and psammoma-type calcifications were frequently present. Fig. 1a–d. ADH “mimicking DCIS.” a Tufts and short micropapillations with a broad base. b Pseudo-cribriform spaces. c Microlumen with incomplete polarization of surrounding epithelial cells. d Cellular bridges without cellular polarization. Cells are parallel to the axes (arrows) ADH corresponding to “mini DCIS” (Fig. 2). In this type, architectural and cytologic atypia corresponded to a low-grade DCIS but were quantitatively insufficient to allow a diagnosis of DCIS, therefore this “mini” DCIS lesion was classified as ADH when ≤2 mm. Tufts and short micropapillations had a tight base, were present on over all the periphery of the duct, and were non-cohesive with small free papillary tufts in the lumen. There were true cribriform patterns with a polar organization of cells around glandular spaces and/or variants of cribriform patterns (i.e., trabecular bars, cartwheel formations, and Roman bridges, Fig. 3) with polarized cells arranged perpendicular to the axes. Some solid areas with regular arrangement of cells were also present. Cells were often small, monomorphous, sometimes without a columnar change, with a distinct cytoplasmic membrane and a spaced regular round nucleus with uniformly dispersed chromatin without prominent nucleoli. Intraluminal secretions and calcifications (amorphous or psammoma-type) were also frequently present (Fig. 4). When one mini DCIS focus was found in one partially or completely involved duct/ductular cross-sections in one terminal ductal lobular unit (TDLU), it was classified as ADH when it measured ≤2 mm and as DCIS when it measured >2 mm. When there were several foci of “mini” DCIS in close duct/ductular cross-sections in the same TDLU or in TDLUs located in the same field at low power magnification (2.5), the lesion was classified as ADH when its size, i.e., its largest diameter, was ≤2 mm and as DCIS when >2 mm. When there were several foci of “mini” DCIS in distant duct/ductular cross-sections in the same TDLU or in close TDLUs, the size of each focus was assessed separately. FEA, rare and scattered single micropapillations, and cribriform variants were not taken into consideration for sizing, even if located in the same TDLU. Fig. 2a–e. ADH corresponding to “mini DCIS.” a A solid mini DCIS focus measuring less than 2 mm in one TDLU. b Tufts and short micropapillations over the entire periphery of the duct with small free papillary tufts in the lumen. c Short micropapillations with a tight base. d True cribriform spaces. e Microlumen with complete polarization of surrounding epithelial cellsFig. 3a–c. Variants of cribriform patterns. Polarized cells arranged perpendicular to the axes. a Trabecular bars. b Cartwheel formations. c Roman bridgesFig. 4a and b. a Mild cytologic atypia. b Columnar cells with uniform ovoid nuclei, intraluminal calcifications FEA were present either as a single lesion or in association with ADH in the same TDLU and since 1997 have been included in the ADH group. The distinction of FEA from columnar change without atypia was based on the criteria given by the WHO for the definition of FEA. Furthermore, columnar change without atypia was characterized by one or two layers of columnar cells without nuclear atypia, i.e., no increase in the nuclear/cytoplasmic ratio, no prominent nucleoli. Nevertheless, some cases of columnar change with progesterone impregnation, especially in the second part of the cycle, might display a lobular distension with a secretory material and large nuclei with prominent nucleoli. In such cases, myoepithelial cells displayed the same alterations with clarified cytoplasms, thus facilitating the diagnosis. The distinction of ADH mimicking DCIS from usual hyperplasia (UDH) was based on morphological criteria. Architectural pattern and cytologic criteria of usual ductal hyperplasia were easy to identify in most cases. UDH corresponded to a proliferation of epithelial cells in solid or fenestrated areas without any polarization of surrounding cells. Cells were haphazardly arranged with overlapping nuclei or were parallel with characteristic streamings. They were elongated or pseudo epithelioid, but there was no columnar metaplasia. Cytoplasms were more or less abundant with indistinct borders. Nuclei had irregular size and shape and sometimes contained a prominent eosinophilic inclusion. In some rare cases, immunohistochemical staining with cytokeratin 5/6 [41] was used and was negative in ADH mimicking DCIS and strongly positive in UDH. In some lesions, differential diagnosis between ADH and low-grade DCIS was all the more difficult because there were intermediate and intricated morphological aspects in the same TDLU. In practice, diagnosis of micropapillary lesions was often difficult. Extensive micropapillary lesions were classified as DCIS when quantitative and qualitative criteria were simultaneously present, i.e., lesion sizing more than 2 mm corresponding to micropapillations with a tight base over the entire periphery of the ducts. Additional sections could be useful for demonstrating more or less qualitative or quantitative diagnostic criteria. When malignancy remained equivocal, the case was classified as ADH. When a concomitant cancer was diagnosed, histologic size was assessed, and in DCIS, the percentage of blocks with cancer (“positive blocks”) was specified [13]. Presence and topography of microcalcifications were also assessed. Lastly, when FEA and/or “mimicking” DCIS foci were found on excision margins of a SB with DCIS, a further surgical resection was not performed. Follow-up of patients with epithelial atypia as a single lesion There were 443 patients with epithelial atypia in one or several SB, without any previous or synchronous carcinoma in the same or contralateral breast and treated by biopsy alone (median follow-up 160 months, 7 to 315). Only 28/443 (6%) were lost to follow-up. Among the 415 other patients, 180 were monitored at our institute and 235 outside by correspondent specialists working in close relationship with our institute. All patients received a clinical examination and mammography once a year. When a new biopsy was necessary, it was performed at our institute. Statistical analyses Comparison of clinical and histologic characteristics was conducted by using the chi-square test. For women with epithelial atypia, the probability of developing in situ or invasive cancer was calculated from the date of the first biopsy to the earliest event: breast cancer (ipsi- or contralateral), death, or last contact (last consultation for the group monitored at our institute and checkpoint date, i.e., 1 March 2004, for the others). Probabilities were calculated according to the Kaplan–Meier method (SPSSv11). Results Occurrence of epithelial atypia in the 2,833 surgical biopsies Epithelial atypia were recorded in 971/2,833 SB (34%). They were found with and without a concomitant cancer in 301/971 (31%) and 670/971 (69%) of the cases, respectively. Thus, isolated epithelial atypia were found in 23% of the cases (670 out of the 2,833 SB). Calcifications were present at histologic examination in 98.6% of SB with cancer and were located in benign, cancerous, and both lesions in 10, 39, and 51% of the cases, respectively. In several cases, cancerous foci without any microcalcifications were located at points distant from those with calcifications detected by needle localization. Types of epithelial atypia Among the 971 SB with epithelial atypia, there were 101 SB with FEA as a single lesion (11%), 342 (35%) with ADH, 223 (23%) with LN, and 305 (31%) with ADH and LN. Thus, ADH was encountered in 647/971 SB (66%). Types of cancers associated with epithelial atypia Cancers associated with epithelial atypia corresponded to DCIS and micro-invasive carcinoma (DCIS-MI) in 233 cases (77%). Among invasive carcinomas (n = 68), there were 13 (9%) lobular and 11 (6%) tubular carcinomas (Table 1). Cancers were small (≤5 mm in 46% of invasive carcinomas, fewer than half of the blocks positive in 76% of DCIS). They were non-high grade in 78 and 67% of DCIS and invasive carcinoma, respectively. In most cases, ADH and cancer were situated close to each other. FEA alone were less frequently associated with a concomitant cancer than ADH and/or LN (p = 5 × 10−4). Table 1Types of concomitant cancers (n = 301) in the 971 surgical biopsies with epithelial atypiaEpithelial atypiaFEA (n = 101)ADH (n = 342)LN (n = 223)ADH + LN (n = 305)No. of cases (%)No. of cases (%)No. of cases (%)No. of cases (%)Without cancer8483220641396222774With cancer17171223684387826DCIS/DCIS-MI12121033058266020IDC/NOS––164.7178113ILC1110.36352TC44213121FEA Flat epithelial atypia; ADH atypical ductal hyperplasia; LN lobular neoplasia; DCIS ductal carcinoma in situ; DCIS-MI DCIS with micro-invasion; IDC infiltrating ductal carcinoma; ILC infiltrating lobular carcinoma; TC tubular carcinoma Cancers without epithelial atypia (malignancy alone) There were 821 malignant SB without epithelial atypia [590 micro-invasive carcinomas, 206 infiltrating ductal carcinomas (IDC), and 25 infiltrating lobular carcinomas (ILC)]. Subsequent cancer in patients with an initial diagnosis of epithelial atypia as a single lesion At 5 and 10 years, the probabilities of developing invasive breast cancer in the group of 443 patients with epithelial atypia were 2.8% [95%CI = 1.4 to 5.5] and 5.5% [95%CI = 3.3 to 9.9], respectively (Fig. 5). Among the 18 subsequent carcinomas, 15 were invasive (11 IDC and 4 ILC), and 3 corresponded to DCIS. Most subsequent carcinomas were encountered in the homolateral breast (n = 14) and before 10 years (n = 16). Seven carcinomas occurred in the group of patients with an initial diagnosis of LN, in the same (n = 5) or contralateral (n = 2) breast. They corresponded to infiltrating ductal (n = 6) or lobular (n = 1) carcinomas. The interval of development was 4, 5, 6 (n = 2), 9, and 12 (n = 2) years. Seven carcinomas occurred in the group of patients with an initial diagnosis of ADH, in the same (n = 3) or contralateral (n = 4) breast. They corresponded to DCIS (n = 2) and to infiltrating ductal (n = 4) or lobular (n = 1) carcinomas. The interval of development was 1, 2, 3 (n = 3), 9, and 12 years. Four carcinomas occurred in the group of patients with an initial diagnosis of ADH associated with LN, in the same (n = 3) or contralateral (n = 1) breast. They corresponded to DCIS (n = 1) and to infiltrating ductal (n = 2) or lobular (n = 1) carcinomas. In the four cases, the interval of development was 4 years. There was no subsequent carcinoma in the group of patients with FEA. Fig. 5Probability of developing subsequent invasive breast cancer in the group of 415 patients with epithelial atypia (dotted line: confidence interval 95%) Discussion Application of the WHO classification: practical considerations For a long time, DCIS was diagnosed even if the characteristic features were found in only one ductal space [6]. Thereafter, some authors introduced quantitative criteria for distinguishing between ADH and DCIS [46, 67], while others [23] rejected them. More recently, Rosen [57–59] and Schnitt and Vincent-Salomon [61] described CCL comprising CCC and columnar cell hyperplasia (CCH) with and without atypia. Nasser [40] challenged this classification based on columnar shape and limited the group of lesions to proliferations characterized by a low-grade atypicality, “atypical columnar cell lesions,” (ACCL) rather by a columnar cell configuration. In the WHO classification, ADH includes various not clearly defined types of lesions (Table 2). On one hand, there are lesions with arcades, moundings, and micropapillary formations, but without any true cribriform/complex architectural patterns [34]. This type of ADH corresponds to the CCH with atypia of Schnitt and Vincent-Salomon [61] termed category 3 in Simpson’s study [51], to the definition of ADH by Koerner [34] and to ADH “mimicking” DCIS in our study. On the other hand, there are lesions displaying architectural and cytologic atypia. This type of ADH corresponds to the complex architectural pattern with cytologic and architectural atypia of Schnitt and Vincent-Salomon [61] termed category 5 in Simpson’s study [64], to the definition of “microfocus of DCIS” by Koerner [34] and to “mini DCIS” in our study. In Simpson’s study, the ADH/category 5 contained chromosomal changes and the same total mean number of changes to that observed in DCIS/DIN IC, unlike the other CCL. Lastly, because there is still no consensus for measuring ADH, there is no clear-cut distinction between ADH and DCIS, and the cut-off at 2 or 3 [52] mm or at two completely involved spaces [70] seems arbitrary. While awaiting a definitive molecular classification, the simplest attitude could be recommended in routine practice. Only mini foci of ADH obviously similar to low-grade DCIS foci could be measured and classified as ADH when equal to or less than 2–3 mm [52] and as DCIS when more than 3 mm. Although the mode of measurement in our study is not under consensus, it is simple and can been routinely applied. Table 2Terminologies used for intraductal proliferative lesions with low-grade cytologic atypia, so-called atypical columnar cell lesionsSpectrum of lesions1, 3–5 LayersNo polarization*With polarization Occasional mounding Mounding, arcades Cribriform spaces and their variants No or rare arcades and micropapillary formations Cohesive micropapillary tufts with a broad base Non-cohesive micropapillary tufts with a tight base[70]Flat epithelial atypia/DIN 1AADH/DIN 1B ≤ 2 mm; or in two spacesDCIS/DIN 1C[59]Columnar cell hyperplasia with atypiaADH if not extensiveDCIS if extensive[61]Columnar cell change (CCC) with cytologic atypiaComplex structures with architectural and cytologic atypia[34]Columnar cell lesions + ADHMicroscopic focus of DCISDCISInstitut BergoniéEx-clinging carcinoma of monomorphic typeADH “mimicking” DCISADH corresponding to “mini” DCIS ≤ 2 mmDCIS[64]CCC with cytologic atypiaCCH with architectural atypiaCCH with architectural atypiaDCISOrAndCCH with cytologic atypiaCytologic atypiaRegrouping?DIN 1AADH/DIN 1BADH/DIN 1CDCIS/DIN 1CNot measuredMeasured: ≤3 mm>3 mm*Or incomplete polarization Occurrence of epithelial atypia and their association with a concomitant cancer: practical considerations In our study, the proportion of epithelial atypia is high (23%), a result difficult to compare to others in the literature, as the methodologies used by teams are different. In the Page and Dupont case-control studies [15, 46], ADH and ALH were found in 2.1 and 1.6% of the cases, respectively. In mammographic screening programs, epithelial atypia and cancers increase as the number of biopsies performed for microcalcifications increases, especially as ACR4/ACR5 lesions are more often excised than ACR3. However, as underlined by Page [46], “the most direct relationship of epithelial atypia incidence is to slide rating.” The number of slides per SB in our study (median 26) was higher than in the other studies on benign breast lesions: 1–5 in 93% of the cases in the study of Page et al. [46] (n = 283), 3 (range 1–25, n = 674) in the study of Shaaban et al. [62], and a mean of 1.6 slides per cm of tissue (n = 199) in the study of Tavassoli and Norris [67]. In a recent study conducted in the south west of France in women aged between 50 and 75 with mammographically detected non-palpable breast lesions, a similar proportion of atypical lesions were found when biopsies were serially sectioned [39]. Furthermore, this high slide rating allowed the detection of small concomitant cancers in the vicinity of epithelial atypia in 31% of our cases, with a skew towards low-grade lesions (high proportions of DCIS and low-grade invasive carcinomas, especially tubular carcinomas). Our results strengthen the hypothesis that FEA and ADH are risk markers of low-grade cancers. This has been confirmed by the study of Simpson et al. [64] on molecular genetic profiles of CCL. In some of them, there are both a morphological and a molecular continuum in the degree of proliferation and atypia, supporting the hypothesis that “CCL are a non-obligate, intermediary step in the development of some forms of low grade in situ and invasive carcinoma.” The association of epithelial atypia with a concomitant cancer in nearly one third of the cases in our study parallels previous findings concerning the frequency of cancers found in SB performed for atypia in CNB. Thus, approximately 30% [20, 29] and 15 to 21% [5, 8, 14, 18, 35, 45, 53, 63, 76] of excisions after CNB with ADH and LN, respectively, were proven to have cancer. Consequently, excision is recommended [45] for all patients in whom ADH is identified on CNB and may be justified in patients with FEA, as they are included in the spectrum of ACCL. Excision remains a controversial issue in patients with LN. Some authors have advocated it [3, 18, 33, 63], while others have rebutted it [55], especially when LN is an incidental non-extensive finding [48] with no radiologic–pathologic discordance [18] and without any synchronous mass lesion [37]. SB corresponding to re-excision should be processed in its entirety by serial macroscopic sectioning [32, 65]. When pathologic examination is exclusively focused on mammographic calcifications, the risk is to underestimate the DCIS size/extension because cancerous foci without any calcification (10% in our study vs 6% in Owing’study) [42] may be located at points distant from those with benign breast tissue containing calcifications. Subsequent cancer after an initial diagnostic of epithelial atypia as a single lesion: practical considerations In the literature, 4 to 22% (average interval 8.3 years follow-up) [6, 42, 67] and 15 to 20% [16, 30, 56] of patients developed invasive carcinomas after a diagnosis of ADH and LN, respectively. The risk of developing cancer increases with extended follow-up, but many cancers after a diagnosis of LN have a good prognosis and a low mortality [36]. These results are difficult to compare to ours because the methodologies are different. The low probabilities of subsequent invasive cancer in our study could be due to the high slide rating, allowing the detection of small concomitant cancer that might have been missed with a low slide rating and inadequate patient management [11, 54]. In conclusion, when epithelial atypia are present, they are associated in nearly one third of the cases with a concomitant close cancer and are found as isolated lesions in nearly 23% of SB performed for microcalcifications. In practice, ADH should be more clearly defined with simple guidelines for measuring lesions. When malignancy remains equivocal and/or when sizing is difficult, it is better to classify the lesion as ADH. Epithelial atypia could be more a “risk factor” of a concomitant geographically small close cancer than a risk marker for a subsequent cancer, as they form part of a spectrum of lesions [64].

J_Antimicrob_Chemother-1-1-2386080

HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4°C

Background Dried blood spots (DBSs) are an attractive alternative to plasma for HIV-1 drug resistance testing in resource-limited settings. We recently showed that HIV-1 can be efficiently genotyped from DBSs stored at −20°C for prolonged periods (0.5–4 years). Here, we evaluated the efficiency of genotyping from DBSs stored at 4°C for 1 year. Introduction The availability of clinical specimens that can be easily collected, stored and transported is advantageous in areas that lack appropriate infrastructure for blood processing. Whole blood samples collected by finger stick and dried onto a filter paper [dried blood spots (DBSs)] represent an attractive alternative to the conventional collection of blood in tubes. DBSs have been extensively used for HIV-1 antibody testing,1 molecular diagnostics2 and virus load (VL) quantification3–5 and are now considered a convenient alternative to plasma for HIV-1 drug resistance testing. We recently found that drug resistance genotypes generated from DBSs were similar to those derived from plasma in antiretroviral-naive and -experienced patients.6,7 We also found that resistance testing from DBSs can be as sensitive as with plasma when using a genotypic assay that amplifies a large (1.8 kb) pol fragment.6 The high success of amplification was noted using DBSs stored at −20°C in the presence of desiccant, suggesting that −20°C may represent a feasible storage temperature for DBSs. Although these findings were encouraging, storage at −20°C is not always possible in resource-limited settings where 4°C or room temperature may represent a more realistic alternative. Studies on HIV-1 VL determinations using DBSs have reported the efficient amplification of short HIV-1 RNA sequences from filter papers stored at 4°C or room temperature.3,8,9 However, genotypic assays usually rely on the amplification of large pol fragments and are thought to be particularly sensitive to HIV-1 RNA degradation that may occur under suboptimal storage conditions. Here, we report on the efficiency of amplification and drug resistance genotyping from DBSs stored at 4°C for 1 year. Methods Preparation and storage of DBSs We prepared a total of 40 DBSs from residual diagnostic specimens collected from highly antiretroviral-experienced HIV-1 subtype B-infected persons. A more detailed description of the study population including antiretroviral drug treatment has been reported elsewhere.6 Drug resistance genotypes were available for the matched plasma specimens.6 The median plasma VL in these patients was 13 680 RNA copies/mL (range 518–676 694, Versant HIV-1 RNA 3.0 Assay, Bayer HealthCare Diagnostic Division, Tarrytown, NY, USA) (Table 1). DBSs were prepared by pipetting 50 µL of whole blood onto pre-marked circles on 903 filter paper cards (Schleicher & Schuell, Keene, NH, USA). Cards were dried overnight at room temperature (25°C), placed in a gas-impermeable, sealable plastic bag (Fisher Scientific Company, Pittsburgh, PA, USA) containing a silica gel desiccant (Mini Pax Sorbent, Multisorb technologies, Buffalo, NY, USA) and stored at 4°C until they were shipped to CDC in dry ice. Upon arrival, individual plastic bags were put in a larger bag containing three extra desiccants and stored at 4°C. All desiccants were evaluated for humidity at 6 months; 21 of the 40 bags had evidence of humidity as indicated by the colour indicator in the desiccant. Therefore, a new desiccant was added to each individual bag after allowing the spots to equilibrate at room temperature for 30 min. None of the specimens had mould after 12 months of storage at 4°C. Table 1 Frequency of amplification of HIV-1 pol from plasma or DBSs stored at −20°C for 6 months or at 4°C for 12 months Specimen ID Plasma VL (log10 copies/mL) CD4 cells/mm3 Plasmaa DBS (−20°C, 6 months, ViroSeq assay)b DBS (4°C, 12 months, ViroSeq assay) DBS (4°C, 12 months, in-house assay) 1 676 694 32 + + + + 2 266 612 195 + + + + 3 214 330 195 + + + + 4 191 674 48 + + + + 5 170 289 663 + + + + 6 90 758 324 + + + + 7 63 261 169 + + + + 8 51 598 324 + + + + 9 52 342 457 + + + + 10 49 805 450 + + + + 11 37 010 152 + + + + 13 30 267 525 + + + + 14 28 742 378 + + + + 16 23 352 221 + + + + 17 23 321 168 + + + + 18 21 896 391 + + + + 19 17 563 289 + + + + 20 16 359 609 + + n.s + 21 14 622 483 + + + + 22 13 952 396 + + + + 23 13 407 224 + + + + 24 12 452 374 + + − + 25 12 307 465 + + + + 26 12 264 1320 + + + + 27 11 999 81 + + − + 28 11 663 336 + + n.s + 29 10 998 442 + + n.s. − 30 9955 342 + + − + 31 8316 288 + + − + 32 4717 + + − + 33 4349 990 + + − + 34 4213 1080 + + − + 36 3880 434 + + n.s. + 37 3455 468 + + − + 38 2240 154 + + + + 39 1929 416 + + − + 42 1254 858 + + − n.s. 44 1045 360 + + − + 45 929 63 + + n.s. + 46 518 240 + + − + n.s., HIV-1 RT and protease were amplified but failed to generate full-length sequences. aPlasma genotypes were generated using the Viroseq assay except for 1, 3, 18, 32, 33, 39 and 42 (TrueGene assay). bAmplification from these DBSs stored at −20°C has been reported elsewhere.6 Nucleic acid extraction Total nucleic acids were extracted from one spot after 1 year of storage at 4°C using a modification of the Nuclisens assay described previously.7 Briefly, a whole spot containing 50 µL of blood was cut with scissors and added into 9 mL of Nuclisens lysis buffer (bioMérieux, Inc., Durham, NC, USA). Special care was taken to avoid contact of the scissors with the blood spots; scissors and forceps were sprayed with 70% ethanol after each use and wiped dry. After 2 h of incubation with lysis buffer at room temperature under gentle rotation, the supernatant was clarified by centrifugation at 250 g for 5 min and then transferred to a clean 15 mL conical tube. Nucleic acids were then extracted following the manufacturer’s instructions, resuspended in 45 µL of elution buffer and stored at −80°C until use. Drug resistance testing Resistance testing from DBSs was performed using the ViroSeq HIV-1 Genotyping System (Abbott Molecular, Des Plains, IL, USA) and an in-house reverse transcriptase (RT)-nested PCR method described previously.7 The ViroSeq assay amplifies a 1.8 kb pol fragment and has a sensitivity of detection of 2000 RNA copies/mL plasma. The in-house assay amplifies a 1023 bp fragment of HIV-1 pol comprising amino acids 15–99 of the protease and 1–256 of the RT. This assay has been validated only for HIV-1 subtype B viruses and has a sensitivity of detection of 1000 RNA copies/mL of plasma.7 Genotypes were interpreted using the Stanford Genotypic Resistance Interpretation Algorithm (version 4.2.6) available at http://hivdb.stanford.edu/pages/algs/HIVdb.html. Results When the ViroSeq assay was used, only 23 of the 40 (57.5%) DBS specimens stored at 4°C were successfully genotyped; 22 of the successful amplifications were from specimens that had plasma viraemia >10 000 RNA copies/mL (Table 1). An additional five specimens produced amplicons that did not generate full-length RT and protease sequences. Visual inspection of the desiccant bags after 6 months of storage at 4°C showed evidence of humidity in 21 of the 40 specimens. Of these 21 specimens, only those with plasma VL higher than 14 000 RNA copies/mL (12 specimens) were successfully genotyped by the ViroSeq assay at 12 months (data not shown). As we previously noted using parallel DBSs stored at −20°C, resistance genotypes generated from DBSs and matched plasma specimens were highly concordant. Of the 163 drug resistance mutations identified in these 23 plasma sequences, 158 were also found in sequences generated from DBSs. Of the five mutations absent in DBSs, three were protease mutations, two were major (V82A and I54M) and one minor (L10IT), and two were polymorphisms at codon 333 of the RT (G333E and G333D) (data not shown). We also compared the efficiency of amplification seen in these DBSs with that previously seen in DBSs prepared in parallel from the same patients and stored at −20°C for 6 months.6 Under storage at −20°C, all of the 40 DBS specimens were successfully amplified and genotyped (Table 1). The ViroSeq assay amplifies a large (1.8 kb) pol fragment in a single round of PCR and may be particularly sensitive to the degradation of HIV-1 nucleic acids that may occur during long-term storage. We, therefore, assessed if the rate of amplification from DBSs stored at 4°C could be improved by using an in-house assay that amplifies a smaller (1023 bp) fragment and uses a nested PCR step.7 Using this method, viruses from 38 of the 40 DBSs (95%) were successfully genotyped (Table 1). Despite the use of a different assay to genotype viruses from these 38 DBS specimens, resistance genotypes were highly concordant with those generated from plasma using the ViroSeq assay; 275 of the 291 mutations found in plasma viruses were also found in viruses from DBSs (data not shown). To investigate if the decreased amplification success rate observed with the ViroSeq assay was due to insufficient or degraded HIV nucleic acids, we performed serial dilutions of RNA extracts from high VL specimens and tested them by both the in-house and the ViroSeq assays (Figure 1). Although both assays have comparable sensitivity, amplification signals were only maintained when the shorter pol fragment was amplified by the in-house method. Figure 1 shows that such signals were consistently lost when the larger pol fragment was amplified using the ViroSeq assay, suggesting the possibility of nucleic acid degradation during storage at 4°C. Such a possibility was further supported by parallel testing of serial dilutions of RNA extracts prepared from specimens stored at −20°C. Figure 1(c) shows that amplification signals generated from these specimens using the in-house assay were generally stronger than those obtained from the same DBS specimens stored at 4°C (Figure 1b). Figure 1 Agarose gel showing the HIV-1 pol amplification signals obtained from DBS specimens stored at 4 or −20°C. Two serial 10-fold dilutions (1/10 and 1/100) of specimens 2 (266 612 RNA copies/mL), 3 (214 330 RNA copies/mL), 4 (191 674 RNA copies/mL) and 10 (49 805 RNA copies/mL) were prepared in Nuclisens elution buffer and were tested in parallel using the ViroSeq assay (1.8 kb) or the in-house RT-nested PCR method (1023 bp). (a) DBS specimens stored at 4°C and amplified using the ViroSeq assay. (b) DBS specimens stored at 4°C and amplified using the in-house method. (c) DBS specimens stored at −20°C and amplified using the in-house method. Discussion Our study on a small number of specimens suggests that storage at 4°C may represent a feasible alternative to −20°C for long-term storage of DBSs. However, we noted that the efficient genotyping from these DBS specimens required the use of an in-house nested PCR assay and that the ViroSeq assay failed to genotype a substantial proportion of specimens. The results with the ViroSeq assay differed from our previous findings with this assay showing a high success of genotyping from DBSs stored at −20°C and suggest that some degradation of RNA may have occurred during long-term storage at 4°C possibly due to suboptimal storage temperature, humidity or both. Such a possibility was suggested by the weaker amplification signals observed in RNA extracts from specimens stored at 4°C compared with those stored at −20°C upon serial dilutions. Although different efficiencies of RNA extraction could partially explain these differences, we found that a high proportion of the specimens stored at 4°C had evidence of humidity at 6 months, whereas none of the specimens stored at −20°C did. High humidity conditions are thought to be detrimental to resistance testing from DBSs given the extreme sensitivity of HIV nucleic acids to degradation in the presence of humidity.10 Nonetheless, we showed that it is possible to overcome potential losses in HIV-1 RNA integrity and efficiently genotype from DBSs stored at 4°C by using an in-house nested PCR protocol that amplifies a smaller fragment using stringent, quality-controlled reagents. These results are encouraging and expand on our earlier findings showing the efficient genotyping of HIV-1 pol from DBSs stored at −20°C for up to 4 years.6,7 A high success of genotyping has also been noted from DBSs stored for 3 months at 37°C and 85% humidity in the presence of desiccant.10 Under these extreme conditions, viruses from ∼85% of the DBS specimens could be genotyped by using in-house nested PCR assays that amplified small (700 bp), overlapping pol sequences.10 The use of nested PCR protocols and/or the amplification of small fragments may likely result in higher amplification success rates from DBSs stored under less optimal conditions. Such a possibility was supported by our findings, showing a better maintenance of amplification efficiencies using the in-house method upon serial dilution of RNA extracts. Despite having comparable sensitivity, amplifications were rapidly lost when a larger fragment was amplified using the ViroSeq assay. The main objective of our study was to evaluate the amplification success rate from DBSs stored at 4°C for a prolonged period of time. We used an in-house assay validated for HIV-1 subtype B since our study population included patients infected with subtype B viruses. It is important to note, however, that information on the performance of our assay with non-B subtypes is limited to a small number of specimens.7 Therefore, we caution that resistance testing from DBSs collected in areas with prevalent non-B subtypes will require in-house assays appropriately validated for other HIV subtypes. The ability to collect blood samples on filter paper represents an advantage for HIV drug resistance surveillance and monitoring, particularly in areas that lack the appropriate infrastructure for plasma processing and transport. Much effort is currently underway to define the best conditions that will facilitate resistance testing from DBSs including the most appropriate storage temperature and time. The findings reported here suggest that 4°C may represent a feasible storage temperature for long-term storage of DBSs and add to the promise of DBSs as a convenient specimen type for HIV-1 drug resistance testing. Funding Work at the CDC was done with intramural funding. Work at Hospital Carlos III was supported by grants from the Fondo de Investigación Sanitaria (FIS, CP06/284 and PI06/1826). Transparency declarations None to declare. Disclaimers The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. Use of trade names is for identification only and does not constitute endorsement by the U.S. Department of Health and Human Services, the Public Health Service, or the Centers for Disease Control and Prevention.

Psychopharmacologia-3-1-1915624

Effects of the noradrenergic agonist clonidine on temporal and spatial attention

Rationale Recent theories posit an important role for the noradrenergic system in attentional selection in the temporal domain. In contrast, the spatially diffuse topographical projections of the noradrenergic system are inconsistent with a direct role in spatial selection. Introduction The locus coeruleus–norepinephrine (LC–NE) system is one of several brainstem neuromodulatory nuclei with widely distributed, ascending projections to the neocortex (Berridge and Waterhouse 2003). The conventional view of NE is that it has broad, nonspecific functions such as regulating arousal (Jouvet 1969). However, recent studies suggest that NE may have more specific cognitive functions, which are nevertheless closely related to the concept of arousal (Robbins 1997; Aston-Jones et al. 2000; Yu and Dayan 2005; Chamberlain et al. 2006). In particular, it has been proposed that NE has a key role in facilitating the responses to decision-making processes and in regulating the balance between exploitation and exploration (Aston-Jones and Cohen 2005). Recent views on the role of the LC–NE system in decision making have been based on neuronal recordings of the primate LC during the performance of simple decision-making tasks. These recordings have indicated that under alert conditions (i.e., conditions typical of human subjects in our experiments), LC phasic responses are selectively observed after task-relevant and otherwise motivationally significant (e.g., highly salient or infrequent) stimuli (Aston-Jones et al. 2000). Furthermore, stimulus-elicited phasic LC bursts reliably precede (by ∼200 ms) and are closely coupled in time with the behavioral response (Bouret and Sara 2004; Clayton et al. 2004). The noradrenergic innervation associated with such LC responses results in a system-wide, transient increase in the responsivity of efferent target neurons, which is thought to facilitate processing in response to the eliciting stimulus (Berridge and Waterhouse 2003). These observations have led to the suggestion that the LC phasic response can be thought of as a temporal attentional filter (1) that selects for the occurrence (i.e., timing) of task-relevant (Aston-Jones and Cohen 2005) or unexpected (Dayan and Yu 2006) events, much like cortical attentional systems filter the content (e.g., spatial characteristics or color) of stimuli and (2) that facilitates the processing of and responding to these stimuli to help optimize task performance (Aston-Jones and Cohen 2005; Nieuwenhuis et al. 2005a; see also Coull 1994). In the current study, we evaluated the following two hypotheses: (1) According to the temporal filtering hypothesis outlined above, the LC–NE system should be involved in temporal attention modulations operating at the phasic time scale (e.g., in the order of tens of seconds); (2) In contrast, because of the spatially nonspecific pattern of LC projections to the forebrain, the LC–NE system cannot be directly involved in spatial attention shifts. That is, although the LC–NE system projects to multiple brain areas, including many known to be involved in spatial attention, the diffuse character of these projections is inconsistent with the direct biasing of attention in any specific direction (i.e., the LC does not “know” the relevant spatial location). To test these two hypotheses, we contrasted, in human subjects, the effects of a noradrenergic drug on measures of temporal and spatial attention. Most previous studies examining the functional significance of the LC–NE system have employed drugs acting on α2 receptors. In this study, we used the α2 adrenoceptor agonist clonidine, which at low doses decreases LC firing and attenuates the release of NE from axon terminals (Svensson et al. 1975). Previous studies have examined the effects of clonidine on temporal and spatial attention in a visuospatial cueing task. In this task, subjects are required to respond as quickly as possible to a series of stimuli presented to the left or right of fixation. Each stimulus is preceded by a cue indicating at which location the imperative stimulus is most likely to appear, and subjects are instructed to make use of this information by switching their attention to the location indicated by the cue. The comparison of reaction times on validly versus invalidly cued trials (the “validity effect”) provides an index of the efficiency of cue-induced spatial attention shifts. Fernandez-Duque and Posner (1997) extended this paradigm by including and contrasting reaction times on neutral-cue trials and no-cue trials (the “alerting effect”), which provides an index of the transient increase in responsivity brought about by the mere presentation of the cue. The alerting effect thus serves as a measure of temporal (cue-evoked) changes in attention to the imperative stimulus. Clonidine has been found to reduce the size of the alerting effect in humans (Coull et al. 2001) and monkeys (Witte and Marrocco 1997), suggesting that the temporal attentional effects of cues are mediated in part by the noradrenergic system. In contrast, studies have reported mixed results regarding the influence of clonidine on the validity effect: Two studies have reported a reduced validity effect (Clark et al. 1989; Coull et al. 2001), and one study has reported no effect of clonidine (Witte and Marrocco 1997). However, it is hard to interpret clonidine-related reductions in the validity effect, because they may reflect either a direct involvement of the noradrenergic system in spatial attention processes or an interaction between the alerting and attention-directing (i.e., spatial) effects of cues. Specifically, by increasing the responsivity of cortical representations after cue presentation, LC-induced phasic NE release can enhance the effects of target selection by location (e.g., the top-down modulation by prefrontal cortex), resulting in an increased validity effect. The effects of this amplification mechanism would be diminished by clonidine. In contrast to previous studies, we used separate tasks for measuring the effects of clonidine on temporal and spatial attention: an attentional blink task, which requires the selection of targets in time and has no spatial component, and a visual search task, which requires the selection of targets in space but without the help of alerting cues. In the attentional blink task, each trial starts with a rapid serial visual presentation (RSVP) stream containing two target stimuli (T1 and T2) and multiple distractors, each presented for about 100 ms. At the end of the RSVP stream, subjects are required to report the identity of T1 and T2. The critical finding in this task is that subjects are heavily impaired at the identification of T2 for a few hundred milliseconds after correct identification of T1. This deficit, known as the “attentional blink,” is usually most severe around 200–400 ms (or two to four items) after T1, after which, performance gradually recovers (Raymond et al. 1992; Chun and Potter 1995). In contrast, if T2 follows T1 without intervening distractors (at “lag 1”), performance on T2 is often (partially) spared (Raymond et al. 1992; Hommel and Akyürek 2005); this phenomenon has been labeled “lag-1 sparing.” These and other findings suggest that the attentional blink task reveals important clues about the time course of fine-scale fluctuations in attention (Olivers 2007). Recently, a theory has been proposed that explains the attentional blink in terms of the temporal dynamics of the LC–NE system (Nieuwenhuis et al. 2005b). The theory was inspired by the observation that the timing of the attentional blink coincides with the refractory period in LC activity that follows an LC phasic response to target stimuli. Specifically, although NE potentiates processing in cortical areas, local NE release within the LC is autoinhibitory because of the noradrenergic action at presynaptic and dendritic α2 autoreceptors (Aghajanian et al. 1977). This autoinhibition results in a period after the LC phasic response (∼200–450 ms after the eliciting stimulus), during which subsequent LC phasic discharge is rarely observed. According to the theory, the attentional blink may be mediated by this momentary unavailability of the LC phasic response (and attendant noradrenergic potentiation of information processing) after the LC response to T1. In other words, if T2 is presented during the LC refractory period associated with T1, it will not receive the benefit of NE-mediated facilitation and is therefore more likely to remain unidentified. A computational model implementing this theory was shown to accurately simulate the time course of the attentional blink, including lag-1 sparing (Nieuwenhuis et al. 2005b). In addition, the theory offers an account of the close relationship of the attentional blink to the P3 component of the event-related potential, an electrophysiological correlate of the phasic NE release (Nieuwenhuis et al. 2005a). To assess the effect of clonidine on spatial attention, we used a visual search task. On each trial, subjects searched for a target among multiple distractors in a visual search array and indicated as quickly as possible whether the target was present or absent. The number of distractors (i.e., set size) was systematically varied across trials. In one condition (single-feature search), the target was defined by a salient basic feature, such that search was efficient and the number of distractors had little influence on reaction times. In another condition (conjunction search), the target was defined by a conjunction of two features, such that search was inefficient, and reaction times increased linearly with the number of distractors—as if each item needed to be examined in turn. In this task condition, the efficiency of spatial attention processes is expressed in the function relating search reaction times to set size: The more visual attention is impaired, the steeper the slope of this function (as expressed in an increased effect of set size; Tales et al. 2002). Our predictions with regard to the effects of clonidine were the following: First, we expected that clonidine would impair performance on the attentional blink task, in particular through its stimulation of inhibitory α2 autoreceptors in the LC. In accordance with the temporal filtering hypothesis, we expected that the clonidine-induced reduction in noradrenergic activity would lead to an impaired identification of T1 but especially of T2, because it should presumably exacerbate the lack of NE associated with T2 processing under normal conditions (Nieuwenhuis et al. 2005b). Second, aside from the well-documented main effect of clonidine on response speed (i.e., general slowing; e.g., Tiplady et al. 2005), we expected no drug effects on visual search efficiency, as indexed by the effects of distractor set size. This latter prediction was based on the notion that the effects of NE are topographically diffused and hence should not affect the efficiency of selection in the spatial domain. Materials and methods Subjects Thirty-two healthy young adults, aged 18–25 years, took part in a single experimental session in return for 60. Only subjects with a systolic blood pressure above 100 mmHg and a diastolic blood pressure above 70 mmHg were included in the study. All subjects underwent a medical screening and were considered to be in satisfactory health. The use of medication that could interfere with clonidine was stopped the day before. All subjects with color blindness were excluded from the study. Subjects received an oral dose of 150 μg of clonidine or placebo in a double-blind, between-subjects design. Clonidine has well-established antihypertensive properties; therefore, blood pressure and heart rate were monitored for subject safety. Measurements were taken every 15 min starting from t = −15 until t = 120 and every 30 min from t = 120 until t = 240. The clonidine group (N = 16, eight women, M age 21.3) and the control group (N = 16, eight women, M age 21.3) had similar mean ages. We also verified that the groups were similar in terms of intellectual functioning as assessed with the three-subtest version of the Groninger Intelligence Test (GIT), a reliable indicator of the full-scale IQ (Luteijn 1966). Estimated IQ scores were 117.1 for the clonidine group and 116.8 for the control group. The visual-search task data from one subject from the clonidine group were discarded because of an accidental failure to comply with the task instructions. The study was approved by the medical ethics committee of the VU University Medical Center and was conducted according to the Declaration of Helsinki. Informed consent was obtained from all subjects before their inclusion in the study. Procedure Each subject was tested at approximately the same time of the day (afternoon). Subjects were instructed to abstain from caffeine, nicotine, and other psycho-active substances from 15 h before the start of the session and to abstain from alcohol from 15 h before the start of the session until the day after the session. After the medical screening, subjects received a lactose-filled capsule with either clonidine or placebo. Immediately after that, the subtests of the GIT were administered. The two attentional tasks, described in detail below, were performed between t = 60 and t = 120 min posttreatment. Half of the subjects in each group started with the attentional blink task. The other half started with the visual search task. After completion of the tasks, the subjects were debriefed and paid. At t = 240, subjects were reevaluated and returned home by taxi if blood pressure was (near) normal. Attentional blink task Stimulus generation and response recording in both tasks were controlled by E-Prime software (Psychology Software Tools, Pittsburgh, PA). Stimuli were presented in black against a light grey background. Each trial started with a fixation cross measuring 0.5 × 0.5°, presented for 1,000 ms in the center of the display. Subsequently, the fixation cross was replaced by an RSVP stream of 15–22 uppercase letters, each measuring approximately 0.9 × 0.9°. Each letter was randomly drawn (without replacement) from the alphabet and presented for 50 ms, followed by a 30-ms blank interval. This relatively rapid pace of stimulus presentation (i.e., 80 ms between two consecutive stimulus onsets) was chosen to avoid ceiling levels of performance and consequently to increase the sensitivity of the task to individual and group differences. “I,” “O,” “Q,” and “S” were left out as they resemble digits too much. On each trial, two of the letters were replaced with digits, randomly drawn without replacement from the set 2 to 9. T2 was presented three to six temporal positions from the end of the stream. The temporal distance between T1 and T2 was systematically varied from one to five items, corresponding to lags of 80, 160, 240, 320, and 400 ms. The subject’s task was to identify both T1 and T2 by typing the digits in order on a standard keyboard after the end of the RSVP stream. Subjects were instructed to guess whenever they failed to identify a digit. The two keyboard entries were followed by the presentation of a feedback stimulus for 150 ms (e.g., ‘+, −’ to indicate that T1 was correct and T2 was incorrect). After a 1,000-ms blank screen, the next trial started. Each subject started with 15 practice trials, three with each condition, randomly intermixed. This was followed by four blocks of 50 trials each with each block containing ten repetitions of each lag. All task instructions were automated and presented on screen. Visual search task On each trial, subjects searched for a target among multiple distractors in a visual search array. On half of the trials, the target, a vertical red bar, was present in the array. On the other half of the trials, the target was absent. In one condition (single-feature search), the distractors in the search display were vertical green bars of equal size as the target. Because in this case the target is defined by a unique feature (color), it “pops out” from the display, resulting in a fast and efficient search process. In another condition (conjunction search), the distractors were vertical green bars and horizontal red bars. In this case, the target does not consist of a single identifying feature but is defined by a specific conjunction of features (color and orientation), resulting in a more time-consuming and error-prone search process. Each trial started with a white fixation cross measuring 0.9 × 0.9° against a dark background, presented for 500 ms in the center of the display. Subsequently, the fixation cross was replaced by the search display, which consisted of four, eight, or 16 items that were randomly plotted in the cells of an imaginary 6 × 6 matrix (8.7° horizontally × 9.6° vertically) with some random jitter within the cells. The subject’s task was to report whether or not the target (0.7 × 1.3°) was present by giving a response with their left or right index finger using the ‘z’ and ‘m’ keys on the computer keyboard. The keyboard entry was immediately followed a 1,000-ms blank screen after which the next trial started. Subjects performed four blocks of 96 trials each, with each block containing 16 repetitions of the factorial combination of set size (4, 8, or 16) and trial type (target present or absent) presented in random order. Search condition (single-feature search or conjunction search) was varied across blocks in an ABBA-order. Half of the subjects started with single-feature search, and the other half started with conjunction search. Subjects received written instructions and 12 practice trials before entering the experimental phase. The task instructions encouraged subjects to respond as quickly as possible while minimizing the number of errors. Performance feedback was provided at the end of each block. All task instructions were automated and presented on screen. Results Physiological measures Figure 1 presents for both groups the absolute values of heart rate, systolic, and diastolic blood pressure as observed throughout the experimental session. All three measures showed a significant effect of time (all P < 0.001). Systolic [F(1, 30) = 11.8, P = 0.002] and diastolic blood pressure [F(1, 30) = 6.3, P = 0.018] both showed significant main effects of group. The main effects of time and group were qualified by significant time X group interactions for systolic [F(13, 390) = 9.8, P < 0.001] and diastolic blood pressure [F(13, 390) = 6.1, P < 0.001]. Fig. 1Systolic and diasystolic blood pressure and heart rate following administration of placebo or 150 μg of clonidine (time = 0). The grey shaded area marks the time period during which the two cognitive tasks were administered Attentional blink task Figure 2 shows the average T1 accuracy (left panel) and T2 accuracy (right panel; contingent on correct T1 identification) as a function of lag and group. A similar pattern of results was found if T2 accuracy was averaged across correct and incorrect T1 trials. Trials on which T1 and T2 were accurately identified but in the wrong order were treated as correct. As an analysis of variance (ANOVA) showed no significant effects of the order in which the two tasks were performed, this factor was omitted from the reported analyses. Fig. 2Average identification accuracy for the first (T1; left panel) and second (T2; right panel) of the two targets in the attentional blink task as a function of group (clonidine vs placebo) and the lag between T1 and T2. As is usual, T2 accuracy is reported contingent on accurate identification of T1 T1 and T2 accuracy were entered in separate two-way mixed ANOVAs with lag and time-on-task (blocks 1,2 vs 3,4) as within-subject factors and group as a between-subject factor. The variable time-on-task was included to assess the possibility that a group effect emerged over time. T1 accuracy showed an increasing trend with lag [F(4, 120) = 12.3, P < 0.001]. The clonidine group (79.5% correct) performed slightly worse on T1 than the placebo group (84.6%), but the main effect of group did not reach significance [F(1, 30) = 2.0, P = 0.17]. The lag X group interaction was not significant [F(4, 120) = 1.0, P = 0.39]. The two T2 accuracy curves show a pattern that is characteristic of attentional blink research: lag-1 sparing, followed by a drop in performance for lags 2, 3, and 4 (i.e., the attentional blink), and followed by the initial recovery of performance at lag 5, at least in the clonidine group. This pattern was expressed in a significant effect of lag [F(4, 120) = 43.2, P < 0.001]. The most important for the present purposes is the finding that T2 accuracy did not reliably differ between the two groups [F(1, 30) < 1]. Furthermore, although at lag 4 there was a sizeable numerical effect of group in the expected direction, the lag X group interaction was not significant [F(4, 120) = 1.2, P = 0.30]. Time-on-task did not reliably affect T1 or T2 performance or interact with other variables. In a separate analysis, we calculated the average T2 performance across lags 3–5 as a summary measure of attentional-blink magnitude and examined group differences in this measure. The two groups did not differ in attentional-blink magnitude, and performance at lags 3–5 did not improve with time-on-task [both F(1, 30) < 1]. Interestingly, there was a reliable interaction between group and time-on-task [F(1, 30) = 4.9, P = 0.034]: Whereas performance of the placebo group deteriorated over time (66.6 vs 62.2% correct; P = 0.65), performance of the clonidine group improved slightly (60.3 vs 62.6%; P = 0.28). In a final analysis, we examined the percentage of order reversals at lag 1, the phenomenon that the two targets, when immediately succeeding each other, are often identified correctly but reported in the wrong order (Hommel and Akyürek 2005). The percentage of such order reversals was roughly the same in the placebo group (34.4%) and in the clonidine group [35.4%; t(30) < 1]. Visual search task: single-feature search The total number of items in the search display (set size) was varied from trial to trial, allowing us to derive the function relating reaction time (RT) to set size. The slope of this function measures the cost for adding additional items to the display and is often interpreted as “search efficiency,” with steeper slopes indicating slower, less efficient search. As expected and illustrated in Fig. 3, slopes in the single-feature search condition were close to zero [average 1.6 ms/item; t(30) = 2.6, P = 0.014]. Fig. 3Average correct reaction times for the clonidine and placebo groups in the visual search task. Top panels show data from the single-feature search condition, and bottom panels show data from the conjunction search condition. Data for target-present trials are plotted in the left-hand panels, data for target-absent trials are plotted in the right-hand panels Correct RTs were entered in a three-way mixed ANOVA with target presence (target present vs target absent) and set size (4, 8, or 16) as within-subject factors and group as between-subject factor. Despite the relatively shallow RT slopes, the main effect of set size was significant [F(2, 58) = 6.3, P = 0.006]. Furthermore, the clonidine group was overall slower than the placebo group [F(1, 29) = 5.6, P = 0.025]. Group did not interact with target presence [F(1, 29) = 1.2, P = 0.29] or set size [F(2, 58) = 2.5, P = 0.10]. The three-way interaction was also not significant [F(2, 58) < 1]. Error rates were generally low (average 2.3%). An ANOVA yielded only a significant main effect of target presence [F(1, 29) = 8.9, P = 0.006], with errors occurring more often on target-present trials (2.9%) than on target-absent trials (1.8%). Visual search task: conjunction search As expected, slopes in the conjunction-search condition were sizeable [average 25.0 ms/item; t(30) = 13.8, P < 0.001], indicating that visual search was time consuming and inefficient. A three-way mixed ANOVA yielded significant main effects of target presence [F(1, 29) = 31.5, P < 0.001] and set size [F(2, 58) = 152.9, P < 0.001] and a significant interaction of these two variables [F(2, 58) = 26.7, P< 0.001], indicating that set-size effects were larger for target-absent trials. Importantly, the main effect of group was not significant [F(1, 29) = 2.1, P = 0.16], and group did not reliably interact with any of the task variables (all F < 1). Error rates were again low (average 3.3%). An ANOVA yielded the same pattern of effects as the RT analysis, indicating that there was no speed–accuracy trade-off, and that there were no significant group differences. Discussion The main findings of the current study can be summarized as follows. First, in contrast to our predictions, clonidine did not have a deleterious effect on attentional blink performance. Although the numerical group differences in T1 and T2 accuracy were in the expected direction, these differences were small compared to the sizeable performance differences between the individuals within each group (for a discussion of these individual differences, see Martens et al. 2006). The results from the visual search task were as expected: Although clonidine slowed the overall response speed, it did not affect the efficiency of the visual search for a target in a two-dimensional array of stimuli. Below, we will discuss the implications of these principal findings. The absence of a significant drug effect on attentional blink performance appears to be at odds with the temporal filtering hypothesis, which suggests that phasic activity of the LC–NE system is important for the selection of important or infrequent stimuli in the temporal domain (Aston-Jones and Cohen 2005; Dayan and Yu 2006). The attentional blink results also seem inconsistent with the predictions of a recent theory that explains the attentional blink in terms of the temporal dynamics of the LC–NE system (Nieuwenhuis et al. 2005b). One possible explanation for these discrepancies is that the predicted group differences do in fact exist, but that our task and experimental design were not sufficiently sensitive to reveal them. Some critical factors in this regard may be the choice of a between-subject design and the sample size of 16 subjects per group. However, previous studies using between-subject designs, similar group sizes, the same subject population, and the same task have had no difficulty detecting group-levels effect on attentional blink performance of various manipulations aimed at distracting subjects from the RSVP stream (Olivers and Nieuwenhuis 2005, 2006). Indeed, our choice of design was based on these previous studies and on the assumption that the effect of clonidine would be of at least the same size as the effect of these behavioral manipulations. In this context, it is worth noting that the interindividual variance in attentional blink performance was comparable to that in the Olivers and Nieuwenhuis studies, discounting increased variance as a reason for our failure to detect group differences. A more principled reason for the use of a between-subject design was that subjects can learn to adopt a ‘mental state’ that greatly benefits attentional blink performance (Olivers and Nieuwenhuis 2006), and we wanted to avoid the transfer of such learning between drug conditions to exclude a potential contaminating source of variance. In addition, Coull et al. (1995a, b) have found that in within-subject designs, the order of treatments (i.e., clonidine before or after placebo) can show complicated interactions with task performance, another source of variance that we aimed to exclude. Another design feature that may have led to a failure to detect an existing effect is the timing of test administration relative to the treatment (t = 60–120 min). In particular, the systolic and diastolic blood-pressure time series reveal a maximal drug effect after the test period, suggesting that cognitive effects may have peaked later too. However, there are at least three arguments against this possibility. First, Tiplady et al. (2005) have investigated the time course of effects of 150 μg of orally administered clonidine on cognitive test variables and found that such effects were already present at t = 45 min but were decreased at t = 135 min. Second, our task-block comparisons show that, if anything, the group difference in attentional blink performance became smaller over time, a trend opposite from that expected if the effects of clonidine needed more time to develop. A third and related argument is that we found no significant interaction between group and task order, corroborating the notion that group effects did not become larger over time. Alternatively, we cannot rule out the possibility that group effects peaked earlier than our test period. However, our choice of test period seems justified by the finding of significant cognitive effects of clonidine in various previous studies using similar test periods (Halliday et al. 1989, 1994; Coull et al. 2001). Thus, our methods and results do not seem to contain any clear indication that the observed null effects reflect a lack of sensitivity. However, it is of course possible that a higher drug dose would yield more robust group differences. This is a possibility that can be addressed in future research (note that such research should take into account that the primary mode of action of clonidine changes with increasing dose; (Arnsten and Cai 1993). At low doses, presynaptic receptors and LC receptors are stimulated, leading to a reduced NE activity. In contrast, at higher doses, postsynaptic receptors are activated, resulting in a boost of NE activity. This has important implications for the expected effects of clonidine on cognitive function). Another possible explanation for the absence of significant drug effects on attentional blink performance is that the attentional blink may not be mediated by the LC–NE system. This possibility would be particularly bothersome for the theory of Nieuwenhuis et al. (2005b), which directly relates the attentional blink to a refractory period in activity of the LC–NE system. Although there is substantial indirect evidence in support of this theory (e.g., the relationship between the attentional blink and the P3), the basic tenets of the theory are based on cell recordings in animals and are hard to validate directly in humans. Indeed, the current study is perhaps the most direct test to date of a relationship between the attentional blink and the LC–NE system. In this context, it is important to note that another research group has reported preliminary evidence consistent with such a relationship (De Martino et al. 2005). This study, using a between-subject design, found that attentional blink performance was significantly impaired after intake of the ß-adrenergic receptor antagonist propranolol (40-mg oral dose compared to placebo). Although this finding is not predicted by the theory of Nieuwenhuis et al. (2005a, b), which relates the attentional blink to the dynamics of inhibitory α2 autoreceptors in the LC, it will be informative to determine to what extent the current findings generalize to other task designs and other noradrenergic drugs. A further avenue for future research will be to test and evaluate the effects of noradrenergic drugs on temporal attention using alternative experimental paradigms, such as the temporal analogue of the spatial cueing paradigm (Posner et al. 1980; Coull and Nobre 1998; see Coull et al. 2001). Eventually, accumulating evidence obtained in such human psychopharmacological studies can be used to accommodate theories about the role of NE in temporal attention. The results from the visual search task were consistent with our predictions: Although clonidine led to a general slowing of response speed, we found no evidence for drug effects on visual search efficiency as indexed by the effects of the distractor set size. This pattern of results was expected based on the notion that the effects of NE are topographically diffuse (i.e., not selective with regard to any specific spatial representations) and hence should not affect the efficiency of selection in the spatial domain. Previous studies using visuospatial cueing tasks have found that clonidine reduces the cue validity effect (Clark et al. 1989; Coull et al. 2001), which has led to the suggestion that the LC–NE system is directly involved in visuospatial orienting. For example, it has been proposed that the LC–NE system plays an important role in the disengagement of visuospatial attention from invalidly cued spaces (Clark et al. 1989; Posner and Petersen 1990). This proposal is consistent with the little available evidence, which suggests that the reduced validity effect after clonidine administration is mainly due to attenuated performance costs on invalidly cued trials. However, we propose an alternative interpretation for these results to reconcile them with our theoretical framework. According to our interpretation, the presentation of the cue causes a spatially nonspecific, transient LC–NE response that boosts the processing of any stimuli presented for a brief period thereafter (i.e., the alerting effect). Although not supported by the current attentional blink findings, this assumption is based on various sources of evidence in the animal literature (Aston-Jones and Cohen 2005; Witte and Marrocco 1997). The cue-induced LC–NE response speeds up cortical spatial selection processes involved in localizing the cue and/or subsequent imperative stimulus. This improves performance on validly cued trials and impairs performance on invalidly cued trials thus increasing the validity effect. Administration of clonidine counteracts this amplifying effect of phasic LC–NE activity thereby reducing the validity effect (note that this hypothesis predicts both improved performance on invalid trials and impaired performance on valid trials). This type of interaction between the alerting effect and spatial selection processes is absent in the visual search task, which has no clear alerting component. Therefore, the current results are consistent with the notion that the LC–NE system is not directly involved in visuospatial orienting but can only indirectly modulate the effects of spatial attention mechanisms implemented elsewhere in the brain. One drawback of the current study is that we did not include a task that has already been shown to be sensitive to clonidine in previous studies. Therefore, a possible interpretation of the reported null findings is that the administered dose of clonidine failed to affect general cognitive function in our subjects, and that the main effect of drug on visual search RT reflects drug-induced motor slowing rather than a central cognitive deficit. Although we deem this possibility unlikely, given that previous studies using similar doses of clonidine have found significant effects on cognitive function (Coull et al. 2001; Tiplady et al. 2005), a more extensive study with a larger task battery is needed to adequately address this issue. There is rapidly growing interest in the specific role of the LC–NE system in human cognition (Cohen and Aston-Jones 2005). Psychopharmacological data from humans will be of critical importance in testing and further developing the increasingly sophisticated hypotheses that are based on neurophysiological observations in animals (e.g., Yu and Dayan 2005). The current research provides a valuable contribution toward a better understanding of the role of the LC–NE system in human attention, both in the spatial and the temporal domain.

Eur_Child_Adolesc_Psychiatry-2-2-1705530

The effect of birth-weight with genetic susceptibility on depressive symptoms in childhood and adolescence

Low birth-weight has been associated with depression and related outcomes in adults, and with problem behaviours in children. This study aimed to examine the association between low birth-weight for gestation and depressive symptoms in children and adolescents and to examine whether the relationship is moderated by genetic risk for depression. An epidemiological, genetically sensitive design was used including 2,046 twins aged 8–17 years (1,023 families). Data were obtained by parental report and analysed using regression analysis. A small but significant association between birth-weight for gestation and early depressive symptoms was observed. The unit increase in depressive symptoms per unit decrease in birth-weight for gestation was greater for individuals at genetic or familial risk for depression. For low birth-weight children, genetic risk for depression moderated the influence of birth-weight for gestation in predicting early depressive symptoms. Birth-weight for gestation is moderated by genetic and familial risk for depression in influencing early depression symptoms. These observations have clinical implications in that the impact of being small for gestational age on depressive symptoms is greater in children at familial/genetic risk although the association between birth weight and depression does not imply causality. Introduction Several cohort studies have shown associations between indexes of foetal nutrition such as pre-natal famine [6] and low birth-weight (LBW) with depression and related phenomena in adults [3, 7, 49]. However, the association between birth-weight for gestation and depressive symptoms in childhood has not been well examined. Instead, studies of the association between birth-weight and difficulties in childhood have focused on total behavioural problems or externalising problems [20, 52, 56]. Follow-up studies of children who were LBW at birth have reported that these children show a number of difficulties, in particular disruptive behavioural problems and reduced IQ in comparison to children who were born with normal weight at term [4, 20]. Although there have been relatively few studies that have examined the prevalence of depression in LBW children, there are now a growing number of reports illustrating that LBW children also show elevated rates of depressive symptoms [5, 17]. In addition, one recent nested case–control study of adolescent depression found increased rates of depressive disorder in adolescents who were either premature or LBW [32]. Preliminary evidence suggests that early adversity may modify the link between birth weight and depression in adults, with one study finding a stronger association in those adults exposed to more adverse socio-economic conditions during childhood [15]. One other factor that may potentially modify the relationship between birth weight and depressive symptoms is familial or genetic risk for depression. Genetic factors are known to be important in the aetiology of depression [29, 37]. To our knowledge, no study has examined the impact of genetic risk for depression on the association between birth-weight and depression in early life. We used a population-based twin register to first examine the association between birth-weight and depressive symptoms and then to test whether this effect was moderated by familial and genetic risk for depression. Methods Participants A total of 1,581 families, a sub-sample from a systematically ascertained, population-based register of all twin births between 1980 and 1991, the Cardiff Study of All-Wales and North-west England Twins (CASTANET) were invited to participate. Twins on this register were identified through child health databases and the characteristics of the sample have been described in detail elsewhere [38, 47] but are representative of the local population in terms of social class and ethnicity. The percentages of families from social class groups I, II, III, IV and V were 11.4%, 29.3%, 30.1%, 4.9% and 1.4%, respectively, with the remainder classified as at-home or unemployed. These figures are in keeping with those expected from a population from Greater Manchester [36]. The present wave of data collection took place in 2000 including twins from the register aged less than 17 years (range 8–17) and in full-time education born in Greater Manchester. The mean age of participating twins was 11.17 (standard error=0.068). Completed questionnaires were received from 1,023 families, giving an overall response rate of 65% (1,023/1,581). Data on birth-weight, antenatal and perinatal factors and social class had been collected 3 years previously. There were 934/1,545 twin pairs who took part at both assessment points (60%). (Note: There were 934 pairs who took part in both waves of the study but missing computed scores for some variables meant that they were not included in regression analyses). There were no significant differences between those individuals who had complete data available and those who did not take part at Time 2 in terms of birth weight (t=0.545, SE=0.15, P=0.586) or those who did not take part at time 1 in terms of depressive symptoms (t=0.730, SE=0.06, P=0.465). In addition, information on emotional symptoms as assessed by parental report on the Rutter questionnaire (40) at Time 1 did not reveal any significant differences between those with complete data and those who did not participate at Time 2 (t=1.114, SE=0.06, P=0.465). Multi-centre ethical approval was obtained. The study was described to participants and informed consent was obtained following return of the completed questionnaire package. Complete data on birth weight, gestation and depressive symptoms were available for 370 monozygotic (MZ) and 495 dizygotic (DZ) pairs. Zygosity was assigned according to parent responses to the twin similarity questionnaire, which has been found to be over 90% accurate in distinguishing MZ and DZ twins. An algorithm based on previous work was used [8, 47]. Measures Mothers of the twins reported information on antenatal and obstetric factors using a questionnaire adapted from Lewis & Murray [27], which included the twins’ birth-weights, number of weeks of gestation at birth, maternal age at birth and maternal smoking during pregnancy (yes/no). Mother reports of birth weight and gestation have been found to be very accurate in comparison to antenatal records with correlations of over 0.90 on average [16, 51]. Social class information was also collected and classified according to Standard Occupational Classification [31]. Depression in the children and mothers was assessed 3 years later in the year 2000. Child depressive symptoms were assessed using the Mood and Feelings Questionnaire (MFQ) [11]. The MFQ is a 34-item scale based on DSM-III-R symptoms of depression that is a reliable quantitative measure of depression and a useful screening questionnaire for clinical depression in children in the community [11, 48] as well as in clinical populations [57]. Parent reports of children’s depression symptoms were used as child reports are only reliably obtained for those over the age of 11 years. The mean depression score for the sample was 9.15 (range 0–60). Boys’ scores (mean=8.63; range 0–43) were lower than those of girls (mean=9.59; range=0–60) although this difference was not significant (t=−1.45, SE=0.05, P=0.15). Given that there was no significant gender difference in mean symptom counts for the sample, and no evidence for gender differences in the magnitude of the genetic influence on parent-rated depressive symptoms in a previous analysis of this sample [38], the primary analyses were conducted using the combined sample rather than separately for each gender. Parents also completed the depression sub-scale of the Hospital Anxiety and Depression Scale (HADS) [59] about their own depression symptoms in the past week. The mean depression score for parents was 4.46 (range 0–21). The majority of analyses were based on total symptom scores given that there are strong arguments for regarding depression as a continuous dimension of risk rather than as a categorical disorder (affected/unaffected) both in adults and in children. Firstly, studies of adults and children show that depressive symptoms behave as a continuum with no evidence of increased morbidity once clinical diagnostic criteria are met [23, 51]. For example, Pickles & colleagues [33] found that number of depressive symptoms but not functional impairment predicted future depressive episodes. Second, sub-clinical depressive symptoms are associated with impairment [2, 26]—one of the main criteria for fulfilment of disorder. Third, sub-clinical symptoms are associated with service use and significantly increase the risk for depressive disorder in adulthood [21, 34]. Moreover, it appears that a continuous measure of depression is more indicative of genetic risk in children and adolescents. Several studies have found that the aetiology of very high levels of depressive symptoms is less influenced by genetic factors than depressive symptoms across the normal range [13, 14, 38]. Thus, at least in children, symptoms of depression may give a better indicator of genetic risk than categorical diagnoses. Nonetheless, because of the suggestion of aetiological heterogeneity, depressive symptoms were analysed both as a continuous and a categorical measure in this report. Birth weight was examined both as a dimensional measure and as a category since both birth weight across the continuum and LBW have been reported to be associated with depression in adults [7, 15, 32]. Statistical analysis Data were analysed using STATA [45] and SPSS [46]. We adopted two approaches for testing the effect of birth-weight for gestation and genetic/familial risk. First, genetic regression analysis [22, 56] was used to test for genetic effects and for interaction of genetic risk and birth-weight. We made use of the twin design that relies on the observation that identical (monozygotic; MZ) twins share all of their genes in common (coefficient of relationship=1), while on average, non-identical (dizygotic; DZ twins) share only half of their genes in common (coefficient of relationship=0.5). Genetic regression analysis is undertaken using standard forced entry linear regression analysis with one twin’s depression symptom scores being the dependent variable. The twin’s depression score was predicted using the formula T=B0+B1 C+B2 R+B3 I+B4 M+B5 G where T=the twin depression score, B0=the regression constant, C=the co-twin depression score, R=a coefficient of genetic relatedness (0.5 for DZ twins; 1 for MZ twins), I is the interaction term (C*R*birth-weight corrected for gestation), M=the main effect of birth-weight (with the length of gestation regressed out) and G=twin gender. One twin’s depression score was entered as the dependent variable, with co-twin depression, genetic relatedness and the interaction term as independent variables. Regression analyses were carried out using both continuous and categorical measures of birth-weight corrected for gestational age. Low birth-weight for gestation was classified as ≤10th centile since standard published cut points were not available for birth weights of twins. Next, linear regression analysis was used to test for a main effect of birth-weight on depressive symptoms. The survey commands in STATA were used to take account of clustering within twin pairs by likening the twin data to a two-stage cluster design with the twin pairs as the primary sampling unit. A robust variance matrix calculation is utilised that relaxes the assumption of independence within groups. Birth-weight was corrected for number of weeks gestation at birth by regressing out the effect of length of gestation and using the unstandardized residuals for further analysis. As boys tended to be heavier at birth than girls, and the association between birth-weight and depressive symptoms may differ by gender. The effect of sex was controlled for by including sex as a predictor variable in the regression model. A number of factors have been reported to be associated with LBW [12]. Information on three potential influences was available—maternal smoking during pregnancy, social class and maternal age at birth. Social class was not associated with birth weight for gestation (b=−4.20, SE=3.32, P=0.206) or depressive symptoms (b=0.147, SE=0.107, P=0.169) in the present sample. Maternal pre-natal smoking (yes/no) was inversely associated birth weight for gestation (b=−141.62, SE=18.32, P=0.001) and positively associated with depressive symptoms in the offspring (b=2.16, SE=0.618, P=0.001). Maternal age at birth of child was negatively associated with child depressive symptoms (b=−.143, SE=0.053, P=0.007) and positively associated with twin birth weight (b=6.68, SE=1.65, P=0.001). These three variables were entered as predictor variables in regression models in addition to child gender. The second approach to test the effects of birth-weight and genetic/familial risk was to use standard regression analysis but this time defining (1) genetic and (2) familial risk according to cut points on the depression questionnaires for (1) the co-twin and (2) the mother. Regression analysis was used to estimate the unit increase in depressive symptoms per unit decrease in birth weight for those individuals at genetic risk or family risk of depression and those not at risk. Both familial risk and genetic risk for depression was used for this analysis because to estimate the unit increase in symptoms by genetic risk meant restricting the analysis to MZ twins and thus a substantial reduction in sample size. Using familial risk allowed results to be replicated using the much larger full sample. Furthermore, using mother’s depression to index familial risk rather than the DZ twins allowed the entire sample to be double entered i.e. each twin to be entered as an individual in the analysis. This is because maternal depressive symptoms is a variable that is obligatory shared for each member of a twin pair. Appropriate correction for the non-independence of twins was undertaken using the survey commands in STATA as described above. For genetic risk, birth-weight and gestation were entered as independent variables to predict MZ twin depressive symptoms for MZ twins individuals without an affected co-twin (MZ co-twin depressive symptoms <90th centile) and those with an affected co-twin (MZ co-twin depressive symptoms ≥90th centile). This analysis was then repeated for the whole sample using parent depression symptoms as the cut point (parental depression score ≥90th centile) as a measure of familial risk. Dummy coded groups [9] were then analysed to assess whether there was an interaction between genetic or familial risk for depression and birth weight in predicting early depressive symptoms. These dummy variables allow the difference between the beta coefficients for the low-risk and high-risk groups to be tested. Results Descriptives Parent-rated MZ mean depression symptoms were lower than those of DZ twins but this was not significant (MZ mean=8.97, standard deviation=9.49; DZ mean=9.14, standard deviation=8.92; t=0.980, df=967, P=0.327). There were no significant differences on the ante-natal and labour complications assessed according to zygosity (admission to hospital for high blood pressure χ2=0.328, P=0.609, vaginal bleeding χ2=0.088, P=0.800, labour less than 3 h χ2=1.035, P=0.345, emergency Caesarean, χ2=2.270, P=0.151, maternal smoking during pregnancy, χ2=0.192, P=0.697, forceps or ventouse delivery? χ2=1.398, P=0.237). Descriptive statistics for the birth-weight and weeks gestation of twins are shown in Table 1. It can be seen that second-born twins were lighter at birth than first-born twins. MZ twins were lighter than DZ twins although they also tended to be born earlier in pregnancy. The average birth-weight for the entire sample was 2469.81 g (standard deviation=553.38) and the average length of gestation was 36.36 weeks (standard deviation=2.87). Table 1Birth-weight in grams and gestation in weeks by zygosity, gender and birth order for twins with complete data pointsNMeanStandard deviationFirst-born twin birth-weight8892484.55544.03Second-born twin birth-weight8892457.62562.17MZ first-born birth-weight3812404.32531.55MZ second-born birth-weight3812392.41545.44DZ first-born birth-weight5082544.65546.01DZ second-born birth-weight5082506.10570.11Girl first-born birth-weight4892445.43544.59Girl second-born birth-weight4892426.00560.75Boy first-born birth-weight4002532.18540.06Boy second-born birth-weight4002494.76562.45MZ duration of gestation (weeks)37136.443.00DZ duration of gestation (weeks)49735.992.75Girls weeks gestation48336.312.87Boys weeks gestation38536.182.87 Main effect of birth-weight for gestation—standard regression analysis Linear regression showed that birth-weight for gestation significantly predicted parent-rated child depression symptoms when controlling for child gender (b=−0.001, SE=0.001, P=0.003, 95% CI −0.003, −0.0006) (n=1,740). This remained significant when child gender, maternal age at birth, pre-natal smoking and social class were also included in the regression model (b=−0.001, SE=0.001, P=0.015, 95% CI=−0.003, −0.0003). This illustrates that children who were small for gestation had higher depressive symptoms in childhood and adolescence. Genetic regression analysis The results of genetic regression analyses with depression as a continuous variable are shown in Table 2. It can be seen from Table 2 that birth-weight for gestation, genetic risk and maternal smoking during pregnancy showed main effects on depressive symptoms. When birth-weight for gestation was included as a continuous variable there was no birth-weight by genetic risk interaction effect although there was a slight trend (β=−0.048, P=0.096) (n=861). Data were also analysed using LBW as a categorical variable. Significant main effects of birth-weight for gestation, genetic risk, pre-natal smoking and a significant genetic risk by LBW interaction were found (β=0.394, P=0.001) (n=861). This relationship is illustrated graphically in Fig. 1 and shows a stronger relationship between genetic risk (C*R) and depressive symtpoms for those individuals who were small for gestational age at birth (≤10th centile). This suggests that it is LBW for gestational age that interacts with genetic risk for depression when depression is assessed as a continuous variable. Table 2Genetic risk for depression and birth-weightBeta (birth-weight as a continuous variable)P valueBeta (low birth-weight for gestation as a categorical variable)P valueInteraction between birth-weight corrected for gestation & genetic risk−0.048a0.0960.394*0.001 Birth-weight for gestation−0.081*0.006−0.075*0.032Co-twin depressive symptoms0.541*0.001 0.212*0.002Twin gender0.044a0.1300.049a0.080Zygosity0.0270.349−0.096*0.010Maternal pre-natal smoking (yes/no)0.063*0.0330.067*0.021Social class0.0110.7050.0110.700Maternal age at birth−0.0240.411−0.0180.529aP<0.1*P<0.05Fig. 1Genetic risk and depressive symptoms by birth-weight for gestation Analyses of depression as a categorical variable—standard regression analysis When twins were classified at genetic risk of depression if they had an affected MZ co-twin, the coefficients derived showed that for those twins at genetic risk for depression (N=32), for each 1 kg decrease in birth weight corrected for gestation, gender, social class, maternal age at birth and pre-natal smoking there was an increase of 2 depressive symptoms. As birth weight was measured in grams, this was calculated by the regression coefficent multiplied by 1000 (−0.002×1,000=−2). For those identical twins (N=338) not at genetic risk, the increase in depressive symptoms for each 1 kg decrease in birth weight was 1 symptom (−0.001×1,000=−1). Thus, there was a two fold increase in the number of depressive symptoms for each 1 kg decrease in birth-weight for twins at genetic risk (2 vs. 1 symptom) when controlling for covariates. Similarly, for each decrease in birth-weight of 1 kg there was an increase of 7 depressive symptoms for those at familial risk of depression (N=196) (−0.007×1,000=−7) and an increase of 1 depressive symptoms for those not at familial risk of depression (N=1478) (−0.001×1,000=−1) representing a seven-fold increase in number of symptoms. Regression with dummy coded variables showed that the effect of birth-weight on depressive symptoms was not significantly different for those at high genetic risk of depression compared to those not at genetic risk of depression (for interaction term, β=0.009, P=0.853). It should be noted that regression with dummy coded variables for genetic risk of depression required restriction to MZ twins only and thus a substantial reduction in sample size. There was, however, a significant interaction according to familial risk (β=−0.086, t=−2.381, P=0.017). Discussion Depressive symptoms in young people show strong similarities with depression in adulthood [18, 19] and are associated with a number of deleterious health outcomes including increased risk for depressive disorder in adulthood; impaired educational and social functioning; increased service use and deliberate self-harm [2, 18, 21, 26, 34]. We find that genetic risk interacts with LBW for gestation in influencing depression. Standard regression analysis treating birth-weight for gestation as a continuous variable also showed that familial risk but not genetic risk interacts with birth-weight. These results suggest that being at genetic or familial risk for depression increases the influence that being small for gestational age has on depression. This result suggests that there may be public health implications for the future mental health of infants who are small for gestational age if there is also a family history of depression. Results from genetic and standard regression analysis showed broadly similar results. However, results from genetic regression analysis suggested that LBW for gestation has the strongest effect on depressive symptoms in the context of genetic risk, rather than birth-weight for gestation across the continuum. It is possible that defining genetic risk according to cut points on the depression questionnaire may not be as robust an approach as examining depression as a continuum. Many studies of childhood depressive symptoms have reported estimates of genetic aetiology substantially lower for high depression scores than for scores within the normal range [13, 14, 38]. The results of studies reviewed earlier which suggest that depression may be viewed as a continuum [2, 21, 22, 26, 33, 34, 51] suggest that in the present study, results from analyses where depression is included as a continuous variable may therefore be more robust. It is unclear whether birth-weight is a ‘causal’ risk factor for depression in children and adolescents, although some studies suggest that LBW is an important environmental risk factor for behavioural and cognitive problems [25, 52, 56]. However, the purpose of this paper was not to assess causality, but rather, to examine the association between birth-weight and depression and whether it was modified by genetic and familial risk for depression. Birth-weight has several properties that make it a useful risk factor for examining potential gene-environment interaction effects [25, 42]. First, birth-weight is primarily influenced by environmental rather than genetic factors. For example, in this sample, examination of the MZ and DZ correlations for birth-weight showed that birth-weight was influenced primarily by shared environmental rather than genetic factors as the MZ and DZ correlation coefficients are similar (rMZ=0.781; rDZ=0.697) (full model fitting results available from first author). Moreover, there was little evidence that there were common genes influencing both depressive symptoms and LBW as the cross-twin cross-trait correlations in this sample were near zero for both MZ and DZ twins. This is not true of other risk variables, indeed, there is consistent evidence that many important environmental risk factors (e.g. smoking in pregnancy) are not independent of genetic predisposition [42]. As many risk factors or indicators are influenced by genetic factors this can cause difficulty in detecting and interpreting gene by environment interactions [41, 44]. Second, LBW for gestation does not appear to be associated with maternal depression during pregnancy in developed countries [1, 35, 43, 58]. Indeed, there was no association between current maternal depressive symptoms and birth-weight corrected for gestation in this sample (rtwin 1 birth-weight=0.021, P=0.540; rtwin 2 birth-weight=0.001, P=0.975). This observation reduces the possibility that the influence of birth-weight on depressive symptoms observed in the present study is due to an association with maternal depression although it does not rule out the possibility. Birth weight is influenced by a number of psychosocial and maternal health characteristics including social class, pre-natal smoking, maternal height, weight, age and parity. There were three such factors that have previously been found to influence birth weight, for which data were available in this study (social class, pre-natal smoking, maternal age at birth of child). These were included as predictor variables in regression models in addition to child gender, thus effects for birth weight adjusted for gestation in the present study are independent of these variables although we were not able to control for all variables that have previously been associated with birth weight (e.g. maternal weight at birth). In the present study, depression was assessed according to current symptoms. It is likely that this will underestimate the genetic risk for depression as life time ever rates of depression are significantly higher than point prevalence estimates [10, 24, 39]. We also relied on symptoms rather than diagnoses of depression. However, there is compelling evidence that depression appears as a continuum. Moreover, in particular for tests of interaction, statistical power is low [9, 42] and thus power is substantially increased with the inclusion of continuous rather than categorical measures. The reliance upon parental reports of birth-weight and antenatal risk factors in the present study is not ideal—information from antenatal records would have been preferable. However, the retrospective recollection of birth-weight of children by parents has been found to be accurate in comparison to medical records with correlations of over 0.90 between the two data sources reported [16, 54]. In addition, retrospective recall of pregnancy related events has been shown to be reliable [50]. The reliance on parental reports of the twins’ emotional state may have influenced results. Maternal reports of child psychopathology have been reported to be influenced by the mother’s own mental state. For the most part, it has been found that this results in inflated levels of emotional problems in children being reported by mothers who have emotional difficulties themselves. This is consistent with evidence that depression runs in families, but also raises the possibility that familial risk for children may be partly attributable to rater effects. However, there is some evidence that depressed mothers can be more sensitive to depression in their children [55]. In addition, maternal reports of anxiety and depression are predictive of outcome in young adulthood, which lends some predictive validity to maternal reports of emotional problems in children [53]. Assortative mating has been reported for depression, though reported spousal correlations for depression have been relatively small [28]. Information on the father’s mental state was not available in this study. Assortative mating for depression would result in an inflated DZ twin correlation and thus reduced evidence for genetic influences [30] and we have previously found a genetic influence for depressive symptoms in this sample [38]. One final potential criticism is that birth-weight in twins may not mean the same as in singletons. However, the twin design was necessary to allow for the effects of genetic influences and until susceptibility genes for depression are identified, molecular genetic indicators of risk for depression are not feasible. In summary, the present results suggest that LBW for gestation and genetic/familial factors co-act and interact in influencing early parent-rated depressive symptoms. These observations suggest that it may be useful to monitor children in cases where there is a family history of depression and the child was small for gestational age.

Crit_Care-8-6-1065076

Tight blood glucose control: a recommendation applicable to any critically ill patient?

The issue of tight glucose control with intensive insulin therapy in critically ill patients remains controversial. Although compelling evidence supports this strategy in postoperative patients who have undergone cardiac surgery, the use of tight glucose control has been challenged in other situations, including in medical critically ill patients and in those who have undergone non-cardiac surgery. Similarly, the mechanisms that underlie the effects of high-dose insulin are not fully elucidated. These arguments emphasize the need to study the effects of tight glucose control in a large heterogeneous cohort of intensive care unit patients. Until the end of the past millenium, relatively little attention was given to control of blood sugar levels. In critically ill patients, hyperglycaemia was considered to be physiological because it results from the metabolic and hormonal changes that accompany the stress response to injury. In most intensive care units (ICUs), blood sugar was checked every 4–6 hours and hyperglycaemia (defined as blood sugar levels >10–12 mmol/l [180–216 mg/dl]) was corrected by subcutaneous or intravenous insulin. The presence of pre-existing diabetes mellitus or post-neurosurgical status often prompted more intense control of hyperglycaemia. Furthermore, the issue of glucose control was discussed in few sessions or satellite symposia during intensive care meetings. The deleterious effects of hyperglycaemia during critical illness have been characterized over the past few years, and include an increased susceptibility to infections and thromboses, macrovascular and microvascular changes, and delayed wound healing, among other effects (for review [1]). Renewed interest in control of hyperglycaemia in critically ill patients (Fig. 1) followed the publication of a study conducted by Van den Berghe and coworkers in 2001 [2]. Those investigators reported a 43% decrease in relative intensive care mortality as well as consistent decreases in several surrogate markers of disease severity in patients randomly assigned to tight glucose control by intensive intravenous insulin therapy. A post hoc multivariate logistic regression analysis of these data suggested that control of hyperglycaemia played a more important role than did the amount of insulin administered [3]. Interestingly enough, at least two recent retrospective, large-scale studies [4,5] confirmed that outcome was improved in patients whose average blood glucose was maintained below 8 mmol/l (144 mg/dl; Table 1). Although the findings reported by Van den Berghe and coworkers are impressive, some concern arose regarding the applicability of these results to other types of patients. Of the patients studied, 63% were admitted for follow up after cardiac surgery; this high proportion was felt to be consistent with a particular benefit from tight glucose control with intensive insulin in these patients, but there is uncertainty regarding whether tight glucose control is beneficial in patients who have not undergone cardiac surgery. Fear of life-threatening hypoglycaemia and increased workload and costs probably underlie the reluctance of many intensivists to launch systematic protocols of tight glucose control. Indeed, many intensivists still use a high glucose threshold (10 mmol/l [180 mg/dl]) [6]. In a European survey (unpublished data) we found considerable variation in the glycaemic thresholds employed in ICUs, which ranged from 6 to 11.1 mmol/l (108–200 mg/dl). Some arguments against generalized use of tight glucose control are reported in the present issue of Critical Care by Vriesendorp and coworkers [7]. In a retrospective study performed at one centre in Amsterdam, those authors found that, after oesophageal surgery in patients without significant cardiovascular compromise (ASA class I–II), postoperative hyperglycaemia was not a risk factor for infectious complications. Only by univariate analysis were they able to find an improvement in patients with blood glucose levels below 9.3 mmol/l (167 mg/dl) in terms of length of ICU stay. These findings differ strikingly from those of other studies [2,4,5]. Although the report by Vriesendorp and coworkers challenges the concept of tight glucose control, it can hardly be considered a major piece of evidence against it. Indeed, blood glucose concentrations were presented as means of values recorded only over 48 hours, whereas the ICU stay extended up to 71 days, with a median of 3 days. Insulin was administered to only 9% of the patients during the 48-hour period of observation. In addition, patients received a mean of only 22.5 g glucose/day, and were fed early after surgery with an enteral solution of 'immunonutrients' – a potential confounding factor with respect to infectious morbidity. However, despite these limitations, as well as others that are acknowledged by the authors, the findings of the study support the hypothesis that tight glucose control could be of greater benefit to patients with cardiovascular disease than to those without. In conclusion, as recently suggested by Van den Berghe [8], further studies are needed to confirm the benefits of tight blood glucose control with intensive insulin therapy in a heterogeneous population of ICU patients. Hence, a large randomized prospective multicentre trial is warranted. Such study will also help in determining the physiological importance of the effects of insulin and, more importantly, will provide intensive care workers with key information for guiding the management of blood glucose in critically ill patients. Abbreviation ICU = intensive care unit. Competing interests The author(s) declare that they have no competing interests.

J_Mol_Biol-1-5-2291452

Crosstalk between the Protein Surface and Hydrophobic Core in a Core-swapped Fibronectin Type III Domain

Two homologous fibronectin type III (fnIII) domains, FNfn10 (the 10th fnIII domain of human fibronectin) and TNfn3 (the third fnIII domain of human tenascin), have essentially the same backbone structure, although they share only ∼ 24% sequence identity. While they share a similar folding mechanism with a common core of key residues in the folding transition state, they differ in many other physical properties. We use a chimeric protein, FNoTNc, to investigate the molecular basis for these differences. FNoTNc is a core-swapped protein, containing the “outside” (surface and loops) of FNfn10 and the hydrophobic core of TNfn3. Remarkably, FNoTNc retains the structure of the parent proteins despite the extent of redesign, allowing us to gain insight into which components of each parent protein are responsible for different aspects of its behaviour. Naively, one would expect properties that appear to depend principally on the core to be similar to TNfn3, for example, the response to mutations, folding kinetics and side-chain dynamics, while properties apparently determined by differences in the surface and loops, such as backbone dynamics, would be more like FNfn10. While this is broadly true, it is clear that there are also unexpected crosstalk effects between the core and the surface. For example, the anomalous response of FNfn10 to mutation is not solely a property of the core as we had previously suggested. Introduction Studies of the folding of structurally related proteins have been a powerful tool for investigating conservation of folding pathways,1 identifying structurally important residues,2 and examining the role of highly conserved residues.3 One of the most common folds in the SCOP database4 is the immunoglobulin (Ig)-like fold. Over 40,000 Ig-like domains have been identified in the current PFam database5 and the fold is found in a number of different superfamilies, where there is no apparent sequence identity between superfamilies.4 In this study, rather than seeking to understand what related proteins have in common, we ask a different question—can differences between closely related proteins be explained? Many members of the Ig-like fold have been well characterised, leading to: identification of the key residues essential for formation of the Greek key structure,6,7 the observation of a correlation between stability and folding rate,8–11 the identification of a common folding pathway,12–16 and the identification of the role of conserved proline residues3 and the conserved tyrosine corner motif.2 However, when two Ig-like domains from the fibronectin type III (fnIII) superfamily, the 10th fnIII domain of human fibronectin (FNfn10) and the third fnIII domain of human tenascin (TNfn3), were studied in detail and compared, they were found to differ markedly in several respects. FNfn10 and TNfn3 are essentially structurally identical (backbone RMSD is 1.2 Å), but have low sequence identity (24%). They differ in their stabilities17,18 and response to mutation19; they differ in their folding kinetics17,18,20 (although they share a common folding mechanism12,16); they display different backbone and side-chain dynamics21–23`; and, finally, they differ in their response to mechanical force.24–26 Why do proteins that are structurally almost identical behave so differently? Which components of a protein are responsible for the various aspects of its behaviour? How independent are the properties of the surface and hydrophobic core? We have addressed these questions by making a chimera of these two fnIII domains. The chimera, FNoTNc, was created with the “outside” (surface and loops) of FNfn10 and the core of TNfn3.26 Fifteen mutations were made in the core of FNfn10 so that all buried residues (with < 10% solvent-accessible surface area) are identical with those residues in the core of TNfn3 (total number of core residues = 27) (Fig. 1). Thus, we can test how transferable the properties of the core and surface of the respective parents are when combined in this way. FNoTNc is a stable, folded protein that is structurally almost identical with the parent proteins (Supplementary Fig. 1). FNoTNc has also retained the cell-adhesion activity of the FNfn10 parent protein mediated by specific integrin binding.26 We have previously used this chimera to demonstrate that resistance to mechanical force is a core property: FNoTNc has mechanical unfolding properties indistinguishable from TNfn3 and very different from FNfn10.26 Here we investigate the stability, folding and dynamics of the new, chimeric protein. Most protein engineering analyses concentrate on the hydrophobic core of proteins, since it was shown in 1959 that the hydrophobic interaction is the major factor involved in protein folding27 and surface mutations rarely affect protein stability by more than ∼ 1 kcal mol− 1. Our results suggest, however, that the surface and loops can play a key (and sometimes unexpected) role in determining the biophysical properties of a protein. Results Thermodynamic stability Wild-type FNoTNc FNoTNc has a free energy of unfolding (ΔGD–N) of 7.5 kcal mol− 1, intermediate between the stabilities of FNfn10 and TNfn3 (9.4 and 6.7 kcal mol− 1, respectively) at pH 5.0. We infer that the surface interactions and loop and turn regions of FNfn10 make a significant contribution to the overall stability of FNoTNc. As was shown for FNfn10, the ΔGD–N of FNoTNc is independent of pH between pH 5.0 and pH 7.0, whereas TNfn3 is less stable at pH 7 (5.7 kcal mol− 1). The dependence of the stability of TNfn3 on pH has been shown to be the result of the presence of patches of acidic residues on the surface of TNfn3, which are, of course, not present in FNoTNc.17 Anomalous response of certain peripheral mutations A number of core residues were mutated in FNoTNc to investigate the response of the protein to mutation. These were positions that had previously been investigated in the parent proteins FNfn10 and TNfn3. The thermodynamic stability of the mutant proteins was determined by chemical denaturation in guanidinium chloride (GdmCl). The mutations can clearly be divided into two categories. A few peripheral mutations (mutations in the A and G strands and the B–C loop) have little effect on stability as was previously observed in FNfn10 (Fig. 2a), whereas most other mutations were more typically destabilising. In the latter case, the ΔΔGD–N was similar to (but in general slightly lower than) what had been observed previously in TNfn3 (Fig. 2b). The residues in each category are mapped onto a backbone ribbon representation of FNoTNc in Fig. 2c. ΔΔGD–N values for all mutations are compared to those of FNfn10 and TNfn3 in Supplementary Table 1. Equilibrium hydrogen exchange The rates of hydrogen–deuterium exchange were measured at pD 7.0. Measured rate constants for exchange, kex, ranged between 9.3 × 10− 2 and 1.6 × 10− 4 min− 1. Values of kex and the apparent free energies of exchange, ΔGexapp, are listed in Supplementary Table 2. The ΔGexapp of FNoTNc, FNfn10 and TNfn3 are compared in Fig. 3. The hydrogen exchange behaviour of FNoTNc clearly resembles that of FNfn10: both have many residues that exchange in the experimental dead time, in particular in the edge strands (A, C′, E and G strands) that are more protected in TNfn3. Note that this does not result from differences in the intrinsic stabilities of the domains—TNfn3 is significantly less stable than either FNoTNc or FNfn10 under these experimental conditions. Folding kinetics Wild-type FNoTNc The rate constants of folding and unfolding were determined using stopped-flow measurements monitored by changes in fluorescence > 320 nm. The logarithm of the folding and unfolding rate constants was plotted against concentration of denaturant (Fig. 4). Both unfolding and refolding arms show a linear dependence on denaturant concentration. There is an additional refolding phase, which we attribute to proline isomerisation and do not consider further. (Both FNfn10 and TNfn3 have proline-isomerisation limited phases, and FNoTNc has eight Pro residues.) To compare the kinetics of FNoTNc with those of FNfn10 and TNfn3 (which have been studied using different denaturants due to large differences in stability), the logarithm of the observed rate constants is plotted against stability in Fig. 4. The free energy of unfolding calculated from the ratio of the folding and unfolding rate constants extrapolated to 0 M denaturant (7.7  kcal mol− 1) and the kinetic m value (2.1 kcal mol− 1 M− 1) are the same as the equilibrium ΔGD–N (7.5 kcal mol− 1) and m value (2.1 kcal mol− 1 M− 1) within error—consistent with the folding being a 2-state process, with no stable intermediates being populated. The relative compactness of the transition state can be determined from the folding and unfolding m values. FNoTNc has a βT value of 0.6, similar to those of TNfn3 and FNfn10. Φ-value analysis The folding kinetics of 18 variants of FNoTNc with nondisruptive deletion mutations were measured as for the wild type. Few of these mutants were in the A, B and G strands due to very small changes in stability with mutation in general. In a few of the mutants, the unfolding arm of the chevron plots shows negative curvature at high denaturant concentrations, which we attribute to the Hammond effect: the simplest model that fits the data.28–30 All chevrons were fitted therefore to a two-state equation with a quadratic term in the unfolding arm. (All the chevron plots are shown in Supplementary Fig. 2.) Note that the model chosen to fit the kinetic data does not affect our results because equilibrium values of ΔΔGD–N were used to determine Φ. In Table 1, the Φ values for each mutant are compared to those in TNfn3 and FNfn10. The Φ values are generally low, closely resembling those of TNfn3. Dynamics Backbone dynamics 15N T1 and T2 relaxation time constants and 1H15N nuclear Overhauser enhancement parameters were measured using 1H–15N correlation spectroscopy (Supplementary Table 3). The generalised order parameter, S2, and a conformational exchange broadening parameter, Rex, were determined for each backbone amide (Fig. 5 and Supplementary Table 4). The S2 values are similar to those of both the parents. However, the high values of Rex that were seen in the A/B region of FNfn10, have decreased in FNoTNc. Side-chain dynamics The relaxation of the operator terms IzCzDz, IzCzDy and IzCz was measured to give deuterium relaxation time constants T1(D) and T2(D)31 (Supplementary Table 5). Order parameters, S2, were determined for each methyl group using the standard model-free formalism as previously described22 (Supplementary Table 6). The dynamics data for those methyl groups that had overlapping peaks were treated with caution, as the contribution from the overlapped peaks cannot be separated. Nevertheless, we have some confidence in these results due to the agreement of the order parameters for residues that have both overlapped and well-resolved methyl groups. S2 ranges from zero to unity, with higher values of S2 indicating greater conformational restriction. The methyl S2 values are shown projected onto the protein structure and compared to the parent proteins in Fig. 6. Within the core of FNoTNc is a cluster of deeply buried residues, which have unusually low order parameters, as has previously been observed in TNfn3. Discussion The core of FNoTNc is similar to that of TNfn3, but apparently less closely packed. Evidence from mutations FNoTNc is close in structure to both the parent domains (backbone RMSD, excluding the mobile C–C′ and F–G loops, is 0.95 and 0.89 Å compared to FNfn10 and TNfn3, respectively). From the crystal structure we would deduce that the core of FNoTNc is essentially the same in terms of core packing and number of core contacts as TNfn3, the parent protein that donated the core residues (Fig. 7a). However, the calculated free volume in the interior of FNoTNc is larger than for TNfn3 (128.4 Å3 versus 118.1 Å3, respectively). When the response of FNoTNc to mutation is compared to TNfn3, it is clear that the ΔΔGD–N values of FNoTNc are slightly lower, on average ∼ 80% of those in TNfn3 (Fig. 7b). Since the loss of free energy on mutation is strongly correlated with the number of side-chain contacts deleted,32,33 we infer that the less tight packing of the core of FNoTNc accounts for the difference in ΔΔGD–N compared to TNfn3. Evidence from side-chain dynamics The same conclusion can be drawn from the analysis of the side-chain dynamics. The core of TNfn3 has been shown to be exceptionally mobile, with several of the most buried residues falling several standard deviations below the expected order parameter for their residue type.22,23 FNfn10 has a core that is much more conformationally restricted, with order parameters within the usual range for buried residues. Our previous analysis of the side-chain dynamics of FNfn10 and TNfn3 led us to suggest that the differences in core dynamics between FNfn10 and TNfn3 could, at least in part, be ascribed to the slightly lower core packing in TNfn3; the residues in TNfn3 with unusually low order parameters are also found to have packing volumes that are larger than expected. The buried side chains of FNoTNc were found to have, on average, even lower order parameters than the same side chains in TNfn3 (Fig. 6d). Behaviour of peripheral regions of the protein is modulated by the surface and loops Evidence from mutation A number of sites in FNfn10 were identified where upon mutation the ΔΔGD–N is significantly lower than for the same (or an equivalent) mutation in TNfn3.19 These sites are at the periphery of the protein and include residues Pro5 and Leu8 (A strand), Pro25 (B–C loop) and residues Ser85 and Phe92 (G strand). Unexpectedly, in FNoTNc we see the same anomalous response to mutation for Pro5, Pro25 and Ser85, although Ile8A and Phe92A now have similar ΔΔGD–N values to TNfn3 (Fig. 2a). Pro5, Pro25 and Ser85 are all found in the same region of the molecule (Fig. 2c). The anomalous response to mutation in this peripheral region of the core of FNoTNc and FNfn10 is intriguing: apparently the “plastic” response of the protein to mutation of these three buried residues is modulated by the surface of the protein and is not determined by the core alone. The additional plasticity at this end of the hydrophobic core may be due to its proximity to the longer FG loop in FNfn10 and FNoTNc, which restricts motion less than the corresponding loop in TNfn3. Evidence from hydrogen exchange FNoTNc, FNfn10 and TNfn3 have very similar structures and hydrogen bonding patterns. Further evidence for “plasticity” of FNfn10 compared to TNfn3 came from the observation that the peripheral A, C′, E and G strands were significantly less well protected against amide exchange in FNfn10 than in TNfn3, despite the fact that FNfn10 is considerably more stable than TNfn3.19 Under the experimental conditions, hydrogen exchange is in the EX2 regime,34,35 meaning that exchange reflects local stability; that is, these peripheral regions of FNfn10 have lower local stabilities than TNfn3. Our experiments clearly show that the exchange behaviour of FNoTNc is similar to FNfn10—with low protection of residues in the same peripheral strands (Fig. 3). Again surprisingly, local instability appears to be a function of the surface of the protein and not a property of the core; however, the plasticity inferred from response to mutation and local instability still appear to be related, as was previously inferred. This is not, however, related to slow exchange motions of the backbone, as we had previously suggested, since the millisecond time scale motions observed in the A/B region of FNfn10 are not found in FNoTNc. The stability of FNoTNc is modulated by both the core and the surface In summary, despite the evidence for FNoTNc having a less well packed core than TNfn3, with the mutation of buried side chains having less effect on the overall stability (Fig. 7b), FNoTNc is significantly more stable than TNfn3, suggesting again that the surface and loops of FNfn10 contribute significantly to the stability of FNoTNc. This is not a new observation; it has been previously demonstrated that surface interactions can play a significant role in the stabilisation of proteins.36 Interestingly, however, the “local stability” of the peripheral regions of FNoTNc is lower than the local stability of TNfn3, as manifested in the hydrogen exchange experiments. The surface residues of FNfn10 are modulating the stability of these peripheral regions. A concurrent study by Siggers et al.37 investigated the stability and dynamics of a series of loop-swap mutants of FNfn10 and TNfn3: essentially these were mutants where the C–C′ and F–G loops were exchanged. Interestingly, both TNfn3 domains had lower thermal stabilities than the wild-type protein, whereas the thermal stability of FNfn10 was unaffected by the exchange. We infer that in our chimeric protein, interactions of the surface residues are most important in the increase in stabilisation of FNoTNc beyond that of TNfn3, and not the new C–C′ and F–G loops. The TNfn3 core governs the folding kinetics Wild-type kinetics In order to compare the kinetics of the three proteins directly, the logarithm of the rate constant has been plotted against stability (Fig. 4). This unusual scale is used as each protein was characterised using a different denaturant. FNoTNc folds at a rate intermediate between FNfn10 and TNfn3. In broad terms, the chevron of FNoTNc resembles that of TNfn3. It has previously been pointed out that the folding rates of Ig-like domains in water do not correlate with contact order (the average sequence separation of native contacts).8,38 For the present three proteins, which have almost identical contact order (absolute contact order 15–16%, relative contact order 16–17%), the rate constants at the folding midpoint span 2.5 orders of magnitude. Thus, the variation in the folding rate cannot be explained only by differences in contact order—protein stability plays a key role.8,39 FNfn10 shows clear rollover in both the folding and unfolding arms. The rollover in the folding arm has previously been ascribed to the presence of a populated folding intermediate.18 Neither FNoTNc nor TNfn3 show any evidence for population of a folding intermediate—the presence of a stable folding intermediate in FNfn10 appears to be the result of core interactions. Curvature in the unfolding arm of a chevron plot has been ascribed to the presence of a high-energy intermediate, to Hammond effects, or to population of an unfolding intermediate.30,40,41 This has not been investigated for any of these fnIII domains; indeed, it is often not possible to distinguish between the first two cases.42 Both FNfn10 and some mutants of FNoTNc display curvature in the unfolding arm that is not seen in wild-type TNfn3. It should be noted, however, that unfolding curvature has been seen in a less stable form of TNfn3 (missing the final two C-terminal residues) and may simply not be observed in this case because the unfolding arm in the stable form of TNfn3 used here is relatively short. In summary, FNoTNc has similar folding characteristics to TNfn3. This suggests that the core of these proteins plays the major role in determining the folding behaviour. However, FNoTNc is more stable than TNfn3. When we compare the folding and unfolding rate constants in water we find that FNoTNc folds some 10 times faster than TNfn3 (kfH2O = 60 and 6 s− 1, respectively) but unfolds at approximately the same rate as TNfn3 (kuH2O = 2 × 10− 4 and 5 × 10− 4 s− 1, respectively). Thus, the stabilising surface interactions apparently stabilise the transition state of FNoTNc as much as the native state, while still not causing a folding intermediate to be populated. This, perhaps, allows us to pinpoint further which surface interactions are responsible for the added stability of FNoTNc (over TNfn3). At the transition state, the loops and the peripheral A and G strands are largely unstructured, but there is structure in the B, C, C′, E and F strands, particularly towards the centre of the core (see Φ value section below). Interactions between surface residues in these strands are therefore the most likely candidates for providing additional stability to the FNoTNc protein (both native and transition states), above those interactions between residues that are packing in the core. In contrast, FNoTNc folds some three times more slowly than FNfn10 and the unfolding rate constant of FNfn10 in water is approximately an order of magnitude lower (∼ 2 × 10− 5 s− 1). Φ-value analysis The Φ-value analysis reveals the extent of formation of structure in the transition state.43 The pattern of Φ values in FNfn10 and TNfn3 are similar, suggesting that they have a common folding mechanism. There are differences, however. The Φ values of TNfn3 tend to be much lower than those of FNfn10: TNfn3 has five Φ values greater than 0.4,12 while FNfn10 has eight16; in FNfn10 the folding nucleus appears to be more extensive than in TNfn3, with more than one residue in the central C, E and F strands having high Φ values. FNoTNc has even lower average Φ values than TNfn3 with only two Φ values greater than 0.4 (Table 1) Both FNoTNc and TNfn3 have less extensive formation of structure in the transition state than FNfn10. However, Φ-value analysis is at its most powerful when patterns of Φ values are compared in different proteins rather than by direct comparison of Φ values. Previous analysis of TNfn3 has identified a ring of interacting residues in the B, C, E and F strands as the folding nucleus, with residues in the C′ strand packing onto these.12 The Φ values of the peripheral A and G strands are all close to 0, suggesting that they are unformed at the transition state. A similar pattern of Φ values was seen in FNfn10, although this Φ-value analysis was less complete.16 The Φ values for FNoTNc were divided into three categories of low (Φ < 0.25), medium (0.25 < Φ < 0.35) and high (Φ > 0.35) (Fig. 8) and compared to the pattern of Φ values for the identical residues in TNfn3. The pattern of Φ values is very similar to that of TNfn3, although there are slight qualitative differences. Again, the highest Φ values are found in the B, C, C′, E and Φ strands and the Φ values in the A and G strands are ∼ 0. The folding mechanism of FNoTNc is unperturbed and the same as both the parents, although from the magnitude and extent of the residues with higher Φ values the transition-state structure appears to be closer to that of TNfn3 than FNfn10. Dynamics from NMR is determined by local interactions Side-chain dynamics The side-chain dynamics appear to reflect core packing, as was discussed above, and so FNoTNc resembles TNfn3 more closely than FNfn10. Backbone dynamics The S2 values of FNoTNc are generally very similar to those of both the parents (Fig. 5). It is interesting to compare our study with that of the loop swap mutants of FNfn10 and TNfn3 from Palmer and coworkers,37 where the F–G loop of FNfn10 was grafted into TNfn3. In that case, the S2 values in the loop region were more similar to those in FNfn10, than those in the equivalent positions in TNfn3. A similar result was seen in the protein where the C–C′ loop was grafted in. It is difficult to compare our results directly to those of Siggers et al.37: their method of analysis leads to greater differences between the S2 values of FNfn10 and TNfn3, resulting in more visible effects on the S2 values when the loops are swapped than we see in FNoTNc compared to the parent proteins. However, there is a general agreement that the behaviour of the loops is a local property, rather than being a direct effect of either core or surface interactions. Conclusion We have grafted the core of one fnIII domain (TNfn3) into the homologue FNfn10, creating a chimera, FNoTNc, which has retained the structure of the parent proteins. Using several different probes, we have shown that FNoTNc does not behave like either one of the parent proteins alone. Instead, it has retained a number of properties of each. We find that each property investigated clearly resembles the behaviour of one of the parents, enabling us to separate the contribution of the core and the surface of the protein in determining the behaviour of the domain. Some of these are unsurprising, such as the pH dependence of stability, the core side-chain dynamics and the dependence of folding on the composition of the core. However, the surface of the protein confers significant stability not only on the native state, but also on the transition state for folding. Others properties are less predictable. The surface of the protein confers “plasticity” in peripheral regions of the proteins as detected by the anomalous response of some regions of the core to mutation and hydrogen exchange protection patterns. This suggests that the surface of a domain may have a more significant coupling with the core than we had previously considered. Since most biophysical studies tend to focus on the core of a protein, this coupling is a relatively unexplored area of research. Materials and Methods Chemicals GdmCl was purchased from MP Biomedicals Inc., guanidine isothiocyanate from Gibco-BRL and urea from BDH Laboratory Supplies. Protein expression and purification Site-directed mutagenesis reactions were performed with the QuickChange kit from Stratagene using the FNoTNc plasmid. The identity of the mutants was confirmed by DNA sequencing. The mutants studied in this work are listed in Table 1 and Supplementary Table 1. The nature of the mutation is indicated with the wild-type residue first (single-letter code), the position of the mutation second and the mutant residue third. Expression and purification of FNoTNc and mutants was performed as described earlier for TNfn3.20 Measurements of protein stability All biophysical measurements were performed in 50 mM sodium acetate buffer, pH 5.0 at 25 °C unless otherwise stated. The stability of FNoTNc and FNoTNc mutants were determined by equilibrium denaturation experiments using GdmCl and by standard methods using 1 μM protein.20 Kinetic measurements Kinetics were measured using fluorescence stopped-flow measurements in 50 mM sodium acetate buffer, pH 5.0, at 25 °C and were monitored by changes in fluorescence above 320 nm. Refolding measurements for FNoTNc were made in 0 M denaturant by stopped-flow fluorescence using pH jumps from pH 12.4 to pH 5.0 as previously described.12 (FNoTNc is acid stable.) NaOH (25 mM, pH 12.4) unfolds FNoTNc completely (data not shown). Φ-value analysis Φ values were determined from refolding data at 1.0 M GdmCl, to avoid the errors associated with long extrapolation, from Eq. (1):43whereand kfWT and kfmut are the rate constants for folding of wild-type and mutant proteins, respectively. NMR sample preparation FNoTNc was expressed and purified by affinity chromatography as previously described.17 Uniformly 15N labelled and 13C and 15N labelled samples were expressed in M9 minimal media containing 15NH4Cl and [U-13C]6-glucose as the sole nitrogen and carbon sources. Samples for side-chain dynamics were expressed as previously described.22 Chemical shift assignments Backbone assignment experiments were carried out on a double-labelled (13C, 15N) sample of FNoTNc, at an approximate concentration of 1–2 mM, in 50 mM imidazole buffer at pH 7.0 in 10% D2O. Sodium azide (0.05%) was added to prevent microbial growth. The sample was centrifuged to remove insoluble protein and degassed. Spectra were acquired at 298 K on a Bruker DRX500 spectrometer with an inverse triple-resonance cryogenic probe. Backbone assignments were based on HNCACB, HNCO and CBCA(CO)NH experiments together with the 1H–15N heteronuclear single quantum coherence (HSQC) spectrum. Side-chain 1H and 13C resonance assignments were obtained from 3-D HCCH–total correlated spectroscopy (TOCSY) H(CCCO)NH and (H)CC(CO)NH preceding TOCSY spectra. The spectra were processed and analysed using NMRpipe and Sparky.44,45 The 1H–15N HSQC has excellent resolution, although resonances from 12 residues in loop regions cannot be detected at either pH 5 or pH 7 (Supplementary Fig. 3 and Supplementary Table 7). All samples for side-chain methyl assignment were prepared in 50 mM sodium acetate buffer at pH 5.0 in 10% D2O, at a concentration of 1–2 mM. Sodium azide was added to prevent microbial growth. The sample was centrifuged to remove insoluble protein and degassed. All experiments were carried out as previously described.22,46,47 Chemical shift assignments of the side chains were made using standard triple-resonance experiments. Many of the signals in the 1H–13C HSQC are overlapped, meaning that 5 of the 64 methyl groups in the 1H–13C HSQC could not be assigned with confidence. Assignment of the leucine and valine methyl groups was made stereospecifically, based upon the phase of peaks in a 1H–13C HSQC acquired for a sample with 10% 13C enrichment (Supplementary Fig. 4 and Supplementary Table 7). Hydrogen exchange Hydrogen exchange experiments were carried out under EX2 conditions on a 15N-labelled sample of FNoTNc, at an approximate concentration of 1–2 mM, in 50 mM imidazole buffer at pD 7.0 in 10% D2O. Sodium azide (0.05%) was added to prevent microbial growth. The exchange of amide protons was followed by the decay of intensity of peaks in HSQC spectra.48 The apparent free energy of exchange, ΔGexapp, was determined from the rate constant of exchange, kex, and the intrinsic rate constant, kint, determined from peptide data to take account of the primary sequence of the protein and exchange conditions49,50 using Eq. (2) and intrinsic rate constants determined using the software Sphere†.51 Backbone 15N relaxation measurements Backbone dynamics were determined from 15N T1 and T2 relaxation times and the steady-state heteronuclear 1H15N nuclear Overhauser enhancement at 500 MHz as previously described.22 The data were analysed using standard protocols for backbone dynamics with the program TENSOR2. Side-chain methyl 2H relaxation measurements Side-chain deuterium relaxation times T1(D) and T1ρ(D) were determined by measuring the relaxation of the two- and three-spin operator terms, IzCz, IzCzDz and IzCzDy and analysed as previously described.22,31

Int_J_Cardiovasc_Imaging-4-1-2233708

3.0 T cardiovascular magnetic resonance in patients treated with coronary stenting for myocardial infarction: evaluation of short term safety and image quality

Purpose To evaluate safety and image quality of cardiovascular magnetic resonance (CMR) at 3.0 T in patients with coronary stents after myocardial infarction (MI), in comparison to the clinical standard at 1.5 T. Methods Twenty-five patients (21 men; 55 ± 9 years) with first MI treated with primary stenting, underwent 18 scans at 3.0 T and 18 scans at 1.5 T. Twenty-four scans were performed 4 ± 2 days and 12 scans 125 ± 23 days after MI. Cine (steady-state free precession) and late gadolinium-enhanced (LGE, segmented inversion-recovery gradient echo) images were acquired. Patient safety and image artifacts were evaluated, and in 16 patients stent position was assessed during repeat catheterization. Additionally, image quality was scored from 1 (poor quality) to 4 (excellent quality). Results There were no clinical events within 30 days of CMR at 3.0 T or 1.5 T, and no stent migration occurred. At 3.0 T, image quality of cine studies was clinically useful in all, but not sufficient for quantitative analysis in 44% of the scans, due to stent (6/18 scans), flow (7/18 scans) and/or dark band artifacts (8/18 scans). Image quality of LGE images at 3.0 T was not sufficient for quantitative analysis in 53%, and not clinically useful in 12%. At 1.5 T, all cine and LGE images were quantitatively analyzable. Conclusion 3.0 T is safe in the acute and chronic phase after MI treated with primary stenting. Although cine imaging at 3.0 T is suitable for clinical use, quantitative analysis and LGE imaging is less reliable than at 1.5 T. Further optimization of pulse sequences at 3.0 T is essential. Introduction The combination of functional cardiovascular magnetic resonance (CMR) and late gadolinium enhancement (LGE) is evolving as an important diagnostic [1, 2] and prognostic [3–5] modality in patients with ischemic heart disease. Because the availability of high field MR systems is increasing, the need arises to evaluate the performance and clinical value of these systems in cardiovascular disease. Earlier reports already suggested that 3.0 T MR systems offer higher temporal and spatial resolution, due to an increased signal-to-noise ratio, which would be especially advantageous in CMR scanning [6–8]. However, these studies were all assessed in healthy volunteers and in patients with non-ischemic heart disease or suspected coronary artery disease, and without the presence of coronary stents [6–10]. There is limited to no data about safety and image quality at 3.0 T CMR scanning in the acute or chronic phase after myocardial infarction (MI), in patients treated with percutaneous coronary intervention and primary stenting. In these patients B0 inhomogeneities (induced by the heart-lung interface or from coronary stents), flow artifacts, inhomogeneity of normal myocardial suppression in LGE (induced by B1 inhomogeneity) and poor cardiac triggering may interfere with the gain offered by the higher magnetic field. The aim of this study was to test whether CMR scanning at 3.0 T is safe and feasible in patients with coronary stents in the acute and chronic phase after MI, and to prospectively compare image quality at 3.0 T with the current clinical standard at 1.5 T. Furthermore, the presence and significance of different image artifacts are considered. Methods Patient population Patients were eligible for the study if they had been admitted with a first ST-elevation acute MI, according to standard electrocardiographic and enzymatic criteria [11], and had undergone successful primary PCI with stent implantation. Exclusion criteria were electrocardiographic evidence of reinfarction, haemodynamic or other clinical instability or (relative) contraindications for CMR such as claustrophobia, pacemakers, intracerebral aneurysm clips or very irregular heart rhythm. Patients were treated with aspirin, heparin, abciximab, clopidogrel, statins, beta-blockade and ACE-inhibitors, according to ACC/AHA practice guidelines [12]. The study was approved by the local ethics committee and all patients gave written informed consent. CMR parameters Eighteen CMR scans were performed with a 3.0 T MR system (Intera, Philips, Best, The Netherlands), with a gradient performance of 30 mT/m and slew rate of 150 T/m/s, using a six element cardiac phased array surface coil. Another 18 CMR scans were acquired with a 1.5 T MR system (Magnetom Sonata, Siemens, Erlangen, Germany), with a gradient performance of 40 mT/m and slew rate of 200 T/m/s, using an eight element cardiac phased array surface coil. Cine imaging was performed at both field strengths using a SSFP pulse sequence, without parallel imaging. Long axis views, as well as short axis views covering the entire left ventricle were acquired during repeated breath-holds in expiration. At 3.0 T, cine SSFP sequence parameters were a temporal resolution between 25 ms and 50 ms, excitation angle of 45°, receiver bandwidth 868 Hz/pixel, TR/TE of 3.8/1.9 ms, matrix 192 × 155 and voxel size of 1.5 × 1.8 × 6.0 mm3. At 1.5 T, the temporal resolution was between 35 ms and 50 ms with an excitation angle of 60°, receiver bandwidth 930 Hz/pixel, TR/TE of 3.2/1.6 ms, matrix 256 × 156 and voxel size of 1.4 × 1.9 × 6.0 mm3. LGE images were acquired in mid-diastole using a 2D segmented inversion-recovery gradient-echo pulse sequence, 10–15 min after intravenous injection of 0.2 mmol/kg of a gadolinium chelate (Dotarem, Guerbet, Roissy, France). The LGE images were obtained in exactly the same orientation as the cine images. Sequence parameters at 3.0 T were an excitation angle of 25°, receiver bandwidth 434 Hz/pixel, TR/TE of 3.5/1.3 ms, matrix 192 × 119, voxel size of 1.7 × 2.1 × 6.0 mm3, triggering every other heart beat, and an inversion time between 250 ms and 350 ms to null remote myocardium. At 1.5 T, sequence parameters were an excitation angle of 25°, receiver bandwidth 130 Hz/pixel, TR/TE of 9.6/4.4 ms, matrix 256 × 166, voxel size of 1.4 × 1.7 × 6.0 mm3, triggering every other heart beat, and an inversion time between 220 ms and 300 ms to null remote myocardium. Safety and quality analysis To evaluate patient safety, a physician was present at the CMR scanner throughout the scan. Heart rhythm was monitored continuously. The patient was asked to report any discomfort and symptoms during the scan procedure. Additionally, a repeat catheterization was performed in 16 patients who underwent CMR examination in the acute phase after MI (9 at 3.0 T, 7 at 1.5 T). Stent position and patency after CMR scanning were visually assessed on the repeat catheterization and compared to the primary PCI. Occurrence of repeat intervention or hospitalization within 30 days of the CMR examination was recorded. Different types of artifacts were reviewed and marked as being clinically relevant if the artifact interfered with visualisation of the myocardium. Flow related artifacts in SSFP imaging [13], and artifacts due to static field inhomogeneities such as the heart-lung interface were scored on cine images. Separately, we looked at artifacts caused by the coronary stent on the cine images, and measured the maximum artifact diameter, perpendicular to the length of the stent, using an appropriate cardiac view. In addition, the potential effect of B1 inhomogeneity on the homogeneous suppression of viable myocardium was visually assessed on LGE images. Furthermore, image quality of the cine and LGE images were scored on a separate workstation (Centricity Radiology v6.1, GE Medical Systems, Zeist, the Netherlands) by four independent observers, who were blinded for MR system and clinical history. To distinguish between image quality that is satisfactory for clinical use or high quality images for research purposes, images were scored on a scale from 1 to 4: 1 not clinically useful; 2 clinically useful, but of insufficient quality for quantitative analysis; 3 clinically useful and of sufficient quality for quantitative analysis; 4 excellent quality. The following definitions were used for evaluation of the cine images: 1 poor quality, extensive artifacts, completely obscuring endocardial borders; 2 moderate quality, assessment of global function is possible, partly using assumptions, but regional wall thickening is not possible in all segments, due to interfering artifacts; 3 good quality, assessment of global and regional function is possible, despite some small artifacts; 4 excellent quality, functional analysis is possible and there is no interference of artifacts. And for the evaluation of LGE images: 1 poor quality, extensive artifacts, infarcted myocardium is not visible; 2 moderate quality, infarcted myocardium is visible, but delineation is not possible in all segments; 3 good quality, infarcted myocardium is easy to distinguish from viable myocardium, despite some small artifacts; 4 excellent quality, no artifacts. Two scores were given for each scan: 1 for the set of cine images and 1 for the set of LGE images. After completing the independent image quality assessment, all 4 observers exchanged their scores for each case and agreed on a consensus score. Statistical analysis Continuous variables with normal distribution are expressed as mean ± SD, or as median (25th–75th percentile). Comparison of the baseline characteristics and stent artifact diameter was done by using an unpaired Student’s t-test. The consensus score of image quality of both field strengths was assessed for statistical difference by a Mann-Whitney U test. All statistical tests were two-sided with a significance level of P < 0.05. SPSS 12.0.1 for Windows (SPSS Inc., Chicago, USA) was used for analysis. Results Twenty-five consecutive patients with 36 CMR studies were included in the study. One patient was studied on both MR systems in the acute phase, 9 patients (5 on 3.0 T, 4 on 1.5 T) were studied both in the acute phase and in the chronic phase, and 1 patient (3.0 T) was studied twice in the chronic phase after MI. The baseline characteristics of all 25 patients are listed in Table 1. On each system, 12 CMR scans were performed in the acute phase after MI, at 4 ± 2 days after primary PCI, and 6 CMR studies in the chronic phase, at 125 ± 23 days after primary PCI. One patient refused contrast injection during scanning in the chronic phase. Table 1Patient characteristicsNumber of patients25Age (years)55 ± 9Men21 (84)BMI (kg/m2)25.5 ± 1.9Risk factorsDiabetes mellitus0 (0)Hyperlipidaemia3 (12)Hypertension6 (24)Smoking16 (64)Family history of CAD8 (32)Maximum peak CK-MB (U/L)308 (197–464)Infarct-related arteryLAD18 (72)RCx3 (12)RCA4 (16)Values are presented as number (ratio in %), mean ± standard deviation or median (25th–75th percentile) ECG electrodes were positioned and displaced until an optimal ECG signal was acquired. Although it took more time to obtain a stable ECG waveform with a clearly delineated R wave at 3.0 T than at 1.5 T (10–15 min vs. <2 min respectively), it did not affect image quality, since scans were repeated when trigger problems occurred. Stent safety and artifacts at 3.0 T No patient reported any discomfort or symptoms during the CMR scan procedure. In addition, there were no clinical events during, or shortly after scanning at 3.0 T, and none of the patients underwent a repeat intervention or hospitalization for any reason within 30 days. The mean number of stents implanted per patient per scan at 3.0 T was 1.3 ± 0.7, with a mean stent length of 19 ± 5 mm and diameter of 3.2 ± 0.4 mm. Further specifications of the implanted coronary stents are listed in Table 2. Repeat catheterization was performed 2 (1–4) days after scanning in 9 of the 12 patients who underwent 3.0 T CMR scanning in the acute phase after MI. Angiographic evaluation revealed no differences in stent position and patency compared to the initial result after primary PCI. Table 2Type and number of coronary stents used per CMR examinationField strength and timingNumber of patientsNumber and type of coronary stents present during CMRa3.0 T acute phase91× Multi-Link Vision12× Multi-Link Vision21× Driver3.0 T chronic phase31× Multi-Link Vision21× Driver/2x TAXUS Libertéb11× Driver1.5 T acute phase41× Prokinetic12× Prokinetic21× Lekton Motion11× Lekton Motion/2× Multi-Link Vision11× Multi-Link Vision11× AVE11× Multi-Link Zeta11× CYPHERb1.5 T chronic phase21× Lekton Motion12× Lekton Motion11× AVE11× Multi-Link Zeta11× CYPHERbaStent material and manufacturer—Multi-Link Vision: cobalt chromium alloy, Abbott Vascular; Driver, cobalt chromium alloy, Medtronic; TAXUS Liberté, 316L stainless steel, Boston Scientific; Prokinetic, cobalt chromium alloy, Biotronik; Lekton Motion: 316L stainless steel, Biotronik; Multi-Link Zeta, 316L stainless steel, Abbott Vascular; AVE: 316L stainless steel, Medtronic; CYPHER: 316L stainless steel, CordisbDrug-eluting stent During cine imaging, the coronary stent was visible in 14 of the 18 scans (78%), with a susceptibility related signal loss of 12.8 ± 4.3 mm in diameter. Due to severe interference of the coronary stent in 6 scans (33%), analysis of surrounding myocardium was impossible (Fig. 1A). Through-plane flow artifacts were present in 14 scans (from the aorta in 9 scans, from the pulmonary trunk in 7 scans, from the left ventricle in 3 scans), and hindered visualization of the myocardium in 7 scans (39%). In-plane flow artifacts were present in 4 scans, but were not clinically relevant. In the majority of the scans (94%) dark band artifacts appeared at the transition between myocardium and lung, with a predilection for the anterolateral wall, which resulted in severe image distortion in 8 scans (44%). Overall, artifacts were responsible for clinically relevant image deformation in 12 scans (67%), of which half was caused by the coronary stent (in 6 scans). Fig. 1Short axis cine SSFP images in different patients, demonstrating a signal void from a coronary stent with myocardial interference at 3.0 T (A, white arrow head), and a smaller signal void without interference at 1.5 T (B, white arrow head) No effect could be observed of B1 inhomogeneity on the suppression of viable myocardium on LGE images at 3.0 T. Stent safety and artifacts at 1.5 T Also during scanning at 1.5 T, no patient reported any discomfort or symptoms, and there were no clinical events during, or shortly after scanning. None of the patients underwent a repeat intervention or hospitalization for any reason within 30 days after CMR scanning. The mean number of implanted stents per patient per scan at 1.5 T was 1.2 ± 0.5 (see also Table 2). The mean stent length was 19 ± 5 mm with a mean diameter of 3.2 ± 0.4 mm, which was comparable with the stent length and diameter scanned at 3.0 T (p = ns). In 7 of the 12 patients who underwent CMR scanning at 1.5 T in the acute phase, repeat catheterization at 2 (2–4) days after scanning revealed no differences in stent position and patency compared to post-PCI. During cine imaging, the coronary stent was visible in 6 of the 18 scans (33%), with a susceptibility related signal loss of 5.5 ± 0.3 mm in diameter, which was significantly smaller than at 3.0 T (P < 0.01). Furthermore, visible stents did not cause clinically important image deformation (Fig. 1B). Besides the small signal void from coronary stents, no other artifacts were observed at 1.5 T. Also at 1.5 T there was no effect of B1 inhomogeneity on the suppression of viable myocardium on LGE images. Evaluation of image quality With scanning at 3.0 T, localized shimming was compulsory in every scan to obtain better image quality (Fig. 2). At 1.5 T, there was no need for adjustments in sequence parameters or the use of local shimming to optimize image quality. At 3.0 T, in 44% of the cases, image quality was not sufficient for quantitative analysis and assessment of regional function of the left ventricle (Table 3). For clinical purposes and global assessment of left ventricular function, image quality at 3.0 T and 1.5 T were comparable. Image quality of LGE images was not sufficient for quantitative analysis in 53% of the cases at 3.0 T, and 12% of the images were not useful for clinical purposes. Both image quality of cine and LGE imaging at 3.0 T were significantly lower compared to 1.5 T (P < 0.001). Fig. 2Four chamber cine SSFP images at 3.0 T in end-diastole (A & C) and end-systole (B & D), at the same slice position in one patient. Upper panels are without and lower panels with localized shimming. Dark band artifacts (apex) and flow artifacts (around the atrioventricular valves) are reduced with localized shimmingTable 3Consensus score of image quality of cine and late gadolinium-enhanced images for 1.5 T and 3.0 TCine SSFP imagesLate gadolinium-enhanced images1.5 T3.0 T1.5 T3.0 T1. Not clinically useful0 (0%)0 (0%)0 (0%)2 (12%)2. Clinically useful, no quantitative analysis0 (0%)8 (44%)0 (0%)7 (41%)3. Clinically useful, quantitatively analyzable0 (0%)4 (22%)1 (6%)8 (47%)4. Excellent quality18 (100%)6 (33%)17 (94%)0 (0%)Values are presented as absolute numbers (percentage) Discussion In this paper we report on our initial experience with CMR scanning at 3.0 T in a clinical situation. We found that it is safe and feasible to perform CMR scanning at 3.0 T in the acute and chronic phase after MI in patients treated with primary stenting. Image quality of cine imaging at 3.0 T is of sufficient quality for global assessment of left ventricular function, however, quantitative analysis is not possible in almost half of the patients, due to dark band, flow and stent artifacts. Image quality of LGE studies were significantly better at 1.5 T. Therefore further optimization of pulse sequences at 3.0 T is essential. MR imaging is considered to be contraindicated in patients with ferromagnetic implants, primarily because of the potential risks associated with migration, the induction of an electrical current and heating of the implant [14, 15]. According to earlier reports and the American Society for Testing and Materials International, the overall magnetic field interaction and heating for coronary stents is limited or absent at 1.5 T and 3.0 T, as tested in vitro [16–18]. In vivo studies concerning stent safety at 1.5 T demonstrated that CMR scanning is safe in patients early after coronary artery stent placement [19–21]. In this in vivo study using both 1.5 T and 3.0 T, no clinical signs were observed by the attending physician, and no substantial side effects or clinical events occurred during or within 30 days of CMR scanning. In addition, in the patients who underwent a repeat catheterization there was no angiographic evidence of stent migration, confirming the in vitro data. As discussed by Schär et al. [7], dark band and flow artifacts in SSFP cine imaging can be solved using optimized sequence parameters, localized shimming and correct water resonance frequency adjustment. In the clinical setting of the present study, it was not always possible to solve these artifact problems despite sequence optimization and localized shimming, especially in the presence of stent artifacts. The larger and diagnostically interfering artifacts of coronary stents at 3.0 T as opposed to the smaller stent artifacts at 1.5 T are an important issue in patients after MI, since primary PCI with stent implantation is the method of choice to re-establish coronary flow [22]. For clinical application, it may be more advantageous to return to spoiled gradient-echo cine imaging at 3.0 T, which includes longer acquisition times and lower contrast between blood and myocardium, but is less sensitive for off-resonance artifacts and equally accurate as SSFP cine imaging (Fig. 3) [9, 23]. Fig. 3Short axis cine SSFP image (A) and cine spoiled gradient-echo image (B), at the same slice position in one patient at 3.0 T. The coronary stent artifact on a SSFP image is larger than on a spoiled gradient-echo image (white arrows heads). Flow artifacts (A, asterisk) are less visible with spoiled gradient-echo imaging at 3.0 T We initially intended to evaluate differences in signal- and contrast-to-noise ratios (SNR and CNR) of both pulse sequences as well. At 1.5 T good noise estimates could be made using the technique described by Constantinides et al. [24]. However, this method did not work at 3.0 T due to a different reconstruction algorithm, even without parallel imaging, and by switching off clear and image enhancement filters. Subtraction methods with two consecutive scans to estimate noise are inaccurate in a mobile tissue as the heart [25]. Other methods were beyond the scope of this initial study. Despite the fact that SNR’s and CNR’s were therefore omitted from this study, it is interesting to mention that an important reason for inferior quality of LGE images at 3.0 T was that infarcted myocardium was sometimes difficult to delineate from the left ventricular cavity (Fig. 4). Fig. 4Short axis LGE images 15 min after injection of contrast at 3.0 T (A) and 1.5 T (B), in different patients. Visual assessment of infarct extent and location was more difficult at 3.0 T than at 1.5 T, since it was sometimes difficult to delineate infarcted myocardium from the left ventricular cavity at 3.0 T We used the same contrast agent and dose at both field strengths. The contrast dose has been optimized for 1.5 T in the past [26, 27], but might be different at 3.0 T as T1 relaxation rates are in general field strength dependent. However, a recent study of Sharma and colleagues showed that there were only minor differences in post contrast myocardial T1 relaxation times between 1.5 T and 3.0 T, using a contrast dose of 0.2 mmol/kg [28]. As long as inversion times are set appropriately at each field strength as done in this study, it is not to be expected that significant differences in LGE image quality are caused by contrast dose effects. In conclusion, our study demonstrates that it is safe and feasible to perform CMR scanning at 3.0 T in the acute and chronic phase after MI in patients treated with primary stenting. Although cine imaging at 3.0 T is of sufficient quality for clinical use, quantitative assessment is less reliable compared to 1.5 T, mainly due to dark band, flow and stent artifacts. Further optimization of pulse sequences at 3.0 T is essential to make 3.0 T CMR scanning suitable for clinical cardiology. Limitations The use of different MR systems, from different vendors, with different coils and sequence parameters, of course introduces confounding factors for a comparison. However, the sequences were optimized for their field strength to evaluate safety, feasibility and image quality rather than technical differences. A second limitation is that patients did not undergo a CMR examination at both 1.5 T and 3.0 T. Because the MR systems were on two different locations, it was not feasible to study the same patient twice in the acute phase after MI.

Diabetologia-4-1-2292420

Brisk walking compared with an individualised medical fitness programme for patients with type 2 diabetes: a randomised controlled trial

Aims/hypothesis Structured exercise is considered a cornerstone in type 2 diabetes treatment. However, adherence to combined resistance and endurance type exercise or medical fitness intervention programmes is generally poor. Group-based brisk walking may represent an attractive alternative, but its long-term efficacy as compared with an individualised approach such as medical fitness intervention programmes is unknown. We compared the clinical benefits of a 12-month exercise intervention programme consisting of either brisk walking or a medical fitness programme in type 2 diabetes patients. Introduction Regular exercise has been identified along with diet and medication as one of the three components of good diabetes therapy [1]. Structured exercise intervention programmes have been reported to be as effective as pharmaceutical strategies in improving glycaemic control [2–5] and/or cardiovascular risk profile [6, 7] in type 2 diabetes patients. Despite the growing body of scientific evidence on the health benefits of exercise intervention, most meta-analyses report a lack of studies that have tried to assess the long-term efficacy of exercise intervention in type 2 diabetes patients [3–5, 7–9]. In general, exercise intervention studies implement endurance and resistance type exercise, supervised by a physical therapist [10–17]. The long-term adherence to these (so-called) medical fitness programmes has been shown to range between 10 and 80% [11, 18–20]. Financial costs per patient are considerable in such programmes. Brisk walking exercise has been proposed as a less expensive alternative, with a good clinical outcome when patients are frequently counselled by motivated, supportive physicians [6, 21, 22]. However, brisk walking programmes mainly consist of endurance type exercise activities. As combined endurance and resistance type exercise training has been reported to be of greater clinical benefit [3, 23], it is conceivable that the long-term efficacy of a medical fitness programme is greater than that of a brisk walking programme. In the present study, we compared the changes in glycaemic control and cardiovascular risk profile following the prescription of 12 months of either supervised group-based brisk-walking or of a more individualised medical fitness programme in a large population of type 2 diabetes patients (n = 92) in a primary healthcare setting. We report our results for changes in HbA1c values as a primary outcome parameter, with blood pressure, plasma lipid levels, insulin sensitivity by homeostasis model assessment (HOMA), body composition, physical fitness, programme adherence and health-related quality of life as secondary outcome parameters. Given the greater improvements in glycaemic control following combined endurance and resistance type exercise training as opposed to each separately [3, 23], we hypothesised that a medical fitness programme would result in a significantly greater decline in HbA1c than brisk walking. Methods Participants From June till December 2005, 493 patients with type 2 diabetes were preselected from a population of 12,197 patients attending a primary healthcare centre that offers structured and guideline-based [24] diabetes care programmes. After 5 months of active recruitment by two nurse practitioners and four general practitioners, a total of 76 (=15.4%) patients were willing to participate in supervised exercise sessions three times a week for a period of at least 1 year. The type 2 diabetes patients selected had been diagnosed more than 3 months prior to screening according to the WHO criteria [25]. Exclusion criteria were: presence of (silent) cardiac or peripheral vascular disease, orthopaedic limitations and/or diabetic foot ulceration. To increase sample size, the exercise intervention programme was subsequently advertised in local newspapers, which yielded another 26 potential study candidates. In total, 99 type 2 diabetes patients agreed to participate in either brisk walking or medical fitness programmes. Potential study candidates performed a graded exercise test on a cycle ergometer (Medifit; Medifit Systems, Maarn, the Netherlands) using a ramp protocol [26]. Cardiac function was monitored using a 12-lead electrocardiogram and blood pressure was measured to detect malignant hypertension. Of the 99 selected patients, three did not show up and four were excluded because of an abnormal stress-ECG. The remaining 92 volunteers were randomly allocated to either brisk walking (n = 49) or medical fitness (n = 43) programmes, by subsequently drawing a sequentially numbered opaque, sealed envelope (Fig. 1). Laboratory personnel and the physician in charge of the graded exercise tests and randomisation procedure were blinded to the selection outcome of each participant. Drug prescriptions of each patient were recorded through an electronic database system operated through a network of local pharmacies. As part of the diabetes protocol in the healthcare centre, all type 2 diabetes patients consulted a dietitian and/or diabetes nurse educator annually for dietary guidance. Baseline participant characteristics are provided in Table 1. The nature and the risks of the exercise intervention were explained to all participants before written informed consent was obtained. This study was approved by the local Medical Ethical Committee of the Máxima Medical Centre, Veldhoven, the Netherlands. Since the study protocol was planned before 2005, official pre-trial registration at international trial registers was not performed. Fig. 1Study flow diagramTable 1Participants’ baseline characteristicsCharacteristicTotal (n = 92)Brisk walking (n = 49)Medical fitness (n = 43)Age (years)60 ± 961 ± 959 ± 9Men/women (n)47/4527/2220/23Duration of diabetes (years)5 ± 55 ± 45 ± 5BW (kg)94.3 ± 18.494.1 ± 18.194.5 ± 19.0BMI (kg/m2)32.3 ± 5.532.1 ± 5.232.5 ± 6.0Wmax (W)156 ± 47155 ± 49157 ± 45Estimated (l/min)2.1 ± 0.62.1 ± 0.62.1 ± 0.6%Pred. 82 ± 1182 ± 1083 ± 11Participants treated with: Diet only (n)1156 Hypoglycaemic agents (n)  Total814437  Metformin432419  Sulfonylurea251312  Thiazolidinedione1046  Insulin1367 Antihypertensive agents (n)  Total593227  ACEI/ARB392019  Beta-blocker17710  Diuretic19109  Calcium-channel blocker954  Other312 Lipid-lowering agents (n)  Total543024  Statin532924  Fibrate110  Other000Data are n (number of participants) or means ± SD.Estimated was based on Wmax during cycling ergometry and the equation according to Storer et al. [30].Relative age-, height- and sex-adjusted cardio-respiratory fitness (%Pred. ) was based on the equation by Fairbarn et al. [31].ACEI, ACE inhibitor; ARB, angiotensin receptor blocker; BW, body weight Exercise intervention Overview Two different exercise intervention programmes were implemented over a 12-month period, during which three brisk walking or medical fitness sessions were performed each week. The exercise load was progressive in nature and had components of resistance and endurance type exercise according to the American Diabetes Association/American College of Sports Medicine guidelines [27]. Brisk walking programme The weekly volume of brisk walking consisted of three 60 min exercise sessions. During the first 3 months participants were supervised by certified exercise trainers and a physical therapist. Group size varied between 15 and 25 patients. After 3 months, certified trainers guided and supervised the training sessions, while the physical therapist was visited on a consultation basis. The endurance type exercise consisted of brisk walking (5–6 km/h), with a focus on interval type endurance exercise training. During the intervention period the intensity was gradually increased and averaged 75 ± 5% of maximum heart frequency, as determined during the maximal cycle ergometer test. The resistance type exercise training consisted of resistance and floor exercises using individual body weight and/or elastic bands (The Hygenic Corporation, Akron, OH, USA). Further details on the brisk walking programme are provided in Electronic supplementary material (ESM) Table 1. Medical fitness programme The medical fitness programme consisted of three exercise sessions per week. Endurance type exercise consisted of interval type exercise on a home trainer, elliptical trainer or rowing ergometer with an average intensity of 73 ± 2% of maximum heart frequency. All training sessions were tailored to individual performance capacity. Resistance type exercise consisted of a selection of eight different exercises targeting upper and lower body muscle groups. Over a period of 6 months the training volume of the medical fitness programme was progressively increased from three times 30 min per week (90 min/week) towards a total of 180 to 225 min per week. Further details on the medical fitness programme are provided in ESM Table 1. Energy expenditure and monitoring blood glucose Based on average heart rate and indirect calorimetry measurements performed during either brisk walking [28] or circuit resistance training in elderly [29] participants, energy expenditure in both exercise interventions was estimated to range between 0.23 and 0.33 kJ kg−1 min−1. Given the participants’ bodyweights, the exercise interventions should be regarded as moderate intensity. Participants were provided with blood glucose monitor systems and test strips (A. Menarini Diagnostics, Valkenswaard, the Netherlands) and during the first month of brisk walking or medical fitness were advised to assess capillary blood glucose levels ∼30 min before and after exercise. Patients were instructed to report recurrent hypoglycaemic events to their diabetes nurse educator or general practitioner. For logistic reasons no systematic enquiries on mild hypoglycaemic events were performed. Financial cost of the intervention programmes Direct yearly costs for the individualised medical fitness programme were Euro 853 per participant and consisted of an ECG-stress test, supervision and consultations by a physical therapist, and the use of fitness centre facilities. The direct costs of the group-based brisk walking programme averaged Euro 396 per participant. Costs consisted of an ECG-stress test, supervision and consultation by a physical therapist, and a brisk walking membership fee. Other healthcare-related costs were not assessed. Outcome variables Resting heart rate and blood pressure Resting heart rate and blood pressure were determined in a supine position before and at 6 and 12 months after initiating the exercise programme. Heart rate was stored and averaged through a heart rate monitoring system (CO2ntrol; Tildesign, Zeewolde, the Netherlands). Average systolic and diastolic blood pressure were determined (HEM-907; Omron Healthcare, Hoofddorp, the Netherlands) from five successive measurements. Physical fitness Before and at 12 months after the start of the exercise programme, peak oxygen uptake capacity () was estimated based on maximum workload capacity (Wmax) during cycling ergometry according to Storer et al. [30]. Relative age-, height- and sex-adjusted cardio-respiratory fitness (%Pred. ) was defined as the ratio between estimated and predicted as determined in a healthy non-diabetic population [31]. Blood analyses Two weeks before and at 3, 6, 9 and 12 months after initiating the exercise programme, fasting blood samples were collected. Blood samples collected before and after 3 and 9 months of intervention were analysed for HbA1c and basal glucose concentration. On the evening prior to blood sampling, participants remained fasted from 00:00 hours onwards. They arrived at the laboratory at 08:00 hours, having travelled by car or public transport. After 5 to 10 min of rest, a venous blood sample was collected from an antecubital vein. Blood samples were collected in tubes containing a glycolytic inhibitor (sodium fluoride) and an anticoagulant (potassium oxalate), immediately centrifuged for 5 min at 1,000×g and 4°C, after which aliquots of plasma were immediately frozen in liquid nitrogen and stored at −80°C until analyses. Plasma glucose (dehydrogenase assay), triacylglycerol (lipase/peroxidase assay) concentrations, and total serum cholesterol (peroxidase/cholesterol-esterase assay), HDL-cholesterol (peroxidase esterase assay) and LDL-cholesterol (according to the Friedewald formula if triacylglycerol was <4.5 mmol/l, otherwise through direct peroxidase esterase assay) were analysed (Synchron LX20; Beckman Coulter, Fullerton, CA, USA). Plasma insulin was determined in duplicate by electrochemiluminescence-immunoassay (Elecsys 2010; Roche, Mannheim, Germany). Because of cross-sensitivity in the latter assay, exogenous insulin users were excluded from this analysis. HOMA insulin resistance index [32] was assessed to monitor changes in insulin sensitivity [33]. To determine HbA1c, 3 ml blood samples were collected and analysed by high-performance liquid chromatography (HA8160 Menarini; A. Menarini Diagnostics, Florence, Italy). Quality-of-life assessment Health-related quality-of-life was measured with the RAND 36-Item Health Survey 1.0 [34, 35] before and after 12 months of exercise intervention. Statistical analyses We calculated that 74 persons (37 per group) were needed to have 80% power to detect a moderate 0.65 SD difference in HbA1c as primary endpoint parameter between brisk walking and medical fitness programme, with an α of 0.05. Sample size was exceeded to allow for an estimated withdrawal of ∼15%. Data were analysed according to the intention-to-treat (ITT) principle. To minimise type I errors and loss of power [36], endpoint analyses according to the last observation carried forward principle were performed for missing values at 12 months follow-up. To assess whether physiological differences existed between the long-term application of brisk walking and medical fitness programme, post hoc analyses were performed for individuals actively participating for 10 months or more. Data are expressed as means ± SD. ANOVA repeated measures was used to determine differences between baseline and status after 12 months of exercise intervention. Unpaired Student’s t, χ2 and Mann–Whitney U tests were used to test whether long-term changes observed in brisk walking differed from those seen in the medical fitness programme. Significance was set at the 0.05 level of confidence. Pearson’s correlation calculation was used to test for linear relationships between long-term changes in dependent variables. All statistical calculations were performed using SPSS 10.1 (SPSS, Chicago, IL, USA). Results Characteristics of the study population Following randomisation there were no significant differences in baseline characteristics, sex and anthropometry between brisk walking and medical fitness programme groups (Table 1). Programme adherence, follow-up and adverse events After 6 months of intervention, 22 (45%) brisk walking participants and 13 (30%) medical fitness programme participants (p = 0.15) were no longer participating (ESM Table 2). After 12 months, 18 (37%) brisk walking and 19 (44%) medical fitness participants respectively were still actively participating with mean adherence levels of 75 ± 16% and 68 ± 13% (p > 0.05) for the 156 available exercise sessions (ESM Table 2). Besides motivational reasons (25%), orthopaedic-related co-morbidities, such as overuse injuries and/or subclinical osteoarthritis of the lower extremities, formed the main reason for dropout in 48 and 50% of the brisk walking and medical fitness programme participants, respectively (Fig. 1, Table 2). The distribution of missing data points was similar and not statistically different between groups (p > 0.05). Table 2Adverse medical eventsEventsBrisk walking (n = 49)Medical fitness (n = 43)Adverse events related to exercise intervention1512 Shoulder pain/chronic tendinopathy of rotator cuff01 (Aggravation of) low back pain22 Aggravation of pre-existing osteoarthritis in hip or knee joint64 Shin splints/lower leg pain10 Chronic tendinopathy of Achilles tendon/plantar fascia31 Other/generalised musculoskeletal discomfort33Adverse events not related to exercise intervention46 Medical event requiring hospitalisationa33 Other serious medical eventb13Participants with adverse event resulting in withdrawal1917Data are number of participating diabetes patients that experienced an adverse event throughout the 12 months follow-up periodaHospitalisations were related to: arthroscopic knee surgery (brisk walking, n = 1), elective surgery for chronic abdominal aortic aneurysm (brisk walking, n = 1), elective cataract surgery (brisk walking, n = 1), elective knee joint replacement (medical fitness, n = 1), bacterial pneumonia (medical fitness, n = 1), elective varicose veins surgery (medical fitness, n = 1, did not result in withdrawal from study).bOther serious medical events requiring medical attention were: otitis externa (brisk walking) n = 1, atrial fibrillation (medical fitness) n = 1, newly diagnosed myocardial ischaemia (medical fitness) n = 1, and newly diagnosed carcinoma of the mammary gland (medical fitness) n = 1 Glycaemic control According to ITT analyses, changes in HbA1c values following the prescription of brisk walking or medical fitness intervention were identical (95% CI −0.42, 0.43; p = 0.99) (Table 3). A total of 12 patients (brisk walking, n = 5; medical fitness, n = 7) had been prescribed higher doses of blood glucose-lowering medication throughout the follow-up period. When these participants were excluded from our ITT analysis, HbA1c increased by 0.05% (95% CI −0.41, 0.51; p = 0.82) in brisk walking as compared with medical fitness programme. Post hoc analyses of long-term active participants (≥10 months with mean adherence level of 62 ± 17 and 70 ± 18% in brisk walking and medical fitness groups, respectively) showed that, independently of changes in prescribed blood glucose-lowering drugs, HbA1c in the brisk walking group increased by 0.53% (95% CI 0.07, 1.00; p = 0.025) as compared with the medical fitness group (ESM Table 3). Table 3Changes in diabetes outcome (ITT analysis)VariableChange from baseline to 12 monthsMean ± SDnMean ± SDnDifference95% CIp ValueHbA1c (%) Total7.13 ± 1.36926.99 ± 1.2671   Brisk walking group7.18 ± 1.42497.08 ± 1.3738    Medical fitness group7.08 ± 1.29436.89 ± 1.1333    Intragroup comparisons         Intragroup comparison total    −0.14−0.39, 0.030.153  Intragroup comparison brisk walking    −0.11−0.51, 0.150.464  Intragroup comparison medical fitness    −0.18−0.45, 0.090.176 Brisk walking vs medical fitness    0.00−0.42, 0.430.985BMI (kg/m2) Total32.3 ± 5.59231.8 ± 5.489    Brisk walking group32.1 ± 5.24931.9 ± 5.048    Medical fitness group32.5 ± 6.04331.7 ± 5.941    Intragroup comparisons         Intragroup comparison total    −0.5−1.1, 0.10.108  Intragroup comparison brisk walking    −0.2−0.6, 0.30.471  Intragroup comparison medical fitness    −0.8−2.0, 0.30.152 Brisk walking vs medical fitness    0.7−0.5, 1.90.237Fasting plasma glucose (mmol/l) Total8.44 ± 2.81928.21 ± 2.3771    Brisk walking group8.64 ± 2.83498.33 ± 2.6438    Medical fitness group8.21 ± 2.80438.06 ± 2.0533    Intragroup comparisons         Intragroup comparison total    −0.23−0.72, 0.250.345  Intragroup comparison brisk walking    −0.31−0.96, 0.340.344  Intragroup comparison medical fitness    −0.15−0.91, 0.610.699 Brisk walking vs. medical fitness    −0.16−1.14, 0.820.744HOMAa Total5.55 ± 3.68685.60 ± 3.3854    Brisk walking group5.86 ± 3.56375.75 ± 3.2030    Medical fitness group5.19 ± 3.58315.43 ± 3.6424    Intragroup comparisons         Intragroup comparison total    0.05−0.50, 0.620.833  Intragroup comparison brisk walking    −0.12−1.11, 0.850.794  Intragroup comparison medical fitness    0.24−0.19, 0.740.241 Brisk walking vs medical fitness    −0.40−1.52, 0.720.479Resting heart rate (bpm) Total73.4 ± 11.89169.6 ± 13.977    Brisk walking group72.6 ± 11.84868.3 ± 15.041    Medical fitness group74.4 ± 11.84371.0 ± 12.436    Intragroup comparisons         Intragroup comparison total    −3.9−5.9, −1.80.000  Intragroup comparison brisk walking    −4.3−7.4, −1.10.009  Intragroup comparison medical fitness    −3.4−5.9, −0.90.010 Brisk walking vs medical fitness    −0.9−4.9, 3.20.678Systolic blood pressure (mmHg) Total148.5 ± 18.191137.5 ± 15.577    Brisk walking group150.3 ± 18.948138.9 ± 16.341    Medical fitness group146.3 ± 17.143135.9 ± 14.636    Intragroup comparisons         Intragroup comparison total    −10.9−14.0, −7.80.000  Intragroup comparison brisk walking    −11.4−15.9, −6.80.000  Intragroup comparison medical fitness    −10.4−14.7, 6.20.000 Brisk walking vs medical fitness    −0.9−7.1, 5.3)0.768Diastolic blood pressure (mmHg) Total81.9 ± 10.69176.6 ± 8.377    Brisk walking group81.3 ± 10.84875.9 ± 8.841    Medical fitness group82.6 ± 10.44377.4 ± 7.736    Intragroup comparisons         Intragroup comparison total    −5.3−7.1, −3.60.000  Intragroup comparison brisk walking    −5.4−7.8, 2.90.000  Intragroup comparison medical fitness    −5.2−7.8, −2.70.000 Brisk walking vs medical fitness    −0.1−3.6, 3.40.949Total cholesterol level (mmol/l) Total4.70 ± 0.86884.53 ± 0.9465    Brisk walking group4.83 ± 0.89474.61 ± 0.9235    Medical fitness group4.54 ± 0.12414.44 ± 0.9630    Intragroup comparisons         Intragroup comparison total    −0.16−0.32, −0.010.035  Intragroup comparison brisk walking    −0.22−0.45, 0.010.064  Intragroup comparison medical fitness    −0.10−0.30, 0.100.307 Brisk walking vs medical fitness    −0.12−0.42, 0.190.453LDL-cholesterol level (mmol/l) Total2.83 ± 0.76882.70 ± 0.7964    Brisk walking group2.90 ± 0.82472.72 ± 0.8535    Medical fitness group2.74 ± 0.69412.68 ± 0.7229    Intragroup comparisons         Intragroup comparison total    −0.12−0.26, 0.010.070  Intragroup comparison brisk walking    −0.18−0.38, 0.020.080  Intragroup comparison medical fitness    −0.06–0.25, 0.120.493 Brisk walking vs medical fitness    −0.11−0.38, 0.160.401HDL-cholesterol level (mmol/l) Total1.09 ± 0.28881.10 ± 0.2965    Brisk walking group1.10 ± 0.23471.09 ± 0.2635    Medical fitness group1.07 ± 0.33411.11 ± 0.3230    Intragroup comparisons         Intragroup comparison total    −0.01–0.02, 0.040.570  Intragroup comparison brisk walking    −0.01−0.06, 0.040.629  Intragroup comparison medical fitness    0.03−0.01, 0.080.129 Brisk walking vs medical fitness    −0.05−0.12, 0.020.172Triacylglycerol level (mmol/l) Total1.88 ± 0.99881.79 ± 0.9164    Brisk walking group2.00 ± 1.08471.94 ± 0.9835    Medical fitness group1.74 ± 0.88411.61 ± 0.7929    Intragroup comparisons         Intragroup comparison total    −0.09−0.25, 0.060.239  Intragroup comparison brisk walking    −0.06−0.33, 0.170.646  Intragroup comparison medical fitness    −0.13−0.27, 0.060.120 Brisk walking vs medical fitness    −0.02−0.29, 0.330.892RAND-36 (score 0–100 scale) Total71 ± 158970 ± 1558    Brisk walking group69 ± 154668 ± 1531    Medical fitness group73 ± 154371 ± 1427    Intragroup comparisons         Intragroup comparison total    −0.6−2.4, 1.20.503  Intragroup comparison brisk walking    0.1–2.7, 2.80.969  Intragroup comparison medical fitness    −1.4−3.8, 1.10.261 Brisk walking vs medical fitness    1.4−2.2, 5.00.438Data are means ± SD. n=number of valid measurements. aFor HOMA calculations exogenous insulin users (n = 13) were excluded Resting heart rate and blood pressure Following the 12 month exercise intervention, resting heart rate in the brisk walking group was reduced by 0.9 beats per min (bpm) (CI −7.1, 5.3; p = 0.68) as compared with medical fitness group. No significant differences in blood pressure response were observed between the two interventions (Table 3). After excluding participants who were prescribed higher doses of antihypertensive drugs (brisk walking, n = 5; medical fitness, n = 7), mean arterial blood pressure decreased by 0.9 mmHg (95% CI −5.6, 3.9; p = 0.94) in the brisk walking as compared with medical fitness group. A post hoc analysis of 46 long-term active participants showed a non-significant difference in mean arterial blood pressure of −3.5 mmHg (95% CI −9.2, 2.2; p = 0.22) in the brisk walking compared with the medical fitness programme (ESM Table 3). Blood lipid profile After 12 months, the overall fasting lipid profile did not differ between the two groups (Table 3). When participants (brisk walking, n = 9, medical fitness, n = 7) who were prescribed more blood lipid-lowering agents were excluded, HDL-cholesterol had changed by −0.05 mmol/l (95% CI −0.12, 0.02; p = 0.17) in the medical fitness compared with the brisk walking group. Body composition and workload capacity Body composition (BMI) did not change (Table 3). Workload capacity as measured during cycle ergometry averaged 155 ± 49 and 157 ± 45 W in the brisk walking and medical fitness programme groups, respectively, changing by −4.3 W (95% CI −9.1, 0.5; p = 0.078) in the former and by −2.5 W (95% CI −10, 5.6; p = 0.53) in the latter, with a non-significant difference of −1.8 W (95% CI −10.5, 6.9; p = 0.68) in brisk walking compared with medical fitness programme participants. Maximum heart rates recorded during ergometry averaged 146 ± 22 and 152 ± 19 bpm at baseline, and 152 ± 21 and 149 ± 22 bpm following 12 months of exercise intervention in the brisk walking and medical fitness groups, respectively (p > 0.05). In our long-term active brisk walking participants, Wmax changed by −6.3 W (95% CI −16.5, 3.9; p = 0.22) as compared with their medical fitness programme counterparts. No significant correlations were found between changes in HbA1c values and changes in workload capacity (ΔWmax, Pearson’s R = 0.02, p = 0.93, n = 38) or body weight (Pearson’s R = 0.18, p = 0.225, n = 45). Quality of life assessment A total of 89 and 58 participants completed the RAND-36 questionnaire at baseline and after 12 months of intervention, respectively. Reliability and internal consistency was excellent with Cronbach’s alpha of 0.93 and 0.91 respectively. No significant difference in changes of total RAND-36 scores were observed between the two intervention groups (p > 0.05; Table 3). Discussion It has been firmly established that physical activity counselling [37] and participation in structured exercise intervention programmes [15, 19] improves glycaemic control. However, long-term intervention studies on the clinical benefits of different types of exercise intervention programmes in type 2 diabetes patients are lacking. In the present study, we compared the clinical benefits of 12 months of a group-based brisk walking programme versus a more individualised medical fitness programme. ITT analysis showed that the prescription of a group-based, brisk walking programme is not necessarily inferior to a more individualised medical fitness programme with regard to glycaemic control and markers for cardiovascular risk profile (Table 3). Our data therefore imply that most patients with type 2 diabetes will achieve more or less equal long-term therapeutic benefits from a low-impact brisk walking programme as opposed to a more individualised medical fitness programme. Nevertheless, long-term programme adherence and the possibility of dropout should be taken into consideration when prescribing either type of exercise intervention for a patient with type 2 diabetes. Independently of the provided level of guidance and infrastructure, 60% of the patients dropped out of the exercise programme during the 12 month intervention period, with no differences in adherence and dropout patterns between brisk walking and medical fitness programme. Although motivational factors explained 25% of the dropout in both exercise programmes, almost 50% of the dropout was attributed to orthopaedic-related co-morbidities and overuse injuries of the lower extremities. The latter was not anticipated, since clinically relevant orthopaedic limitations, such as a history of osteoarthritis of the hip or knee joint, were defined as exclusion criteria for participation in either exercise intervention programme. Apparently, subclinical joint disease and/or degeneration of myotendineous structures become apparent when physical activity levels are increased. Stiffening of connective tissue due to non-enzymatic advanced glycation end-product formation [38], as well as decreased collagen turnover due to ageing and chronic disuse [39], could be responsible for the impaired response to increased musculoskeletal loading in these patients. Although more fundamental clinical research is warranted, the high incidence of overuse injuries in the present study adds to the results described in another study [23], suggesting that in unselected, non-study populations dropout will be even greater. Therefore, it might be advisable to implement a more gradual and less intense exercise regimen to allow myotendineous structures to adapt to increased loading. Moreover, before prescribing therapeutic exercise, diabetes healthcare workers should carefully consider obesity- and diabetes-related musculoskeletal deconditioning [40–42]. Such programmes should probably focus even more on resistance type exercise activities [43, 44] to bring patients to a level at which they are able to participate and adhere to more generic diabetes intervention programmes. It has also been suggested that psychological strategies such as motivational interviewing [45] or booster sessions [21, 46] might help to further improve programme adherence. Nevertheless, more long-term, tailored exercise intervention studies are warranted to further define the most effective and feasible exercise interventional strategy. The absence of a significant decline in blood HbA1c levels in brisk walking participants does not imply that the prescription of low-impact, endurance type exercise has no therapeutic value [3, 7, 27, 47–49]. In fact, we observed significant improvements in blood pressure control following either type of exercise intervention (Table 3). Although reliable assessment of actual drug use was not possible in the present study, the improvements in blood pressure occurred independently of changes in the prescription of antihypertensive medication. However, the lack of a non-exercise control group in the present study makes it impossible to speculate on cause and effect, since changes in blood pressure from baseline might also, or at least partly, be explained by the Hawthorne effect, for example, or by familiarisation with blood pressure measurement protocols. Nevertheless, our data suggest that the structured application of group-based brisk walking and supervised medical fitness programmes tends to have a similar impact on cardiovascular risk profile. In conclusion, the prescription of group-based brisk walking represents an equally effective interventional strategy to modulate glycaemic control and cardiovascular risk profile in type 2 diabetes patients compared with a more individualised medical fitness programme. General deconditioning, musculoskeletal overuse injuries and lack of motivation limit the benefits of long-term exercise intervention as an add-on to a comprehensive diabetes care programme. Future diabetes exercise intervention programmes in primary healthcare settings should consider diabetes-related co-morbidities and patient motivation as important factors, which determine both long-term programme adherence and the associated clinical benefits. Electronic supplementary material Below is the link to the electronic supplementary material. Table 1 Exercise programme (PDF 60.7 KB) Table 2 Individual follow-up data for primary outcome variable (HbA1c) and active programme participation (PDF 69.9 KB) Table 3 Changes in diabetes outcome in long-term active participants (PDF 160 KB)

J_Mol_Med-4-1-2262918

Celastrol inhibits polyglutamine aggregation and toxicity though induction of the heat shock response

Heat shock proteins (hsps) are protective against the harmful effects of mutant expanded polyglutamine repeat proteins that occur in diseases such as Huntington’s, prompting the search for pharmacologic compounds that increase hsp expression in cells as potential treatments for this and related diseases. In this paper, we show that celastrol, a compound recently shown to up-regulate hsp gene expression, significantly decreases killing of cells expressing mutant polyglutamine protein. This effect requires the presence of the transcription factor responsible for mediating inducible hsp gene expression, HSF1, and is correlated with decreased amounts and increased sodium dodecyl sulfate (SDS) solubility of polyglutamine aggregates. These results suggest the potential of celastrol as a therapeutic agent in the treatment of human polyglutamine expansion diseases. Introduction A large number of neurodegenerative diseases, including Huntington’s and Kennedy’s diseases, are characterized by expanded polyglutamine repeats in certain proteins leading to their aggregation and toxic effects on the cells expressing them [1, 2]. A number of results suggest that polyglutamine-containing aggregates are important both for the initiation and progression of these diseases [3, 4]. Polyglutamine protein aggregates in brains of patients and transgenic animals have been found to contain various molecular chaperones, ubiquitin, and components of the 20S proteasome [5, 6]. This suggests that neuronal cells recognize the protein aggregates as abnormally folded and try to disaggregate or degrade the mutant proteins by recruiting chaperones and proteasomal components [7]. Consistent with this view, increased expression of hsp70 and other heat shock proteins (hsps) has been found to be effective in reducing the toxicity of mutant polyglutamine proteins, suggesting the potential of pharmacological up-regulation of hsp gene regulation as a means for treating polyglutamine expansion diseases such as Huntington’s [8–11]. Heat Shock Factor 1, HSF1, is the transcription factor responsible for up-regulating the expression of hsp70 and other hsp genes in response to cellular stress [12, 13]. HSF activation involves stress-induced conversion of this factor to its trimeric DNA-binding form [14–17]. The activated trimeric HSF1 then binds to heat shock elements in the promoters of hsp genes to up-regulate their transcription, ultimately leading to elevated levels of cytoprotective hsps in these cells. The function of HSF1 as a key positive regulator of hsp expression, coupled to the observed ability of hsp proteins to protect cells from polyglutamine toxicity prompted the hypothesis that interventions that lead to activation of HSF1 could provide protection for cells against this toxicity and possibly represent agents that could be useful in treating polyglutamine expansion diseases such as Huntington’s [10, 18]. Consistent with this hypothesis, expression in cells of a constitutively active mutant HSF1 protein results in decreased polyglutamine toxicity and aggregate formation in both cell culture and animal models [19, 20]. These and other results have stimulated the search for pharmacological compounds capable of up-regulating hsp gene expression, as such compounds would theoretically have potential as treatments for these and other diseases of protein misfolding. Recently, it was demonstrated that a compound called celastrol, which is found in the Celastraceae family of plants and is already being used to treat diseases such as rheumatoid arthritis, bacterial infection, and fever [21, 22], is able to activate HSF1 and up-regulate hsp gene expression [18]. Celastrol has previously been found to inhibit the aggregation of purified Q58-huntingtin 1-171 amino terminal fragment in an in vitro assay, although this study did not examine effects of celastrol on polyglutamine aggregation in cells [23]. Celastrol has also been found to inhibit the cytotoxicity of expanded polyglutamine form of huntingtin exon 1 [24] and androgen receptor [25], but these studies did not examine the mechanism by which celastrol protects cells from expanded polyglutamine protein cytotoxicity. The results of the experiments presented in this paper demonstrate that celastrol is effective in preventing both the aggregation and toxicity of polyglutamine expression in cells and that it mediates these effects via the HSF1-mediated gene expression pathway. These results support the potential of this drug as a possible therapeutic agent for treating polyglutamine expansion diseases. The results also suggest that other drugs that stimulate HSF1 activity leading to hsp gene expression may also have beneficial activity against these disease states as well as other human diseases that are caused by protein misfolding. Materials and methods Plasmids, cell culture, and celastrol The expression constructs encoding Q19-YFP, Q57-YFP, and Q81-YFP were kindly provided by Dr. James Burke (Duke University). The polyglutamine-YFP (Qn-YFP) vectors were constructed using CAG repeats that were synthesized by polymerase chain reaction (PCR) from human dentatorubral pallidoluysian atrophy (DRPLA) cDNAs containing different CAG repeats. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Cellgro) supplemented with 10% fetal bovine serum (FBS) and 50 μg/ml gentamicin. PC12 cells were cultured in DMEM medium (Cellgro) supplemented with 5% FBS and 10% heat-inactivated horse serum (Gibco), and 100 units/ml Penicillin-Streptomycin (Gibco). HSF1+/+ and HSF1−/− mouse embryo fibroblast (MEF) cells (kindly provided by Dr. Ivor Benjamin, University of Utah) were cultured in DMEM medium (Cellgro) supplemented with 10% FBS, 1× Penicillin-streptomycin (Gibco), and 1× Non-essential amino acids (Gibco). Transfection was performed using Effectene transfection reagent (Qiagen), following the manufacturer’s protocol. Celastrol (Calbiochem) was dissolved in dimethyl sulphoxide at a stock concentration of 5.54 mM. Trypan blue cell viability assay Cells were collected by centrifugation at 1,000 rpm for 10 min at 4°C, and the pellet was washed twice with 1× phosphate-buffered saline (PBS). The cell pellet was then resuspended in 1× PBS to a concentration of approximately 106 cells/ml. A 1:1 dilution of the suspension was prepared using a solution containing 0.4% trypan blue stain (Gibco), and the suspension was then loaded into the counting chamber of a hemocytometer. The number of stained cells as well as the total number of cells was counted, and the percentage of stained cells was taken to represent the percentage of cell death. Experiments were repeated three times. Fluorescence microscopy Cells were seeded on coverslips, and 48 h after transfection, Hoechst 33342 and verapamil were added to the medium to final concentrations of 5 and 50 μg/ml, respectively. After incubation at 37°C for 30 min, the medium was removed, and the coverslips washed with 1× PBS for 5 min. A solution containing 3.7% paraformaldehyde in 1× PBS was added, and after 20 min incubation at room temperature, coverslips were washed with 1× PBS for 5 min. Coverslips were washed briefly three times in distilled water and mounted onto a slide spotted with 15 μl Vectashield (Vector Laboratories). Excess fluid was wicked from the coverslip and the edges of the coverslip sealed with fingernail polish. The fluorescence was then visualized using a Nikon fluorescent microscope and pictures taken with a Nikon Spotcam digital-imaging camera. To quantify the formation of polyglutamine aggregates, visual fields which contained similar numbers of cells (based on the density of nuclei stained by Hoechst) were chosen under 20× objective, and then the number of cells that contained aggregates in each field of vision was counted. Three different visual fields were quantified in each case. There were approximately 300 cells in each visual field for the experiments involving HeLa cells and PC12 cells, and approximately 100 cells in the experiments involving MEF cells. We scored a cell as positive if it had any visible aggregates, and the aggregates in the majority of cells appeared to be intranuclear or perinuclear (example shown in Fig. S1). Extract preparation and Western blot assay Cell lysis was performed on ice for 30 min in 50 mM Tris–HCl [pH 8.0], 100 mM NaCl, 5 mM MgCl2, 0.5% NP40 lysis buffer [26] containing 1× protease inhibitor cocktail (Roche), and 1 mM phenylmethanesulfonyl fluoride added. To examine hsp70 level, the cell lysate was cleared by centrifugation at 1,000 rpm at 4°C for 10 min, and the protein concentration of the supernatant was then determined by BioRad assay. Five micrograms of protein extract was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot following standard procedures. The antibodies and dilutions used to probe the Western blots were as follows. Anti-hsp70 (stress-induced form; Stressgen) was used at 1:10,000, anti-β-actin antibody (Sigma) was used at 1:10,000, and the anti-green fluorescent protein (GFP) polyclonal antibody (Bethyl labs) was used at a dilution of 1:4,000. Hsp70 Western blots were quantified using ImageQuant software. Filtration assay Cell lysis was performed on ice for 30 min in the lysis buffer described above. The insoluble fraction was obtained by centrifugation of the cell lysate at 14,000 rpm at 4°C for 10 min. The pellet was resuspended in DNase buffer (20 mM Tris–HCl [pH 8.0], 15 mM MgCl2, and 0.5 mg/ml DNase I), followed by incubation at 37°C for 1 h. After DNase treatment, the protein concentration was determined by analyzing an aliquot using the BioRad assay. The incubation was terminated by adjusting the mixtures to 20 mM ethylenediamine tetraacetic acid (EDTA), 2% sodium dodecyl sulfate (SDS), and 50 mM dithiothreitol and boiling for 5 min. Thirty micrograms of protein was diluted into 200 μl 2% SDS, and this was then filtered through a 0.2-μm pore size cellulose acetate membrane (Schleicher & Schuell) that was equilibrated with 2% SDS using a Dot-Blot system (Schleicher & Schuell). Filters were washed twice with 200 μl 0.1% SDS and then subjected to anti-GFP Western blot [27]. SDS solubility assay To perform the SDS solubility assay, cell lysates were prepared as described above for the filtration assay, including the 14,000 rpm centrifugation of the cell lysate and DNase treatment and protein concentration determination by BioRad assay. Forty micrograms of protein was then adjusted to 2× SDS-PAGE sample buffer (62.5 mM Tris·HCl [pH 6.8], 10% glycerol, 2% SDS, 5% β-mercaptoethanol) and boiled for 5 min after which the sample was analyzed by SDS-PAGE and Western blot with anti-GFP polyclonal antibody. Statistical analysis Statistical significance was determined using the Student’s t test. A P value of <0.05 was considered to be statistically significant. Results Celastrol protects against polyglutamine toxicity The experimental system we chose to test for effects of celastrol on polyglutamine aggregation and toxicity is transfection of a Q57-YFP fusion protein into cell lines (gift of Dr. James Burke), which is an established model for the aggregation of polyglutamine containing proteins in vivo [28]. Consistent with this, the Q57-YFP protein forms clearly visible aggregates upon expression in HeLa cells, while cells transfected with Q19-YFP exhibit a diffuse pattern (Fig. S1). A previous study showed that 8 h of celastrol treatment leads to increased levels of the hsp70 protein [18]. As the studies described in this paper include cell death and aggregation assays performed at 48 h after transfection with the Q57-YFP construct, we wanted to ensure that cells treated with celastrol treatment for longer times also exhibit elevated hsp70 expression. As shown in Fig. 1a (upper panel), celastrol treatment of HeLa cells for 24 or 48 h both result in a dose-dependent increase in expression of the hsp70 protein. Quantification of these hsp70 Western blot results indicates that the induction of hsp70 by each celastrol concentration is very similar at 24 and 48 h, showing that treatment with this drug results in a sustained increase in hsp70 levels (lower panel). Next, to test whether treatment with this drug confers protection from polyglutamine toxicity, HeLa cells were transfected with the Q57-YFP construct and then incubated in media containing different concentrations of celastrol. The results of this experiment, shown in Fig. 1b, reveal that celastrol treatment is associated with a significant decrease in death of these cells expressing this mutant polyglutamine protein. Fig. 1Celastrol treatment reduces Q57-YFP cytotoxicity. a Hsp70 protein level is increased by celastrol treatment. HeLa cells were treated with the indicated concentrations of celastrol for 24 or 48 h, after which cell extracts were made and subjected to Western blot using antibodies against hsp70 or β-actin (upper panel). These results were quantified using the ImageQuant program, and the values for the two treatment times, grouped by celastrol concentration, were graphed (lower panel). b HeLa cells were transfected with Q57-YFP along with celastrol treatment at the concentrations indicated. After 48 h, the amount of cell death was determined by trypan blue assay. Data are shown as means ± SE (*P < 0.007, **P < 0.004, ***P < 0.0001, for each celastrol concentration treatment vs no celastrol) Celastrol protective effect are correlated with decreased number of cells containing polyglutamine aggregates To determine whether the ability of celastrol to protect cells from polyQ toxicity could be mediated via effects on polyQ aggregates, we then examined whether celastrol treatment alters the number of polyQ aggregates in cells and/or their solubility. The results of these experiments show that treatment with celastrol is associated both with a decrease in the number of cells containing Q57-YFP aggregates, as quantified by fluorescence microscopy (Fig. 2a), and also with an increase in the amount of Q57-YFP that can be solubilized from aggregates by SDS treatment (Fig. 2b). SDS solubility of aggregates is related with their toxicity, and data from previous studies suggests that molecular chaperones may ameliorate the neurodegenerative effect of mutant polyglutamine protein, at least in part, by increasing the solubility of these proteins [9, 29]. Together, the results shown in Figs. 1 and 2 indicate that celastrol treatment decreases the death of cells expressing mutant polyglutamine protein and also decreases the number of cells containing Q57-YFP-aggregates and the insolubility of Q57-YFP-aggregates. Fig. 2Celastrol treatment reduces number of cells containing Q57-YFP aggregates and increases Q57-YFP solubility. HeLa cells were transfected with Q57-YFP along with celastrol treatment at the concentrations indicated. a After 48 h of transfection, the formation of Q57-YFP aggregates was quantified using fluorescence microscopy. Visual fields which contained similar numbers of cells (based on the density of nuclei stained by Hoechst) were chosen under 20× objective, and then the number of aggregates in each field of vision was counted. Three different visual fields were quantified in each case, and data are shown as means ± SE (*P < 0.004, **P < 0.001, ***P < 0.0003, for each celastrol concentration treatment vs no celastrol). b To determine the amount of Q57-YFP monomer that could be solubilized from aggregates in lysates of the transfected cells by SDS treatment, the protein concentration of the insoluble fraction of the cell lysates was determined, and then 40 μg of protein was subjected to SDS solubilization treatment, followed by Western blot using anti-GFP antibody Protective effects of celastrol treatment in PC12 cells As the deleterious effects of expression of mutant polyglutamine proteins in vivo are observed primarily in cells of neuronal origin, we next sought to test the effect of celastrol treatment on polyglutamine toxicity and aggregation in PC12 cells. As shown in Fig. 3a, PC12 cells are very sensitive to killing by expression of the Q57-YFP protein, but as observed for HeLa cells in Fig. 1b above, celastrol treatment results in a significant decrease in death of these cells. As was observed in the experiments using HeLa cells, treatment of PC12 cells with celastrol resulted in a significant decrease in the number of polyglutamine aggregates in the cells (Fig. 3b) and an increase in the amount of Q57-YFP that can be solubilized from aggregates by SDS treatment (Fig. 3c). Interestingly, the concentrations of celastrol required to achieve these effects on polyglutamine toxicity and number and solubility properties of aggregates in PC12 cells were significantly lower than that needed for HeLa cells. Fig. 3Celastrol effects on Q57-YFP toxicity and aggregates in PC12 cells. PC12 cells were transfected with Q57-YFP along with celastrol treatment at the concentrations indicated. After 48 h, the amount of cell death was determined by trypan blue assay (a), the number of cells containing Q57-YFP aggregates was quantified using fluorescence microscopy (b), and the amount of Q57-YFP monomer solubilized from aggregates by SDS treatment visualized by Western blot using anti-GFP antibody (c). In a and b, data are shown as means ± SE [*P < 0.0001 and **P < 0.0001 (a), *P < 0.001 and **P < 0.0001 (b), in each case for each celastrol concentration treatment vs no celastrol) HSF1−/− cells exhibit increased polyglutamine aggregation and toxicity Inducible hsp expression is thought to be important for the ability of cells to decrease polyglutamine toxicity and aggregation [8–11]. Consistent with this hypothesis, HSF1−/− MEFs [30] that are transfected with Q57-YFP exhibit a higher incidence of cell death than HSF1+/+ MEF cells (Fig. 4a). The HSF1−/− MEFs also show increased Q57-YFP aggregation, both as measured by numbers of aggregates using fluorescence microscopy of cells (Fig. 4b) and by amount of aggregated Q57-YFP protein present in cell extracts detected by filtration assay (Fig. 4c). Fig. 4HSF1−/− cells exhibit higher Q57-YFP aggregation and cell death. HSF1−/− and wild-type MEF cells were transfected with Q57-YFP, and after 48 h, cell death was examined by trypan blue assay (a), and the number of cells containing Q57-YFP aggregates was quantified using fluorescence microscopy (b), or filtration assay, in which 30 μg of the insoluble fraction was filtered through 0.2 μm cellulose acetate membrane, and the aggregates retained on the membrane were immunoblotted using anti-GFP antibody (c). In a and b, data are shown as means ± SE. *P < 0.008 (a); *P < 0.003 (b) Protective effects of celastrol require HSF1 Based on previous results showing the ability of celastrol to activate HSF1 leading to increased hsp gene expression [18], we hypothesized that this drug’s ability to decrease polyglutamine toxicity and aggregation shown by the results of Figs. 1, 2, and 3 above could be mediated via the HSF1-regulated gene expression pathway. To test this hypothesis, we compared the effect of celastrol treatment on polyglutamine toxicity and aggregation in the HSF1+/+ vs HSF1−/− MEF cells. Celastrol does not exhibit any toxicity in HeLa cells at 1.6 μM, but we found its optimal concentration in MEF cells to be lower than that of HeLa cells. Thus, for these experiments, we used 0.4 μM celastrol treatment, which has no toxic effect in either HSF1−/− or wild-type MEFs (data not shown). First, Western blot analysis shows that the HSF1−/− cells are unable to up-regulate hsp70 protein expression in response to celastrol treatment, demonstrating that celastrol-induced up-regulation of hsp70 expression is mediated by the HSF1-regulated gene expression pathway (Fig. 5a). Fig. 5Celastrol decreases Q57-YFP toxicity and aggregation in wild-type but not HSF1−/− MEF cells. a Celastrol treatment does not induce hsp70 expression in HSF1−/− cells. Wild-type and HSF1−/− MEF cells were treated with no celastrol or 0.4 μM celastrol, and after 12 h, cell extracts were made, and 5 μg protein was loaded into each lane for immunoblotting with anti-hsp70 antibodies. b and c Wild-type and HSF1−/− MEF cells were transfected with Q57-YFP, with celastrol at the indicated concentrations added at the same time. After 48 h, cell death was examined by trypan blue assay (b), with data shown as means ± SE (*P < 0.002 for HSF2+/+ 0.4 μM celastrol vs no celastrol; *P < 0.002 for HSF2−/− 0.4 μM celastrol vs no celastrol), and amount of aggregated Q57-YFP was determined by filtration assay followed by anti-GFP Western blot (c) The results shown above (Figs. 1, 2, 3, and 5a) indicate that celastrol treatment is effective in preventing polyglutamine aggregation and toxicity and that this drug up-regulates hsp70 expression by an HSF1-dependent mechanism. These results suggest that celastrol treatment protects cells by stimulating HSF1-dependent expression of hsps. However, celastrol could also modulate other cellular activities that could contribute to these protective effects. Thus, to test the contribution of HSF1-regulated pathways in these protective effects, we transfected HSF1+/+ and HSF1−/− MEF cells with Q57-YFP in combination with celastrol treatment and then measured the amount of cell death and levels of aggregated Q57-YFP. The results of this experiment show that, as it did for the HeLa and PC12 cells in the experiments of Figs. 1 and 3 above, celastrol treatment resulted in decreased death of HSF1+/+ MEF cells transfected with Q57-YFP, but treatment with this drug did not protect the HSF1−/− cells (Fig. 5b). Consistent with these results, the HSF1+/+ cells, but not the HSF1−/− cells, showed decreased levels of aggregated Q57-YFP upon celastrol treatment, as measured by the filtration assay (Fig. 5c). These results suggest that the HSF1 protein plays an important role in the protective effects of celastrol against polyglutamine toxicity and aggregation. Discussion The results described above indicate that the drug celastrol is able to decrease polyglutamine toxicity, supporting the proposal that this drug could potentially be useful in the treatment of Huntington’s disease and possibly other human polyglutamine expansion disorders. In support of this possibility, it has been found that celastrol treatment of mice results in elevation of hsp70 levels in neurons in the brains of these mice [31]. The results also show that lower concentrations of celastrol are required for its protective effects against polyglutamine toxicity in PC12 cells, cells with neuronal characteristics, compared to other cell types. A particularly beneficial aspect of this drug is that it is already being used to treat people with other disorders [21, 22], and thus it could likely be adapted more quickly for use in treating polyglutamine expansion diseases than other drugs not currently being used in humans. Results presented in this paper indicate that the protective effect of celastrol against polyglutamine toxicity is associated with decreased numbers of cells containing aggregates as well as increased SDS-solubility of the mutant polyglutamine protein. This might seem to be at odds with findings that formation of polyglutamine aggregates can be protective for cells expressing these mutant proteins [32–34]. However, our results are consistent with the findings of a number of studies which indicate that molecular chaperones can reduce the formation of polyglutamine aggregates and increase the solubility of expanded polyglutamine proteins [8–11, 19, 20]. While one study found that hsp40 overexpression, but not hsp70 overexpression, was associated with reduced aggregation [35], the results of a number of studies do suggest that chaperones are able to reduce aggregation of mutant polyglutamine proteins. Molecular chaperones could be acting at multiple levels to reduce polyglutamine toxicity, but one possibility that has been proposed is that these chaperones, and by extension celastrol through its HSF1-mediated up-regulation of chaperones, may be acting at an early step to prevent formation of toxic intermediates before they can become part of larger aggregates [8–11, 19, 20]. This would explain both how celastrol treatment protects cells from polyglutamine toxicity and the effects we observe on polyglutamine aggregates in the celastrol-treated cells. An alternative explanation that must be considered, in keeping with previous studies indicating that aggregate formation can be protective for cells [32–34], is that the results we observe relating celastrol with the numbers of cells containing polyglutamine aggregates could be due, at least in part, to a potential ability of celastrol to enhance the survival of cells that do not contain aggregates, which would then lead to a perceived decrease in the numbers of cells containing aggregates in our experiments. Our data do indicate, however, the protective effects of celastrol with respect to polyglutamine toxicity. In addition to its ability to its effects on HSF1 activity, celastrol has been found to affect other pathways in the cell, including the NF–κB pathway [18, 21, 22, 36]. However, our results suggest that the beneficial effects of celastrol treatment in decreasing polyglutamine toxicity are mediated through the action of the HSF1-regulated gene expression pathway, based on the finding that celastrol is not protective in Q57-YFP transfected HSF1−/− MEF cells (Fig. 5b). This finding also suggests that other drugs that activate HSF1 could also have potential as candidate therapeutic agents for treating polyglutamine expansion diseases. Electronic supplementary material

Naunyn_Schmiedebergs_Arch_Pharmacol-3-1-2020506

Phospholipase D signaling: orchestration by PIP2 and small GTPases

Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of the versatile lipid second messenger, phosphatidic acid (PA), which is involved in fundamental cellular processes, including membrane trafficking, actin cytoskeleton remodeling, cell proliferation and cell survival. PLD activity can be dramatically stimulated by a large number of cell surface receptors and is elaborately regulated by intracellular factors, including protein kinase C isoforms, small GTPases of the ARF, Rho and Ras families and, particularly, by the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 is well known as substrate for the generation of second messengers by phospholipase C, but is now also understood to recruit and/or activate a variety of actin regulatory proteins, ion channels and other signaling proteins, including PLD, by direct interaction. The synthesis of PIP2 by phosphoinositide 5-kinase (PIP5K) isoforms is tightly regulated by small GTPases and, interestingly, by PA as well, and the concerted formation of PIP2 and PA has been shown to mediate receptor-regulated cellular events. This review highlights the regulation of PLD by membrane receptors, and describes how the close encounter of PLD and PIP5K isoforms with small GTPases permits the execution of specific cellular functions. Introduction The activation of membrane receptors by hormones and growth factors results in the localized generation of intracellular second messengers. The hydrolysis of membrane phospholipids and the generation of biologically active products play important roles in the regulation of cell function and cell fate. Well known is the activation of phosphoinositide-specific phospholipase C (PLC) isoforms, which hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane phospholipid found in all eukaryotic cells (Schmidt et al. 2004). Stimulation of PLC isoforms plays a major role in many early and late cellular responses to receptor activation, including smooth muscle contraction, secretion and neuronal signaling as well as fertilization, cell growth and differentiation (Berridge 2005; Nishizuka 2003). Phospholipase D (PLD) was first described 60 years ago as a distinct, phospholipid-specific phosphodiesterase activity in cabbage leaves (Hanahan and Chaikoff 1948). This pioneering research indicated that PLD hydrolyzes phosphatidylcholine to yield phosphatidic acid (PA) and choline. The recognition that PLD is rapidly and dramatically activated in response to extracellular stimuli in cultured animal cells, now 20 years ago (Bocckino et al. 1987; Cockcroft 1984), has brought PLD signaling to the very forefront of current biological and biomedical research. Meanwhile, phosphatidylcholine-hydrolyzing PLD has been identified in bacteria, protozoa, fungi, plants and animals, and, due to this widespread distribution, is assumed to be involved in the regulation of fundamental cellular functions. Indeed, it has now been established that activation of PLD and the generation of PA by a vast number of membrane receptors modulate such a wide array of cellular responses as calcium mobilization, secretion, superoxide production, endocytosis, exocytosis, vesicle trafficking, glucose transport, rearrangements of the actin cytoskeleton, mitogenesis and survival (Cockcroft 2001; Exton 2002b; Jenkins and Frohman 2005; Liscovitch et al. 2000). PIP2 is a critical cofactor for PLD, and profoundly affects the activity, membrane localization and receptor activation of both PLD isoforms, PLD1 and PLD2 (Brown et al. 1993; Hodgkin et al. 2000; Liscovitch et al. 1994; Pertile et al. 1995; Schmidt et al. 1996d). Thus, reduction of cellular PIP2 levels, for instance via scavenging of PIP2 by the actin-binding protein fodrin (Lukowski et al. 1998) or via forced PIP2 hydrolysis by the phosphatase synaptojanin (Chung et al. 1997), has been shown to inhibit PLD activity. Vice versa, the synthesis of PIP2 by phosphoinositide 5-kinase (PIP5K) isoforms can be directly stimulated by the PLD product PA (Jenkins et al. 1994; Moritz et al. 1992), and this regulation has also been confirmed to occur at the whole cell level (Divecha et al. 2000; Jones et al. 2000b; Skippen et al. 2002). It is now hypothesized that the reciprocal stimulation of PLD and PIP5K enzymes enables rapid feed-forward stimulation loops for a localized and explosive generation of PA and PIP2, which may then govern the recruitment and activation of proteins to execute specific cellular tasks, especially membrane trafficking, and changes in the organization of the actin cytoskeleton. The activity and localization of both PLD and PIP5K are under control of GTPases of the Arf and Rho families, which are well-defined regulators of membrane transport and actin-reorganization processes. The reciprocal stimulation of PIP5K and PLD, and the regulation of these enzymes by ARF and Rho GTPases, point to concerted mechanisms in cellular actions, involving acute, localized PIP2 and PA synthesis (Fig. 1). This review will focus on the regulation of PLD enzymes by membrane receptors and monomeric GTPases, and on how PLD signaling is organized and connected by PIP2 metabolism. Fig. 1Regulation and cellular roles of PLD and PIP5K. Regulation of PLD and PIP5K by ARF and Rho family GTPases is essentially involved in the regulation of intracellular vesicle trafficking and actin cytoskeleton reorganization. Both PLD and PIP5K are stimulated by cell surface receptors and by conventional PKC isoforms, and the latter can become activated after receptor-induced hydrolysis of PIP2 by PLC. Positive feed-forward regulation is achieved by stimulation of PLD by PIP5K-derived PIP2, and of PIP5K by PLD-derived PA. Activation of ARF-GAPs by PIP2 accelerates the inactivation of ARF proteins, and may terminate a round of PA and PIP2 synthesis Phosphatidic acid and PLD isoforms Most cellular responses following PLD activation are probably mediated by the immediate reaction product PA. PA is a multifunctional lipid that can be further metabolized to the bioactive lipids, lysophosphatidic acid (LPA) and diacylglycerol (DAG), can by itself alter membrane curvature, and can serve as a protein attachment site and affect both cellular localization and activity of various proteins, including Raf-1 kinase, protein phosphatase 1, sphingosine kinase 1, and mTOR (mammalian target of rapamycin), a key regulator of cell growth and proliferation (Jenkins and Frohman 2005). PLD enzymes can catalyze a transphosphatidylation reaction in which the phosphatidyl moiety of phosphatidylcholine is accepted by primary alcohols, thereby producing stable phosphatidylalcohol instead of PA. This transphosphatidylation reaction is widely applied to measure PLD activity in biological samples, and quenching of PA synthesis by primary alcohols has proven extremely helpful to identify the involvement of PLD enzymes in cell physiology. In this way, a role for PLD has been demonstrated in a variety of signaling processes, such as activation of phosphoinositide (PI3K, PIP5K) and protein (Akt, ERK1/2) kinases, calcium mobilization, cytoskeleton remodeling, endocytosis, exocytosis, membrane trafficking, superoxide production, glucose transport, cell migration, cell proliferation, and survival signaling (Exton 2002a; Foster and Xu 2003). There are two mammalian PLD genes, PLD1 and PLD2. PLD1 has a low basal activity and is extensively regulated by conventional protein kinase C (PKCα, -β, -γ) isozymes and small GTPases of the ARF (ARF1 - ARF6) and Rho (RhoA, Rac1, Cdc42) families (Henage et al. 2006). PLD2 has a higher basal activity than PLD1, but has been shown to respond to ARF and PKC as well (Chen and Exton 2004). PIP2 is recognized to be the most important cofactor for PLD, and both PLD isoforms are absolutely dependent on PIP2 for activity. Experiments utilizing inactive PLD mutants and RNA interference have discriminated isoform-specific PLD functions, and showed that PLD1 is involved in agonist-induced secretion, actin organization, and cell adhesion and migration (Exton 2002a; Iyer et al. 2006; Kim et al. 2006; Vitale et al. 2001), and PLD2 in endocytosis and recycling of membrane receptors (Du et al. 2004; Koch et al. 2006; Padrón et al. 2006). The PLD isoforms, both with two splice variants, share an ~50% amino-acid sequence identity (Colley et al. 1997; Hammond et al. 1995, 1997; Steed et al. 1998). The catalytic core of both PLD enzymes are composed of four conserved domains (domain I-IV), and the HKD motifs in the domains II and IV probably associate together to form a catalytic centre (Xie et al. 2000). PLD1 is characterized by a 116-amino acid loop region following domain II, which has been proposed to function as a negative regulatory element (Sung et al. 1999). PLD1 and PLD2 further possess N-terminal PH (pleckstrin homology) and PX (phox homology) domains. PIP2 binds to the PH domain (Hodgkin et al. 2000), but also to a polybasic PIP2 binding motif within the catalytic core (Sciorra et al. 1999), and interaction of PIP2 with both domains has been suggested to be involved in membrane targeting of PLD as well as stimulation of PLD catalytic activity (Du et al. 2003; Hodgkin et al. 2000; Sciorra et al. 2002). The PX domain of PLD1 has been reported to preferentially bind to phosphatidylinositol-3,4,5-trisphosphate (PIP3) (Lee et al. 2005; Stahelin et al. 2004), but interaction with PI5P has been observed as well (Du et al. 2003). Recently, it was shown that the PX domain of PLD has GTPase-activating protein (GAP) activity towards dynamin, and that PLD supports EGF receptor endocytosis (Lee et al. 2006). The PH and PX domains probably contribute to the proper localization of the PLD enzymes within cells. In line with a role for PLD enzymes in different cellular tasks, PLD1 and PLD2 show a diverse subcellular distribution. PLD1 is found throughout the cell, but primarily localizes to perinuclear endosomes and the Golgi apparatus (Brown et al. 1998; Freyberg et al. 2001; Hughes and Parker 2001). PLD2 is almost exclusively present at the plasma membrane in lipid raft fractions (Czarny et al. 1999). The localization of PLD1 does not seem to be static, and regulated translocation and recycling of the enzyme between cellular compartments may be crucial to its proper functioning. In an elegant study, coordinated subcellular targeting of the lipid binding motifs has been demonstrated to drive this subcellular cycling of PLD1 (Du et al. 2003). Upon stimulation, PLD1 was found to translocate from the intracellular compartments to the plasma membrane, and this process was probably dependent on the polybasic PIP2 binding site. The PH domain then facilitated entry of PLD1 into lipid rafts, a step critical for internalization of the enzyme, whereafter interaction of the PX domain with PI5P may control the efficient return of PLD1 to the endosomes. PIP2 and PIP5K isoforms PIP2 is an essential and versatile factor in cellular signaling. Hydrolysis of PIP2 by PLC into the second messengers, inositol-1,4,5-trisphosphate (IP3) and DAG, is a general and well-defined answer of cells in response to stimulation of many membrane receptors (Schmidt et al. 2004). Phosphorylation of PIP2 by PI3K results in the rapid accumulation of PIP3, which recruits and activates mediators involved in actin remodeling, mitogenesis and survival (Vanhaesebroeck et al. 2001). But it is now recognized that PIP2, as well as other phosphoinositides, are signaling molecules by themselves and can, by binding to unique phosphoinositide-binding sequences, such as the PH and PX domains, affect the activity and subcellular localization of many proteins, including many actin regulatory proteins, a wide range of ion channels, and PLD (Niggli 2005; Suh and Hille 2005; Yin and Janmey 2003). In this way, PIP2 can modulate a remarkable variety of cellular processes, including cortical actin organization, membrane ruffling, vesicle trafficking, gene expression, cell migration and cell survival (Ling et al. 2006; Oude Weernink et al. 2004b; Toker 2002). Subsequent dephosphorylation of PIP2 by inositol polyphosphate 5-phosphatases, such as synaptojanin, is believed to terminate local PIP2 signaling, for instance in the process of vesicle trafficking (Majerus et al. 1999). To execute this variety of functions, PIP2 may be organized in discrete functional pools within cells, but the existence of PIP2 clusters in the plasma membrane is currently under debate. Using green fluorescent protein-tagged PH domains or antibodies to visualize PIP2, the lipid was found to concentrate in highly dynamic, actin-rich regions (Tall et al. 2000) and lipid rafts (Laux et al. 2000; Parmryd et al. 2003) in the plasma membrane, feeding the idea that spatially organized PIP2 synthesis regulates actin polymerization and other cellular processes. The localization of PIP2 in rafts is supported by biochemical data (Pike and Casey 1996); however, specific PIP2 clustering has been disputed (van Rheenen et al. 2005). PIP2 is generated after phosphorylation of phosphatidylinositol-4-phosphate by PIP5K. In mammals, cDNAs encoding three isoforms of PIP5K (designated Iα, Iβ and Iγ) with alternative splice variants have been cloned and characterized (Ishihara et al. 1996, 1998; Loijens and Anderson 1996). Sequence analysis has shown that PIP5K enzymes are related to PIP4K enzymes, but that they share no identity with most other lipid (PI3K and PI4K) or protein kinases. The sequence similarity between the PIP4Ks and PIP5Ks is clustered in the catalytic core of the kinases (Anderson et al. 1999; Hinchliffe et al. 1998). An activation loop spanning the catalytic domain has been shown to determine both substrate specificity and subcellular targeting of PIP5Ks, which can be swapped by substitution of a single amino acid within this loop (Kunz et al. 2002). In murine PIP5K-Iβ, two dimerization domains were identified, which may contribute to the proper subcellular localization and functioning of the enzyme (Galiano et al. 2002). The identification of three PIP5K isoforms raised the expectation of a differential regulation of the enzymes by cellular signal transduction components, but up to now the regulatory properties of PIP5K-Iα, Iβ and Iγ appear to be remarkably similar. All PIP5K isoforms are stimulated by PA, are extensively regulated by ARF and Rho GTPases, and inhibited by protein kinase A (PKA) and PI-stimulated autophosphorylation (Oude Weernink et al. 2004b). Nevertheless, evidence has been provided that PIP5K isoforms may selectively control functional PIP2 pools, which may support particular processes in different cell types. Thus, actin reorganization down-stream of Rac1 in platelets specifically involves murine PIP5K-Iα (Tolias et al. 2000). Human PIP5K-Iα was found to localize in Rac1-induced membrane ruffles, and the LIM protein Ajuba has been identified to interact with and stimulate PIP5K-Iα in leading-edge membrane ruffles in migrating cells (Kisseleva et al. 2005). Human PIPK-Iβ was detected primarily in cytosolic vesicular structures (Doughman et al. 2003) and may synthesize the PIP2 pool involved in constitutive endocytosis (Padrón et al. 2003). The long-splice variant of PIP5K-Iγ, PIP5K-Iγ90, is enriched in neurons and is implicated in the regulation of clathrin coat recruitment, actin dynamics (Wenk et al. 2001) and focal adhesion formation (Di Paolo et al. 2002; Ling et al. 2002). In contrast, short PIP5K-Iγ87 seems to be the major producer of the PIP2 pool that supports receptor-induced IP3 generation (Wang et al. 2004). The execution of specific PIP2-modulated processes is very probably achieved by an orchestration of appropriate signaling partners within discrete subcellular microdomains, and PLD-derived PA as well as the PLD enzymes by themselves can contribute to this organization. Indeed, both PLD1 and PLD2 interact with PIP5K-Iα, and PLD2 recruits PIP5K-Iα to a submembraneous vesicular compartment (Divecha et al. 2000). PLD2-derived PA was shown to stimulate PIP5K-Iγ splice variants, and the subsequent formation of PIP2 to drive the initial stages of integrin-mediated cellular adhesion (Powner et al. 2005). In many processes, the temporal activation and correct localization of PLD and PIP5K isoforms by monomeric GTPases appears crucial to achieve the spatially organized production of PIP2 and PA (Santarius et al. 2006). ARF GTPases and membrane traffic Although the direct interaction site on PLD for ARF has not yet been unequivocally defined, it is well established that ARF proteins, particularly ARF1 and ARF6, activate both PLD enzymes, but especially PLD1 (Hammond et al. 1995, 1997). ARF GTPases regulate intracellular vesicle trafficking and actin remodeling. ARF1 is localized to the Golgi complex, and is required for proper Golgi structure and function. The use of primary alcohols has also pointed to a role for PLD in vesicle transport to Golgi (Bi et al. 1997; Ktistakis et al. 1996). PLD activity has been shown to stimulate the release of nascent secretory vesicles from the trans-Golgi network (Chen et al. 1997), and to be required for maintaining the structural integrity and function of the Golgi apparatus, but the precise role for PLD in vesicle formation is still controversial. PIP5K is also a direct effector of ARF1, and an ARF1 mutant that selectively activates PIP5K, but not PLD activity, demonstrated that both PLD-derived PA and direct activation of PIP5K by ARF1 contribute to increased PIP2 synthesis (Skippen et al. 2002). In permeabilized cells, ARF1 has been shown to restore secretion by promoting PIP2 synthesis (Fensome et al. 1996), and ARF1-mediated PIP5K activation (Jones et al. 2000a) and recruitment to the Golgi complex (Godi et al. 1999) appears to be critical in Golgi functioning. ARF6 regulates vesicular transport, secretion, and cortical actin reorganization. ARF6 activates PLD, and PA has been implicated in the mediation of the effects of ARF6 in vesicular trafficking events. A critical role for PLD1 in exocytosis has been established in different cell types, including neurons (Humeau et al. 2001), neuroendocrine cells (Vitale et al. 2001) and pancreatic β cells (Hughes et al. 2004). PLD2 has recently emerged as a mediator of ARF-dependent internalization of the μ-opioid receptor (Koch et al. 2003), and both PLD isoforms have been implicated in macrophage phagocytosis (Corrotte et al. 2006; Iyer et al. 2004). In addition, PIP5K colocalizes and interacts with, and is directly activated by ARF6 at the plasma membrane (Honda et al. 1999), and ARF6 and PIP2 colocalize on the plasma membrane and on endosomal structures (Brown et al. 2001). ARF6-organized PIP2 turnover at the plasma membrane is apparently involved in regulated secretion (Aikawa and Martin 2003; Brown et al. 2001; Lawrence and Birnbaum 2003). Focal and transient accumulation of PIP2 by PIP5K is required for phagocytosis as well (Botelho et al. 2000; Coppolino et al. 2002; Wong and Isberg 2003), and PIP2 hydrolysis probably dictates the remodeling of actin necessary for completion of phagocytosis (Scott et al. 2005). The synthesis of PIP2 is essential for priming the exocytotic apparatus, and the recruitment and activation of PLD1 by PIP2 seems the primary mechanism for the functional integration of PLD1 into the exocytotic pathway (Vitale et al. 2001; Waselle et al. 2005). Thus, CD16-induced cytolytic granule secretion mediated by ARF6 was shown to involve PIP5K-Iα membrane targeting and activation of both PIP5K and PLD (Galandrini et al. 2005). PIP2 also recruits additional proteins—for instance the endocytic proteins AP-2, epsin and AP180—to initiate clathrin-coat formation preceding endocytosis (Ford et al. 2001; Itoh et al. 2001; Padrón et al. 2003), and CAPS (Grishanin et al. 2004) to initiate dense-core vesicle exocytosis. Direct activation of PIP5K-Iγ by ARF6 has been shown to stimulate clathrin-coat recruitment to synaptic membranes to allow synaptic vesicle recycling (Krauss et al. 2003). PLD-derived PA may directly contribute to vesicle fusion in a biophysical manner, as PLD cleaves the non-fusogenic lipid, PC, to form the fusogenic lipid, PA. But PA also takes a function as an essential cofactor for PIP5K, and disruption of Golgi membranes (Sweeney et al. 2002), blockade of clathrin-coat assembly (Arneson et al. 1999) and inhibition of ARF1-reconstituted secretion (Way et al. 2000) after quenching of PA production could be attributed to inhibited PIP2 synthesis. Thus, both PLD and PIP2 synthesis seem necessary for membrane trafficking aspects in the endo- and exocytotic machinery. But PLD and PIP5K also mediate other processes down-stream of ARF6. Epidermal growth factor (EGF)-induced membrane ruffling requires ARF6-induced PIP5K-Iα translocation to the ruffles and local PIP2 production. This leads to the recruitment of PLD2, and PLD-derived PA and ARF6 may then synergistically activate PIP5K (Honda et al. 1999). The relationship between ARF and PIP2 is also bidirectional, as phosphoinositides can regulate ARF activity by binding and activating both ARF-specific guanine nucleotide exchange factors (ARF-GEFs) (Klarlund et al. 1998; Paris et al. 1997) and ARF-GTPase-activating proteins (ARF-GAPs) (Kam et al. 2000; Nie et al. 2002) via their PH domains. The fact that ARF-GAPs bind PIP2 with high affinity and specificity offers an attractive feed-back mechanism for terminating ARF activation after a cycle of ARF-induced PIP2 synthesis. Rho GTPases and actin dynamics PA formation, especially by PLD1, has been reported to induce stress fibre formation in specific cell types (Cross et al. 1996; Ha and Exton 1993; Kam and Exton 2001; Porcelli et al. 2002). Rho proteins, in particular RhoA, Rac1 and Cdc42, which control actin cytoskeleton reorganization, exclusively activate PLD1 by direct interaction with its C-terminus (Exton 2002b; Powner and Wakelam 2002). Thus, PLD stimulation by RhoA may happen by direct interaction, but may involve indirect, Rho-dependent mechanisms as well. Inactivation of Rho GTPases, with Clostridium difficile toxin B or Clostridium botulinum C3 exoenzyme, reduced cellular PIP2 levels, resulting in inhibiton of receptor-mediated PIP2 hydrolysis by PLC (Schmidt et al. 1996a) as well as diminished PLD stimulation (Schmidt et al. 1996d). As the inhibition of PLD signaling after Rho inactivation could be largely rescued by the addition of PIP2, Rho proteins do seem to affect PLD via PIP5K regulation (Schmidt et al. 1996c,d). PIP2 is well-known to associate with and regulate the activity of a plethora of actin-binding proteins that organize actin dynamics (Hilpela et al. 2004; Yin and Janmey 2003), and PA and PIP2 may act in concert to mediate Rho-dependent actin cytoskeleton remodeling. PIP5K isoforms are, like PLD, under direct control of Rho GTPases. PIP5K isoforms are markedly stimulated by RhoA, Rac1, and Cdc42 (Chong et al. 1994; Hartwig et al. 1995; Oude Weernink et al. 2004a), and physically associate with both RhoA (Ren et al. 1996) and Rac1 (Tolias et al. 2000), but not with Cdc42 (Oude Weernink et al. 2004a; van Hennik et al. 2003). PIP5K isoforms are now seen as critical mediators of RhoA- and Rac1-induced actin organization and remodeling (Doughman et al. 2003; Shibasaki et al. 1997; Tolias et al. 2000). The established Rho effector Rho-kinase, a serine/threonine kinase, is apparently involved in Rho-dependent regulation of both PLD (Schmidt et al. 1999) and PIP5K activities (Oude Weernink et al. 2000), and PIP5K was found to play an essential role as down-stream effector of Rho and Rho-kinase in neurite remodeling (van Horck et al. 2002; Yamazaki et al. 2002) and platelet cytoskeleton assembly (Gratacap et al. 2001; Yang et al. 2004). But Rho may also directly signal to PIP5K independently of Rho-kinase, as RhoA-induced activation of ERM (ezrin, radixin, moesin) proteins, that cross-link actin filaments to plasma membranes, was found to be mediated by PIP5K, but not by Rho-kinase (Matsui et al. 1999). PLD and PIP5K were also demonstrated to collectively mediate Rho-induced changes in the actin cytoskeleton. Thus, myogenic differentiation induced by arginine-vasopressin, which involves actin fiber formation, is mediated by Rho proteins and PLD1, and involves PLD-induced PIP2 synthesis along the actin fibers (Komati et al. 2005). These findings suggest that PLD and PIP5K enzymes may co-operate down-stream of Rho in processes that depend on actin organization. Another Rho effector, PKC-related protein kinase N (PKN), also directly interacts with PLD (Oishi et al. 2001) and mediates PLD activation by the α1-adrenergic receptor (Parmentier et al. 2002). Interestingly, components of the actin regulatory machinery, β-actin and α-actinin, have been found to directly associate with and inhibit the activity of PLD isoforms (Lee et al. 2001; Park et al. 2000). PLD also binds to and is stimulated by filamentous F-actin, and PLD1 in particular may act as a signal transduction component responsive to dynamic changes of the actin cytoskeleton (Kusner et al. 2002). PKN interacts with α-actinin, and PKN may modulate PLD signaling by reversing the inhibitory effect of α-actinin on PLD1, and by direct interaction with PLD1. Regulation of PLD and PIP5K by membrane receptors In line with the critical role of PA in cellular processes, the enzymatic activity of PLD is tightly regulated by a variety of hormones, neurotransmitters, and growth factors. Regulation of PLD enzymes by membrane receptors, including G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), is complex and mediated by several cytosolic factors, including PKC as well as ARF, Rho and Ras GTPases (Exton 2002b; Liscovitch et al. 2000; López De Jesús et al. 2006; Powner and Wakelam 2002). Most receptors that stimulate PLD also increase PLC activity, leading to activation of the PLD regulator PKC, and it was assumed that PLD activation might be secondary to PLC activation. A physical association between PLD with PKC isoforms has been reported, resulting in strong activation of in vitro PLD1 activity, and the major interaction site was identified within the N-terminus of PLD1 (Park et al. 1998). Indeed, inhibition of PKC was shown to reduce receptor-induced PLD responses, and PLD1 mutants unresponsive to PKC did respond poorly to activation of GPCRs (Zhang et al. 1999) or to active Gαq proteins (Xie et al. 2002). However, stimulation of PLD in several receptor systems, including M3 muscarinic and α1-adrenergic receptors, was actually PKC-independent (Balboa and Insel 1998; Muthalif et al. 2000; Rümenapp et al. 1997; Schmidt et al. 1994), suggesting that PLD stimulation must not necessarily be secondary to PLC stimulation. Brefeldin A, an inhibitor of certain ARF-GEFs, reduced receptor signaling to PLD in several cell types, indicating that ARF proteins participate in receptor-mediated PLD stimulation (Fensome et al. 1998; Mitchell et al. 1998; Rümenapp et al. 1995; Shome et al. 2000). Likewise, sequestration of ARF-GEFs by the ARF-related protein ARP inhibited M3 muscarinic receptor signaling to PLD (Schürmann et al. 1999). Clostridial toxins and enzymes that specifically inactivate Rho proteins and expression of inactive Rho mutants have been used to identify the role of Rho in signaling to PLD. Thus, Rho proteins were found to be involved in PLD stimulation by GPCRs (M3 muscarinic, bradykinin, sphingosine-1-phosphate and LPA), RTKs (PDGF, EGF), and immunoglobulin (FcεRI) receptors (Hess et al. 1997; Ojio et al. 1996; Schmidt et al. 1996c). Stimulation of PLD by GPCRs was shown to be mediated by both pertussis toxin (PTX)-insensitive (Gosau et al. 2002; Schmidt et al. 1994) and PTX-sensitive (Cummings et al. 2002; Fensome et al. 1998) heterotrimeric G proteins. G12 family proteins can stimulate PLD (Plonk et al. 1998), and RGS (regulators of G protein signaling) proteins, that act as α subunit-specific GAPs, have been used to position G12 in PLD activation by the M3 muscarinic (Rümenapp et al. 2001), the PAR1 (Fahimi-Vahid et al. 2002), and the Ca2+-sensing receptor (Huang et al. 2004), as well as mechanical force (Ziembicki et al. 2005). As forskolin and cAMP were shown to cause activation of PLD via PKA and ERK1/2 (Ginsberg et al. 1997; Yoon et al. 2005) or, alternatively, via the cAMP-activated GEF for Ras-like GTPases, Epac and R-Ras (López De Jesús et al. 2006), Gs proteins also mediate stimulation of PLD. PLD activation is also controlled by βγ-subunits, possibly via Src and/or ARF6 (Le Stunff et al. 2000; Ushio-Fukai et al. 1999), but Gβγ can also directly interact with and inhibit PLD (Preininger et al. 2006). As the precise mechanism of PLD stimulation in intact cells was only poorly understood, during the last 10 years our laboratory in Essen has focused on the regulation of PLD activity by membrane receptors. In HEK-293 cells, signaling to PLD by a typical GPCR, the M3 muscarinic receptor, and an RTK, the EGF receptor, was studied and shown to be executed by several distinct pathways (Fig. 2). In addition, by expressing inactive PLD mutants, the M3 muscarinic and the EGF receptors were found to signal to individual PLD isozymes and to selectively stimulate PLD1 and PLD2 respectively (Han et al. 2001). The M3 muscarinic receptor stimulates both PLC and PLD via PTX-insensitive mechanisms (Offermanns et al. 1994; Peralta et al. 1988; Schmidt et al. 1994). Interestingly, stimulation of PLD by the agonist carbachol was not affected by PKC inhibitors, suggesting that activation of PLD by the M3 muscarinic receptor was rather independent of PLC (Rümenapp et al. 1997; Schmidt et al. 1994). Expression of α-subunits of G proteins and of specific RGS proteins was used to identify the G proteins involved in these pathways, and demonstrated that whereas the M3 receptor signals to PLC via Gq proteins, activation of PLD is mediated by G12 family proteins (Rümenapp et al. 2001). PLD activation by the M3 receptor, but not by the EGF receptor, was further found to be under control of ARF (Rümenapp et al. 1995, 1997) as well as Rho proteins, particularly RhoA (Schmidt et al. 1996c,d). Likewise, regulation of mTOR by LPA, but not PDGF, involved PLD1 activation by Rho GTPases (Kam and Exton 2004). Both ARF1 and RhoA were found to become activated after M3 receptor activation (Keller et al. 1997; Rümenapp et al. 1995), and a role for Rho-kinase in RhoA-controlled PLD stimulation could be demonstrated (Schmidt et al. 1999). In further studies, it was shown that activation of PLD by RhoA and Rho-kinase is mediated by G12 and the tyrosine kinase Pyk2, whereas activation by ARF1 is mediated by G13, PI3K and the Arf-GEF ARNO (Han et al. 2003). In cardiomyocytes, Rho proteins were shown to affect signaling to PLD by both endothelin-1 and thrombin, apparently by controlling PIP2 synthesis, whereas ARF selectively affects signaling by the PAR1 receptor (Fahimi-Vahid et al. 2002). Fig. 2Regulation of PLD by the M3 muscarinic receptor and receptor tyrosine kinases in HEK-293 cells. In human embryonic kidney (HEK-293) cells, signaling to PLD by the M3 muscarinic receptor and by typical RTKs (EGF, PDGF, insulin) is organized into rather discrete pathways and channeled by particular heterotrimeric G proteins and small GTPases (orange), specific GEF proteins (pink) and further signaling components (green). AC, adenylyl cyclase; ROCK, Rho-kinase PLD can directly interact with RalA, and a Ras/Ral signaling cascade was shown to regulate PLD responses. In HEK-293 cells, Ras and RalA—but not Rho proteins—were located in RTK signaling to PLD, and this Ras/Ral-dependent signaling cascade was found to be dependent on PKC-α and a Ral-specific GEF (Fig. 2) (Schmidt et al. 1998; Voss et al. 1999). RalA apparently co-operates with ARF (Kim et al. 1998; Xu et al. 2003) and Rho proteins (Frankel et al. 1999; Wilde et al. 2002) to achieve full PLD activation. Likewise, Ras proteins were found to modulate PLD responses by PDGF (Lucas et al. 2000), and RalA to affect EGF receptor signaling to PLD (Lu et al. 2000). It was recently shown that direct activation of Ras-related R-Ras by Epac is involved in PLD stimulation by the M3 muscarinic receptor, apparently by coupling to Gs proteins (López de Jesús et al. 2006), but a contribution of Ral proteins to GPCR-induced PLD activation has not been found (Meacci et al. 2002). Collectively, these data demonstrate that heterotrimeric G proteins as well as small GTPases co-ordinate PLD activation by specific membrane receptors in particular cell types, and these mechanisms probably contribute to the organization of agonist-induced PA production for the execution of diverse cellular signaling tasks. In addition, the synthesis of PIP2 can be directly stimulated by GPCRs (thrombin, LPA, M3 muscarinic) as well as RTKs (Cochet et al. 1991; Nolan and Lapetina 1990; Pike and Eakes 1987). Receptor activation leads to increased association of PIP5K with the actin cytoskeleton (Grondin et al. 1991; Payrastre et al. 1991), and receptor-induced stimulation and cytoskeletal association of PIP5K may be directly involved in actin cytoskeletal regulation and initialize the assembly of enzymes into signaling complexes. GPCR-induced stimulation of PIP2 synthesis was found to be mediated by pertussis toxin-sensitive Gi proteins (Schmidt et al. 1996b; Stephens et al. 1993), but also by G12 and Gq proteins (Oude Weernink et al. 2003). Enhanced PIP2 synthesis is also caused by conventional PKC isoforms, which may increase PIP5K activity by stimulating PIP5K dephosphorylation by the okadaic acid-sensitive protein phosphatase 1 (Park et al. 2001). Concluding remarks In the last decade, PLD has taken a firm position as all-round player in cellular signaling events. It is now appreciated that PLD and PIP5K act together to execute several important cellular functions, including vesicle transport, cytoskeleton dynamics and cell adhesion. Because of the reciprocal stimulation of their activities it seems inappropriate to generally assign a conventional “upstairs-downstairs” relationship to PLD and PIP5K isozymes. The localized generation of the lipid messengers by PLD and PIP5K, PA and PIP2, is clearly co-ordinated by small GTPases of the ARF, Rho and Ras families. The following picture emerges of how PLD and PIP5K may co-operate to execute their cellular tasks. Particular small GTPases, activated by membrane receptors or cellular factors, bind to PIP5K and recruit the enzyme to specific cellular compartments. Subsequent activation of PIP5K catalytic activity triggers the localized generation of PIP2, which now serves as an anchor for specific proteins, including PLD enzymes. The sequestered PLD is activated by PIP2 and the GTPases, and PLD-derived PA now, among other tasks, contributes to the activation of PIP5K. This feed-forward regulation loop depends on both PIP5K and PLD, and quenching of PA formation (by primary alcohols) or reduction of PIP2 levels (by PLC-mediated hydrolysis or dephosphorylation by phosphatases) can interrupt the snowball from rolling. PIP2 dephosphorylation may be important in the cell as a decisive mechanism to terminate the localized reactions before a cellular avalanche develops. Attractive candidates are further specific GEFs and GAPs for the GTPases, some of which have been shown to be directly regulated by PIP2. PIP2-dependent inactivation of the organizing GTPase may then provide the final turn-off signal.

Purinergic_Signal-3-4-2072915

New 2,6,9-trisubstituted adenines as adenosine receptor antagonists: a preliminary SAR profile

A new series of 2,6,9-trisubstituted adenines (5–14) have been prepared and evaluated in radioligand binding studies for their affinity at the human A1, A2A and A3 adenosine receptors and in adenylyl cyclase experiments for their potency at the human A2B subtype. From this preliminary study the conclusion can be drawn that introduction of bulky chains at the N6 position of 9-propyladenine significantly increased binding affinity at the human A1 and A3 adenosine receptors, while the presence of a chlorine atom at the 2 position resulted in a not univocal effect, depending on the receptor subtype and/or on the substituent present in the N6 position. However, in all cases, the presence in the 2 position of a chlorine atom favoured the interaction with the A2A subtype. These results demonstrated that, although the synthesized compounds were found to be quite inactive at the human A2B subtype, adenine is a useful template for further development of simplified adenosine receptor antagonists with distinct receptor selectivity profiles. Introduction Adenosine, a naturally occurring nucleoside, is involved in a wide variety of physiological and pathophysiological processes [1]. Adenosine mediates these effects through the activation of at least four human receptor subtypes (P1), belonging to the superfamily of G protein-coupled receptors, which have been recently cloned [2] and classified as A1, A2A, A2B and A3 [3]. The subtypes are classified on the bases of coupling to second messengers and pharmacological profiles for agonists and antagonists. In fact, A1 and A3 adenosine receptor subtypes are linked to inhibition of adenylyl cyclase and A2A and A2B subtypes are linked to stimulation of the same enzyme [4]. In particular, A2B receptors have been implicated in several physiological functions such as the regulation of mast cell secretion [5, 6], gene expression [5, 7, 8], cell growth [9] and intestinal functions. A2B receptors may also play a role in asthma, since they mediate mast cell degranulation from human mast cells and are present in high density in human blood eosinophils [10, 11]. For this reason A2B antagonists could be considered potential antiasthmatic agents [10–12]. While the A1, A2A and A3 adenosine receptors have been pharmacologically characterized through the use of highly potent and selective agonists and/or antagonists, an accurate investigation of the pathophysiological role of A2B receptors is precluded due to the lack of very selective ligands [13]. On the other hand, only recently radiolabelled adenosine antagonists have been used for binding assays at the A2B receptor subtype [1]. Recently, xanthine derivatives, such as compounds 1 and 2 in Fig. 1, have been proposed as potent and selective adenosine receptor antagonists [14, 15]. On the other hand, in the non-xanthine family poor results have been obtained in recent years. However, mention should be made of the pyrazolo-triazolo-pyrimidine derivative 3, which showed promising binding affinity at the A2B adenosine receptor although the level of selectivity vs the human (h) A3 subtype was still poor [16]. Very recently, a bipyrimidyl derivative 4 has been proposed as an A2B adenosine receptor antagonist, with affinity in the same range of compound 3, while the selectivity vs the other receptor subtypes was found to be significantly better [17] (Fig. 1). Fig. 1Structures and binding profile (Ki nM) of some A2B adenosine receptor antagonists A structural analysis of the derivatives 1 and 3 clearly shows the presence of bulky substituents such as aryloxyacetylamino-phenyl groups at the 8 position (compound 1) and arylacetyl moiety at the N5 position (compound 3). On the other hand, in recent years a number of substituted adenines have been synthesized and tested at the four adenosine receptor subtypes, demonstrating that the introduction of different substituents at the 2, 8 and 9 positions of the adenine core resulted in high-affinity antagonists with distinct receptor selectivity profile [18–22]. At the A2B receptor the derivatives bearing an ethyl in the 9 position and linear chains in the 2 position showed potency in the μM range, while the presence of sterically hindered substituents in the same positions was detrimental for the potency. Furthermore, substitution of the 9-ethyl group with a propyl chain seems to favour the interaction with human A2B receptors [21]. Hence, on the basis of the results obtained with compound 3, introduction of a bulky substituent on the N6 amino group of the adenine and 2-chloroadenine moiety could increase potency and selectivity for the human A2B adenosine receptor subtypes. Hence, bulky substituents such as arylacetyl or aryloxyphenylacetyl moieties were introduced on the N6 amino group of compounds 5 and 6 to obtain derivatives 7–14, with the aim of finding a new class of A2B antagonists (Fig. 2). Fig. 2Structures of designed compounds Chemistry The designed compounds (5–14) have been synthesized as summarized in Schemes 1 and 2. The starting 9-propyladenine (5) [23] was obtained by alkylation of commercially available adenine (15) with propyliodide in the presence of potassium carbonate. Flash chromatography led to the desired 9-substituted isomer 5 as the major product (yield 79%) together with the 7-isomer 5a (yield 9%). Isomeric structure of compounds 5 and 5a was assigned on the bases of 1D-1H-NOE difference spectra. In fact, irradiation of both CH2 groups of the propylic chain in compound 5a gave a NOE at both H-C(8) and 6-NH2 groups, demonstrating the 7 position as the alkylation site. On the contrary, a NOE at both H-C(8) and H-C(2), and not at the 6-NH2 group in compound 5, upon saturation of CH2 groups of the propylic chain, confirmed the 9 position as the alkylation site. Scheme 1a Synthesis of 9-propyladenine; b Synthesis of 2-chloro-9-propyladenine. Reagents: i: DMF, K2 CO3, propyliodide, RT; ii: liq. NH3, sealed tube, RTScheme 2Reagents: i: CDI, dry THF, reflux The 2-chloro derivative 6 was obtained by alkylation of commercially available 2,6-dichloropurine (16) with propyliodide, using the same procedure utilized for compound 5, to afford the 9-substituted isomer 17 as the major product (yield 75%) along with the 7-isomer 17a (yield 10%) [24]. [1H]-NMR spectra of 17 and 17a in CDCl3 are in agreement with those reported in the literature [24]; in the experimental part [1H]-NMR spectra of the same compounds are reported using dimethyl sulfoxide (DMSO) as the solvent. Compound 17 was reacted with liquid ammonia in a sealed tube at room temperature (RT) overnight to give the 2-chloro-9-propyladenine (6) [24] (Scheme 1). Final compounds 7–14 were obtained by condensation of amino compounds 5 or 6 with the appropriate acid 18–22 [25] in the presence of carbonyldiimidazole in tetrahydrofurane (THF) at reflux for 18 h (Scheme 2). Results and discussion All the compounds were evaluated at the human recombinant adenosine receptors, stably transfected into Chinese hamster ovary (CHO) cells, utilizing radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assay (A2B). Receptor binding affinity was determined using [3H]CCPA (2-chloro-N6-cyclopentyladenosine) as the radioligand for A1 receptors, whereas [3H]NECA (5’-N-ethylcarboxamidoadenosine) was used for the A2A and A3 subtypes. In the case of A2B receptors Ki values were calculated from IC50 values determined by inhibition of NECA-stimulated adenylyl cyclase activity. Ki values are in μM, with 95% confidence intervals in parentheses [26]. The results of binding and cyclase activity studies are reported in Table 1. Table 1Biological profile of synthesized compounds 5–14aDisplacement of specific [3H]-CCPA binding at human A1 receptors expressed in CHO cells. bDisplacement of specific [3H]-NECA binding at human A2A receptors expressed in CHO cells. cKi values of the inhibition of NECA-stimulated adenylyl cyclase activity in CHO cells expressing human A2B receptors. dDisplacement of specific [3H]-NECA binding at human A3 receptors expressed in CHO cells. All the tested compounds 5–14 showed affinities at the human A1, A2A and A3 adenosine receptors in the μM range without significant levels of selectivity. At A2B receptors most compounds were found to be inactive when tested at a concentration up to 100 μM (Ki values > 30 μM). It is quite evident that the introduction of phenylacetic or aryloxyphenylacetic moieties at the N6 position of 9-propyladenine (5) or of 2-chloro-9-propyladenine (6) to give compounds 7–14 modifies the binding profile of the derivatives, although without significantly increasing binding affinity (Table 1). On the other hand, the same substitutions were found to be detrimental for the activity at the A2B receptor subtype. In fact, the N6-unsubstituted derivative 6 proved to be the most potent of the series with Ki A2B = 11 μM. The effect of the chlorine at the 2 position on binding affinity at the adenosine receptors it is not univocal, depending on the receptor subtype and/or on the substituent in N6. Analysis of the binding profile of the N6-unsubstituted derivatives in more detail revealed that the presence of a chlorine atom at the 2 position (compound 6) increased the affinity (A1, A2A and A3) and potency (A2B) at adenosine receptors two- to threefold compared with the unsubstituted analogue 5. A quite similar profile could be observed when a 4-bromophenylacetic group was introduced at the N6 position (compare compound 8 with 7). An opposite effect of the chlorine atom was detected at A1, A2B and A3 receptors when a bulkier substituent, such as the 4-aryloxyphenylacetic chain, was introduced at the N6 position. In fact, this kind of combination significantly reduced or did not modify the A1 and A3 affinity and the A2B potency (9: Ki A1 = 1.4 μM, Ki A2B > 30 μM, Ki A3 = 5.3 μM vs 10: Ki A1 = 26 μM, Ki A2B > 30 μM, Ki A3 = 4.9 μM and 12: Ki A1 = 13 μM, Ki A2B = 22 μM, Ki A3 = 10 μM vs 13: Ki A1 = 22 μM, Ki A2B > 30 μM, Ki A3 = 19 μM). However, in all cases, the presence of a chlorine atom in the 2 position favoured the interaction with the A2A subtype (compare A2A affinity of 5, 7, 9 and 12 with 6, 8, 10 and 13, respectively), while the presence of any substituent on the N6 position seems to somewhat reduce the affinity. In fact the compound endowed with the highest A2A affinity proved to be the 2-chloro-9-propyladenine (6: Ki A2A = 2.2 μM). These findings are in agreement with previous observations related to adenosine analogues strongly suggesting that the introduction of substituents in the N6 position dramatically reduces the A2A affinity [27–32]. Nevertheless, it should be underlined that the presence of a bulky chain at the N6 position significantly increased (20- to 70-fold) the affinity at the A1 and A3 subtypes in comparison with 9-propyladenine (9: Ki A1 = 1.4 μM and 11: Ki A3 = 1.4 μM vs 5: Ki A1 = 24 μM and Ki A3 > 100 μM). This increase of affinity seems also to be modulated by the substituent on the aryloxyphenylacetic group; in fact, substitution with a lipophilic bromine (9) or methyl group (11) at the para position is responsible for the increased A1 and A3 receptor affinity, respectively, while the presence of a hydrogen (12) or a methoxy group (14) did not positively influence the binding profile. Conclusions In conclusion the study herein presented, although it did not reach the proposed goal of obtaining A2B adenosine receptor antagonists, increased knowledge of the structure-activity relationships in adenine derivatives. Moreover, it was demonstrated that the introduction of bulky substituents at the N6 position of adenine derivatives significantly increased the affinity at the A1 and A3 adenosine receptors, while the presence of a chlorine atom in the 2 position favoured the interaction with the A2A subtype. These results demonstrated that, although the synthesized compounds were found to be quite inactive at the human A2B subtype, adenine is a useful template for further development of simplified adenosine receptor antagonists with distinct receptor selectivity profiles, opening up new chances to design structurally simplified A1 and A3 adenosine receptor antagonists. Experimental section Chemistry General: melting points were determined with a Büchi apparatus and are uncorrected. 1H NMR spectra were obtained with Varian VXR 300 MHz spectrometer; δ in ppm, J in Hz. All exchangeable protons were confirmed by addition of D2O. Thin layer chromatography (TLC) was carried out on precoated TLC plates with silica gel 60 F-254 (Merck). For column chromatography, silica gel 60 (Merck) was used. Elemental analyses were determined on Fisons Instruments Model EA 1108 CHNS-O model analyser and are within ± 0.4% of theoretical values. 9-Propyladenine (5) and 7-propyladenine (5a) To a solution of adenine (15) (0.5 g, 3.7 mmol) in dry DMF (10 ml), under nitrogen, K2CO3 (0.83 g, 5.97 mmol) and propyliodide (0.433 ml, 4.44 mmol) were added. The mixture was stirred at RT for 16 h, then the solvent was removed under reduced pressure and the crude purified by flash chromatography (CHCl3-MeOH 98:2) to afford 5 [23] and 5a (yield 79 and 9%, respectively) as white solids, after crystallization from CH3OH. 5: m.p. 173–175°C. 1H-NMR (DMSO-d6) δ 0.85 (t, 3H, J = 7.4, CH3); 1.82 (m, 2H, CH2-CH3); 4.11 (t, 2H, J = 7.0, CH2-N); 7.21 (bs, 2H, NH2); 8.15 (s, 2H, H-2 and H-8). Anal. Calcd. for C5H5N5 (177.2) C, 54.22; H, 6.26; N, 39.52; found: C, 54.54; H, 6.71; N, 39.33. 5a: m.p. >250°C. 1H-NMR (DMSO-d6) δ 0.86 (t, 3H, J = 7.3, CH3); 1.91 (m, 2H, CH2-CH3); 4.26 (t, 2H, J = 7.0, CH2-N); 7.75 (s, 1H, H-2); 7.85 (bs, 2H, NH2); 8.34 (s, 1H, H-8). Anal. Calcd. for C5H5N5 (177.2) C, 54.22; H, 6.26; N, 39.52; found: C, 54.45; H, 6.45; N, 39.45. 2,6-Dichloro-9-propyl-9H-purine (17) and 2,6-dichloro-7-propyl-9H-purine (17a) To a solution of 2,6-dichloropurine (16) (1 g, 5.29 mmol) in dry DMF (14 ml), under nitrogen, K2CO3 (1.18 g, 6.61 mmol) and propyliodide (0.59 ml, 6.08 mmol) were added. The mixture was stirred at RT overnight, then the solvent was removed under reduced pressure and the crude purified by flash chromatography (cC6H12-EtOAc 75:25) to afford 17 and 17a as white solids (yield 75 and 10%, respectively) [24]. 17: m.p. 58–59°C; 1H-NMR (DMSO- d6) δ 0.86 (t, 3H, J = 7.5 Hz, CH2CH3), 1.85 (m, 2H, CH2CH3), 4.21 (t, 2H, J = 7.0 Hz, N-CH2), 8.76 (s, 1H, H-8). Anal. Calcd. for C8H8Cl2N4 (231.1) C, 41.58; H, 3.49; N, 24.25. Found: C, 41.85; H, 3.70; N, 24.10. 17a: m.p. 103–105°C; 1H-NMR (DMSO-d6) δ 0.87 (t, 3H, J = 7.4 Hz, CH2CH3), 1.84 (m, 2H, CH2CH3), 4.40 (t, 2H, J = 7.2 Hz, N-CH2), 8.89 (s, 1H, H-8). Anal. Calcd. for C8H8Cl2N4 (231.1) C, 41.58; H, 3.49; N, 24.25. Found: C, 41.75; H, 3.55; N, 24.19. 2-Chloro-9-propyladenine (6) Liquid ammonia (5 ml) and compound 17 (0.46 g, 1.97 mmol) were poured into a sealed tube and the resulting mixture was stirred at RT overnight. Ammonia was evaporated and the crude purified by flash chromatography (CHCl3-MeOH 99:1) to give 6 [24] as a white solid (yield 75%) m.p. 224–226°C. 1H-NMR (DMSO-d6) δ 0.84 (t, 3H, J = 7.3 Hz, CH2CH3), 1.79 (m, 2H, CH2CH3), 4.05 (t, 2H, J = 7.2 Hz, N-CH2), 7.72 (s, 2H, NH2), 8.15 (s, 1H, H-8). Anal. Calcd. for C8H10ClN5 (211.7) C, 45.40; H, 4.76; N, 33.09. Found: C, 45.75; H, 4.80; N, 32.87. General procedure for the preparation of the N6-acylaminoadenine (7–14) A solution in dry THF (4 ml) of the appropriate acid (18–22) (0.46 mmol) and carbonyldiimidazole (83 mg, 0.51 mmol) was poured at reflux under nitrogen for 1 h. Then the amino compound 5 or 6 (0.46 mmol) was added and the resulting mixture was refluxed overnight. The solvent was removed under reduced pressure and the crude purified by flash chromatography to afford the desired final compounds 7–14. 6-[(4-Bromophenyl)acetyl]amino-9-propyladenine (7) Eluent for chromatography CHCl3-MeOH 95:5; yield 59%, white solid; m.p. 149–151°C (dec.); 1H-NMR (DMSO-d6): δ 0.83 (t, 3H, J = 7.2 Hz, CH2CH3), 1.84 (m, 2H, CH2CH3), 3.89 (s, 2H, CH2-CO), 4.19 (t, 2H, J = 7.1 Hz, N-CH2), 7.30 (d, 2H, J = 8.4 Hz, H-Ph), 7.51 (d, 2H, J = 8.4 Hz, H-Ph), 8.47 (s, 1H, H-8), 8.62 (s, 1H, H-2), 10.91 (s, 1H, NH). Anal. Calcd. for C16H16BrN5O (374.2) C, 51.35; H, 4.31; N, 18.71. Found: C, 51.65; H, 4.80; N, 18.50. 6-[(4-Bromophenyl)acetyl]amino-2-chloro-9-propyladenine (8) Eluent for chromatography CHCl3-cC6H12 80:20; yield 26%, white solid; m.p. 164–166°C; 1H-NMR (DMSO-d6): δ 0.84 (t, 3H, J = 7.5 Hz, CH2CH3), 1.83 (m, 2H, CH2CH3), 3.88 (s, 2H, CH2-CO), 4.15 (t, 2H, J = 6.9 Hz, N-CH2), 7.30 (d, 2H, J = 8.4 Hz, H-Ph), 7.53 (d, 2H, J = 8.4 Hz, H-Ph), 8.50 (s, 1H, H-8), 11.25 (s, 1H, NH). Anal. Calcd. for C16H15BrClN5O (408.7) C, 47.02; H, 3.70; N, 17.14. Found: C, 47.49; H, 3.83; N, 17.40. 6-[(4-(4-Bromobenzyloxy)phenyl)acetyl]amino-9-propyladenine (9) Eluent for chromatography CHCl3-MeOH 95:5; yield 58%, white solid; m.p. 154–156°C; 1H-NMR (DMSO-d6): δ 0.85 (t, 3H, J = 7.5 Hz, CH2CH3), 1.85 (m, 2H, CH2CH3), 3.82 (s, 2H, CH2-CO), 4.21 (t, 1H, J = 7.0 Hz, N-CH2), 5.07 (s, 2H, CH2-O), 6.95 (d, 2H, J = 8.8 Hz, H-Ph), 7.27 (d, 2H, J = 8.4 Hz, H-Ph), 7.40 (d, 2H, J = 8.4 Hz, H-Ph), 7.58 (d, 2H, J = 8.4 Hz, H-Ph), 8.48 (s, 1H, H-8), 8.62 (s, 1H, H-2), 10.81 (s, 1H, NH). Anal. Calcd. for C23H22BrN5O2 (480.4) C, 57.51; H, 4.62; N, 14.58. Found: C, 57.99; H, 4.66; N, 14.55. 6-[(4-(4-Bromobenzyloxy)phenyl)acetyl]amino-2-chloro-9-propyladenine (10) Eluent for chromatography CHCl3-cC6H12-MeOH 50:48:2; yield 14%, white solid; m.p. 176–178°C; 1H-NMR (DMSO-d6): δ 0.81 (t, 3H, J = 7.5 Hz, CH2CH3), 1.79 (m, 2H, CH2CH3), 3.76 (s, 2H, CH2-CO), 4.12 (t, 2H, J = 7.4 Hz, N-CH2), 5.03 (s, 2H, CH2-O), 6.92 (d, 2H, J = 8.6 Hz, H-Ph), 7.22 (d, 2H, J = 8.4 Hz, H-Ph), 7.36 (d, 2H, J = 8.4 Hz, H-Ph), 7.54 (d, 2H, J = 8.4 Hz, H-Ph), 8.46 (s, 1H, H-8), 11.11 (s, 1H, NH). Anal. Calcd. for C23H21BrClN5O2 (514.8) C, 53.66; H, 4.11; N, 13.60. Found: C, 53.75; H, 4.25; N, 13.29. 6-[(4-(4-Methylbenzyloxy)phenyl)acetyl]amino-9-propyladenine (11) Eluent for chromatography CHCl3-cC6H12-MeOH 70:28:2; yield 35%, white solid; m.p.131–132°C; 1H-NMR (DMSO-d6): δ 0.83 (t, 3H, J = 7.3 Hz, CH2CH3), 1.84 (m, 2H, CH2CH3), 2.28 (s, 3H, CH3-Ph), 3.80 (s, 2H, CH2-CO), 4.20 (t, 2H, J = 7.1 Hz, N-CH2), 5.02 (s, 2H, CH2-O), 6.93 (d, 2H, J = 8.4 Hz, H-Ph), 7.24 (m, 6H, H-Ph), 8.47 (s, 1H, H-8), 8.62 (s, 1H, H-2), 10.83 (s, 1H, NH). Anal. Calcd. for C24H25N5O2 (415.5) C, 69.38; H, 6.06; N, 16.86. Found: C, 69.74; H, 6.35; N, 16.54. 6-[(4-Benzyloxyphenyl)acetyl]amino-9-propyladenine (12) Eluent for chromatography CHCl3-MeOH 97:3; yield 52%, white solid; m.p. 121–123°C; 1H-NMR (DMSO-d6): δ 0.83(t, 3H, J = 7.5 Hz, CH2CH3), 1.84 (m, 2H, CH2CH3), 3.80 (s, 2H, CH2-CO), 4.19 (t, 2H, J = 7.1 Hz, N-CH2), 5.07 (s, 2H, CH2-O), 6.95 (d, 2H, J = 8.4 Hz, H-Ph), 7.25 (d, 2H, J = 8.8 Hz, H-Ph), 7.37 (m, 5H, H-Ph), 8.47 (s, 1H, H-8), 8.62 (s, 1H, H-2), 10.82 (s, 1H, NH). Anal. Calcd. for C23H23N5O2 (401.5) C, 68.81; H, 5.77; N, 17.44. Found: C, 68.97; H, 5.89; N, 17.35. 6-[(4-Benzyloxyphenyl)acetyl]amino-2-chloro-9-propyladenine (13) Eluent for chromatography CHCl3-MeOH 99:1; yield 17%, white solid; m.p. 150–152°C; 1H-NMR (DMSO-d6): δ 0.84 (t, 3H, J = 7.3 Hz, CH2CH3), 1.82 (m, 2H, CH2CH3), 3.79 (s, 2H, CH2-CO), 4.14 (t, 2H, J = 7.0 Hz, N-CH2), 5.07 (s, 2H, CH2-O), 6.95 (d, 2H, J = 8.6 Hz, H-Ph), 7.25 (d, 2H, J = 8.4 Hz, H-Ph), 7.37 (m, 5H, H-Ph), 8.49 (s, 1H, H-8), 11.16 (s, 1H, NH). Anal. Calcd. for C23H22ClN5O2 (435.9) C, 63.37; H, 5.09; N, 16.07. Found: C, 63.56; H, 5.14; N, 15.76. 6-{[(4-Methoxybenzyloxy)phenyl]acetyl}amino-2-chloro-9-propyladenine (14) Eluent for chromatography CHCl3-MeOH 99:1; yield 29%, white solid; m.p. 174–176°C; 1H-NMR (DMSO-d6): δ 0.84 (t, 3H, J = 7.4 Hz, CH2CH3), 1.82 (m, 2H, CH2CH3), 3.73 (s, 3H, CH3-O), 3.79 (s, 3H, CH2-CO), 4.14 (t, 2H, J = 7.0 Hz, N-CH2), 4.98 (s, 2H, CH2-O), 6.92 (d, 2H, J = 8.0 Hz, H-Ph), 6.93 (d, 2H, J = 8.4 Hz, H-Ph), 7.24 (d, 2H, J = 8.4 Hz, H-Ph), 7.35 (d, 2H, J = 8.4 Hz, H-Ph), 8.49 (s, 1H, H-8), 11.15 (s, 1H, NH). Anal. Calcd. for C24H24ClN5O3 (465.9) C, 61.87; H, 5.19; N, 15.03. Found: C, 61.99; H, 5.33; N, 14.91. Biology All pharmacological methods followed the procedures as described earlier [26]. In brief, membranes for radioligand binding were prepared from CHO cells stably transfected with human adenosine receptor subtypes in a two-step procedure. In a first low-speed step (1,000 g) cell fragments and nuclei were removed. The crude membrane fraction was sedimented from the supernatant at 100,000 g. The membrane pellet was resuspended in the buffer used for the respective binding experiments, frozen in liquid nitrogen and stored at −80°C. For the measurement of adenylyl cyclase activity only one high speed centrifugation of the homogenate was used. The resulting crude membrane pellet was resuspended in 50 mM Tris/HCl, pH 7.4 and immediately used for the cyclase assay. For radioligand binding at A1 adenosine receptors 1 nM [3H]CCPA was used, whereas 30 and 10 nM [3H]NECA were used for A2A and A3 receptors, respectively. Non-specific binding of [3H]CCPA was determined in the presence of 1 mM theophylline; in the case of [3H]NECA 100 pM R-PIA was used. Ki values from competition experiments were calculated with the program SCTFIT [33]. At A2B adenosine receptors inhibition of NECA-stimulated adenylyl cyclase activity was used as a measurement of potency of the new compounds. IC50 values from these experiments were converted to Ki values with the Cheng and Prusoff equation [34].

J_Med_Internet_Res-7-5-1550689

Architecture for Knowledge-Based and Federated Search of Online Clinical Evidence

Background It is increasingly difficult for clinicians to keep up-to-date with the rapidly growing biomedical literature. Online evidence retrieval methods are now seen as a core tool to support evidence-based health practice. However, standard search engine technology is not designed to manage the many different types of evidence sources that are available or to handle the very different information needs of various clinical groups, who often work in widely different settings. Introduction Clinicians need to keep up-to-date with the biomedical literature in order to practice according to the best available evidence. However, this has become increasingly difficult as the amount of medical literature a clinician needs to consider grows exponentially [1,2]. As a result, the effort required to find a specific piece of evidence increases year after year [3]. Clinicians typically work under time pressure, which compounds the problem. The need to develop robust methods and tools to support evidence access is now widely recognized. Online evidence retrieval methods are increasingly seen as a core tool in support of evidence-based health care [4]. In the traditional model of online evidence services, clinicians have access to a number of online information sources, such as journals, databases, and Medline, each with its own idiosyncrasies and search interfaces. This means users need to know which resources are most suitable for their current question and how the search query must be formulated for a given resource. Interoperability standards for the efficient dissemination of content are being developed (eg, the Open Archive Initiative [5]), but until the majority of content adheres to such standards, there is still a need to search through heterogenous data sources. The meta-search engine approach [6,7] addresses many of the limitations of these models by providing a mechanism to search all the available resources at one time and by translating user queries into the respective query languages of each resource. This typically uses a least-common-denominator approach, directly passing on user keywords to different information sources without regard for the specific capabilities or limitations of these resources. For example, a meta-search engine often disregards the rich query language available with some resources in order to simplify the overall meta-search process. Consequently, while the user expects the meta-search to return an integrated set of search results, the reality is that some resources would have been able to perform much better had they been queried individually; the user is unaware of the variations in search quality across the different resources that have been queried for them. Variants of the standard meta-search engine approach have been shown [8] to provide search capabilities beyond the least common denominator but still require users to select the resources they wish to search. One solution to this problem is to “federate” the different resources so that they more genuinely behave as one uniform data source. A federated search system may perform a syntactic reformulation of a user query, translating it into queries that have been optimized for the native query language of individual evidence sources. Semantic reformulation is also possible [9]. For example, user keywords may be translated into equivalent keywords or phrases using a terminological system. However, a federated search can still produce an excessive number of candidate documents, or hits, many of them failing reasonable tests of relevance. One way to improve the chance of retrieving clinically relevant information is to pre-program a search system with specialist bibliographic knowledge using search filters. Search filters capture expert strategies for searching that are known to improve the precision of searches. For example, Medline offers a small set of “clinical queries,” which are pre-defined and validated search filters optimized to retrieve documents that are most likely to be clinically relevant, emphasizing disease etiology, diagnosis, therapy, or prognosis [10,11]. Such search filters are necessarily highly customized to the capabilities of individual information sources and their native search engines. For a federated search system to consistently use search filters, it would need to develop a generalized approach to search filters, or meta-search filters. Quick Clinical (QC) [4] is a federated evidence retrieval system designed to meet the specific needs of clinicians. Its design incorporates the novel use of meta-search filters to optimize search strategies, and it is based upon a wrapper-mediator architecture built around a universal query language. This paper describes the system architecture of QC and the technical challenges to the design of online evidence retrieval systems, and it reports on the technical performance of the system from a clinical trial with primary care physicians. Methods The Quick Clinical System User Interface In the QC user model, a user is presented with a single query interface, which connects to an arbitrarily large number of federated knowledge sources and incorporates query specific meta-search filters called “profiles.” QC guides users to first consider the purpose of their search through selection of a profile, and it then asks them to provide specific keywords related to that search task. As a consequence, users are guided through a process that structures their query for them and improves the chances that they will ask a well-formed query and receive an appropriate answer. Figure 1 depicts the QC search interface. On the left hand is a list of search filters that describe typical search tasks and that are customized to the specific information needs of different user groups. Figure 1 shows filters specifically designed for use by primary care physicians. Figure 1 The QC search query user interface In QC, individual profiles are able to define different keyword types, such as “disease,” which describe the keyword classes typically associated with that profile. Thus, on the right of the interface are four fields where users can provide keywords describing the specific attributes of their search. Selection of a different profile may thus alter the keyword types requested from the user for a given search. QC then translates and submits search queries to the sources specified in the chosen profile, collects and processes the results, and presents them to the user as a list of documents (Figure 2). The title of a document is followed by the link and a short abstract of the content. A user can drill-down into a specific group of results by source type (eg, journal articles or guidelines). Figure 2 Screenshot of a QC results page Quick Clinical System Architecture Overview Most information sources such as websites, online texts, and databases have their own proprietary search interface, including query language and format for the display of results. Therefore, a federated meta-search engine that wishes to query a number of different information sources needs to first represent a user query using some internal query language [13] and then translate that internal query into the specific query languages of the relevant data sources. A well-documented [12] approach to this problem is to use a “wrapper” (Figure 3), which acts as an adapter between the proprietary language of individual information sources and the internal language used within a meta-search system. In QC, the internal query language is called the unified query language (UQL). Each information source known to QC has its own wrapper that translates queries from UQL into the native language of the source. As a result, internal components of QC only need to know UQL and not the individual query languages of the data sources. System maintenance is also simplified since the introduction of a new data source to the system only requires one new wrapper component to be generated. Once the results of a search are returned by an information source, the information must again be translated into a standard output format for presentation to the user, which, in QC, is called the unified response language (UReL). UReL also allows other components in the system to modify the presentation of search results without needing to understand the presentation format of individual sources (eg, to remove duplicate documents). In Figure 3, a search is initiated from the user interface, which forwards a query (in XML) to the mediator. The mediator splits the query into several subqueries and sends these to the appropriate wrapper (via a capability manager if required). Finally, the wrapper translates the query into the native query language of the data source (eg, in HTML for Web data sources). Similarly, the result from the data source gets translated back into the system’s XML representation and sent back to the user interface. Figure 3 Architecture overview of Quick Clinical Unified Query Language UQL is used to represent queries obtained from users in a consistent internal way, and UQL statements identify query elements such as the external information sources to be searched and a set of search attributes used to delimit the search. For example, UQL expressions can store date range delimiters for a search. UQL also contains statements that indicate whether or not QC needs to process the query further. For example, we may wish to remove duplicate items obtained from different sources. In our current implementation, UQL is implemented using XML. To define the structure of the data within the XML document we use a data type definition (DTD), which allows various internal components of QC to validate the XML data received in the UQL query. The following example illustrates how a UQL query might look in XML. <QUERY keyword = "iron AND deficiency" profile = "treatment" duplicateRemoval = "yes" sortBy = "rank" useLexicalVariants = "yes" timeout = "20" dateRangeBeginDay = "1" dateRangeBeginMonth = "1" dateRangeBeginYear = "1999" > <SOURCE name = "PubMed" /> <SOURCE name = "Harrison’s online" /> <SOURCE name = "Merck" /> <SOURCE name = "MIMS" /> </QUERY> Unified Response Language Similarly to the UQL, the unified response language (UReL) is used internally to guide display of information to users, also represented using XML. Each separate result, or “article,” from a source can be broken up into smaller chunks and given meta-data labels to represent the different sections of the data (eg, abstracts from journal articles). Since the majority of sources accessed by QC are journals, the data that are retrieved typically contain document elements such as Title, Author(s), Journal Name, Date of Publication, and the URL where the electronic version of the paper is accessed. Other sources, such as drug descriptions from pharmaceutical compendia, have sections such as Drug Name and Manufacturer. These different document elements, based upon the typical sources QC expects to find, are defined as specific fields in the UReL definition. The following example illustrates how a set of documents retrieved by QC might be represented in UReL. <RESULT> <ARTICLE> <LINK> <HREF> http://www.ncbi.nlm.nih.gov:80 /entrez/query.fcgi?cmd=Retrieve&db=PubMed &list_uids=12198020&dopt=Abstract </HREF> <LINKNAME>Abstract</LINKNAME> </LINK> <AUTHORLIST>Heath AL, Skeaff CM, Gibson RS. </AUTHORLIST> <TITLE> Dietary treatment of iron deficiency </TITLE> <DATE> <YEAR>2002</YEAR> <MONTH>9</MONTH> </DATE> <SOURCE>PubMed</SOURCE> </ARTICLE> <ARTICLE> <LINK> <HREF> http://mims.hcn.net.au /ifmx-nsapi/mims-data/?MIval=2MIMS_abbr_pi &product_code=288 &product_name=Ferrum+H+Injection </HREF> <LINKNAME>More Information</LINKNAME> </LINK> <AUTHORLIST> Sigma Pharmaceuticals Pty Ltd. </AUTHORLIST> <TITLE>Ferrum H Injection</TITLE> <SOURCE>MIMS</SOURCE> </ARTICLE> </RESULT> Wrappers For every information source known to QC, there is a specific wrapper that translates a UQL query into the native query language and format of the source. The wrapper also extracts the relevant information from the HTML result pages returned by the search engine and re-expresses it in UReL. Figure 4 shows the basic architecture of wrappers in our current system. Each wrapper has three main components: a feeder, extraction rules, and a sieve. The feeder converts the user query into the native query language of the data source. The data source responds to the query and returns HTML raw data. The feeder passes the raw data to the sieve, which converts it to UReL in XML format by using the extraction rules for the data source. The UReL is then sent back via other components to the user interface, which can interpret the XML and display the results. Figure 4 Wrapper components Mediator A key requirement of a multisource information retrieval system is the ability to perform concurrent searches on multiple sources with a single query [6,7]. The mediator addresses this requirement. The mediator first analyzes a query and determines how many sources are to be searched. It then creates a separate search job for each of these sources and forwards the search job to other system components. Additionally, the mediator collects individual results as they arrive and amalgamates them for the user into a single result. By introducing parallelism, the time to perform a search across a number of resources should be reduced to the duration of the slowest source. However, the potential drawback of parallel processing is the increased administration overhead of running multiple parallel processes within a system. As a rule of thumb, we would expect the benefits of parallel execution should increase with the number of sources queried, as response times for Web resources can be many seconds long, and computational execution of processes to manage parallel search are typically much less than one second. Connection speed and latency of response time from sources are, for practical purposes, nondeterministic in an Internet environment, and a meta-search engine can therefore experience large fluctuations in responses from the same source under different circumstances. Latency is subject to network traffic conditions, making it impossible to guarantee that all resources that are queried at a particular time will respond predictably and equally. To counter this, the mediator has a time-out feature. If a response is not received within the time-out specified by a profile, the mediator will cancel a subsearch and forward all the results currently available from other sources to the user interface. This effectively guarantees a defined response time irrespective of the state of the individual data sources and provides some control over the speed/accuracy trade-off. Capability Manager Search capabilities vary considerably between the search engines that QC might wish to interrogate, and some sources will have limitations in their ability to process search queries. One approach to this problem is to try to raise all sources to as high a level of common performance as possible by emulating missing capabilities locally, usually by modifying the query and/or search result [13]. A trivial example is mimicking the ability to perform a Boolean search when a data source does not have this capability. To emulate a Boolean AND, a meta-search engine would perform two parallel individual searches on the source and then itself perform the Boolean operation on the two results. In QC, a capability manager (CM) is responsible for mimicking a range of search capabilities and is located between the mediator and wrapper. The CM may modify a query and/or the result depending on the capabilities of the sources about to be queried. Capabilities of the CM within the QC system included the following: Date-CM: search within a date range Duplicate-CM: remove document duplicates Sort-CM: sort results by title, author, document rank, or date Lexical-CM: expand a search term with lexical variants of the term. A lexical variant is a synonym, pluralization, hyphenation, or other modification that changes the text but not its meaning. Lexical variants are particularly important in the medical domain [14] because many concepts can be expressed in Latin or English (eg, cardio vs heart). Moreover, there is a common confusion between terms in American English versus British English (eg, hemoglobin vs haemoglobin, epinephrine vs adrenaline). QC uses a stacking mechanism to insert individual CMs into the processing of queries for wrappers and the processing of results from a source. A component called the search planner, containing simple rules, is responsible for stacking the CMs. This means that the sequence of CMs can be ordered to ensure the correct outcome of query or result translations. Theoretically, this corresponds to a composition of operations. A lexical variant CM, for example, has to replace the search terms in the query before the wrapper executes the search. The Date-CM, on the other hand, can only perform its job after the successful execution of the wrapper. Search Filters Expert searchers typically will use search strategies that are more likely to accurately locate information, based upon an understanding of the specific capabilities of an evidence source. There is an increasing interest in the writing of search filters which capture such strategies, usually focusing on the major evidence repositories like Medline [10,11]. Search filters are designed for typical clinical queries such as “diagnosis” or “prescribing,” and they are crafted to find evidence most likely to satisfy the query by first selectively searching resources identified to be of high quality and, second, by automatically adding specialist keywords to the general question posed by a user. Within QC, search filters are stored in the profiles function. For example, if a clinician selects the “diagnosis” filter and enters the search term “asthma,” QC can add in the additional terms when it queries Medline [10]: sensitivity and specificity [MESH] OR sensitivity [WORD] OR diagnosis [SH] OR diagnostic use [SH] OR specificity [WORD] These terms have been shown to significantly enhance the quality of Medline results, but they are unlikely to be known to a typical clinical user. Unlike standard search filters, QC profiles are meta-search filters because they encode search filters for multiple different sources. Profiles thus encode expert search strategies that are most likely to answer a certain class of query, and they encode, among other things, the most appropriate content sources to search (Table 1). For a primary care physician, these search profiles might be for diagnosis, prescription, review, and treatment [4], but any set of profiles can be created within QC to meet the specific query types and search contexts of different users. In Table 1, the Treatment profile describes a set of nine separate source-specific search filters, which collectively describe the search strategy believed most likely to retrieve an accurate search result from each resource. The # symbol delimits keyword variables that are to be instantiated with user keywords. For example, #1# represents the keyword type “disease,” and QC’s mediator component will substitute the user-provided keywords for “disease” throughout the profile, prior to sending the query to the individual wrappers for the different sources. More than one search string can be created for an individual source (eg, TGL 1 and TGL 2) as a single strategy may not always retrieve all the relevant documents. Table 1 Quick Clinical meta-search filters Source Search String TGL1 (#1# AND #2# AND #3# AND #4#) AND+ ("treatment" OR "therapy" OR "therapeutic use") TGL2 (#1# AND #3#) AND+ ("treatment" OR "therapy" OR "therapeutic use") HealthInsite3 #1# AND #2# AND #3# AND #4# HealthInsite4 #1# AND #3# PubMed5 (#1# ATTR+ [Title] AND #3# ATTR+ [Title] AND #4# ATTR+ [Title] ATTR+ /ther)English 10 years Human PubMed6 #1# ATTR+ [Title] AND (#3# ATTR+ [Title] OR #4# ATTR+ [Title]) ATTR+ /drug ATTR+ therEnglish 10 years Human Merck7 ((#1# AND #3# AND #4#) OR (#1# AND #3#)) AND+ ("treatment" OR "therapy") Harrison’s8 Disconnected Harrison’s9 Disconnected System Platform The system was constructed using Java, the Struts Web application framework, and a MySQL database and is deployed on a RedHat Linux platform. The user interface (JSP, servlet, and HTML pages) is deployed through an Apache Web server connected to a Tomcat servlet engine. The Apache-Tomcat platform incorporates load balancing and fail-over and is suitable for scalability and large-scale deployment. Technical Evaluation QC has undergone a series of clinical evaluations, which have been reported separately [4,15,16]. In total, 227 family physicians from across Australia participated in a trial of QC. Clinicians who had a computer with Internet access in their consulting rooms were recruited and asked to use QC for 4 weeks in routine care. Each participant was given a personal username and password to access the system. All clinicians completed an online pre-trial survey. QC was configured to search a set of eight sources, including remote sites such as PubMed, online journals such as BMJ and the Medical Journal of Australia (MJA), and locally cached sources such as The Merck Manual and Therapeutic Guidelines Australia. For every search, the time from the request arriving at the system to the time when the results were sent back to the user was recorded (Figure 5; search time = system time + slowest source time). Note that there is a cap on search time when the time-out cuts in. Time-outs are search-profile dependent and were set at either 15 or 30 s. The time it took to conduct the search on the individual sources was also recorded. The time taken to send data between QC and the user’s computer (user time) is not incorporated in these measurements. In the following section we report on the technical performance of the architecture and then reflect on its suitability for supporting evidence retrieval in clinical practice. Figure 5 Search time metrics Results In the pre-trial questionnaire, 40% of the clinicians reported having a broadband (ADSL, cable, satellite) connection, while 43% used a 56k or 64k modem connection. The remaining 17% either did not know the type of connection used or had a slower connection. A total of 1662 searches were performed over the trial. Search Speed Under local network conditions (LAN, 100MBit), the user time (from starting the search on a client computer to displaying the results) was approximately 1.5 s. However, since most users accessed the system through the Internet, latency was significantly longer and slowed down the overall search speed. The average search time was 4.9 s, with a standard deviation of 3.2 s (N = 1662 searches). Figure 6 shows the distribution of all search times over the trial. There are four distinctive features in this chart. The first is a small peak at 1 s (ie, searches that took up to 1 s to complete). The second feature is a peak around the mean value. Third, there is a small peak at 15 s, and, fourth, there is a small peak at 30 s. Figure 6 Distribution of search time for all 1662 searches System Time System time for a search was computed by subtracting the duration of the slowest source in every search from the search time (see Figure 5). From the system time histogram in Figure 7, it can be seen that for the majority of the searches the system takes between 100 ms and 130 ms (mean = 117.9 ms; SD = 68.4 ms; N = 1614 [48 searches had missing data, hence 1614 searches]). Figure 7 Distribution of the system time for 1614 searches System Time Versus Number of Individual Sources Involved Depending on the search profile selected, the system will query a certain number of information sources and combine the results. To illustrate the dependency between system time and the number of sources queried, Table 2 shows average system time versus the number of sources queried in a search. The number of sources queried is predefined by the search profile, and none of the search profiles tested queried five, six, or eight sources. Table 2 System time vs number of sources queried Number of Sources Queried N Average System Time (ms) 1 48 18.1 2 9 31.8 3 15 73.3 4 7 59.7 5 0 - 6 0 - 7 1373 122.2 8 0 - 9 162 122.6 Speed and Reliability of Individual Data Sources In addition to the performance measurements of the whole searches, the speed and reliability of the individual data sources was measured. Reliability was measured as the number of error cases (ie, queries that were not answered due to an error condition, such as a network error, an HTTP error, or queries that timed out). Reliability and speed figures are summarized in Table 3. Table 3 Reliability and speed of data sources Source Type Number of Searches Number of Errors Error (%) Mean Speed (s) SD (s) Min (s) Max (s) Merck Local 2144 0 0.0 0.06 0.11 0.01 2.89 TGL Local 2193 0 0.0 0.05 0.12 0.01 2.85 BMJ Remote 73 1 1.4 4.55 3.92 0.99 17.5 HealthInsite Remote 2993 55 1.8 3.09 1.08 1.08 22.3 MedlinePlus Remote 653 0 0.0 1.87 1.36 1.09 12.5 MIMS Remote 650 3 0.5 0.98 1.14 0.28 8.30 MJA Remote 58 1 1.7 0.25 0.31 0.10 1.73 PubMed Remote 3288 39 1.2 3.76 1.69 1.87 15.0 Total 12052 99 Mean 0.8 1.83 0.63* 0.68 10.4 * standard error of the mean The most reliable sources were the locally indexed sources Merck (The Merck Manual) and TGL (Therapeutic Guidelines Australia), both which did not have any error cases. On the other end of the scale are HealthInsite (a national consumer site for health information) and MJA. The slowest source in the trial was BMJ, with an average of 4.55 s to process a query (SD = 3.92 s; N = 73). This was followed by PubMed, which returned results at an average of 3.76 s (SD = 1.69 s; N = 3288). The two locally indexed sources (Merck and TGL) returned search results within an average of 0.061 s and 0.047 s, respectively. However, the two local sources do have a relatively large standard deviation. Figure 8 shows the distribution of query times to the eight individual data sources. Figure 8 Histogram of search times for each of the eight data sources (x-axis is time taken for a search, in ms; y-axis is number of searches) Discussion System Time From the results of the system time versus source time, we can observe that system-processing time is only a fraction of the total search time. However, there are exceptions, namely when local data sources are used exclusively. From a user’s perspective this still would not be an issue as the overall user time is greater by at least a factor of ten. It could, however, become a problem in a situation where many searches are dependant on the result of a previous search and have to be executed in series. System time has thus been kept relatively short, removing the initial reservation that too much parallelism could slow down the system excessively. From Table 3 it can be seen that the system time generally increases in line with the number of sources queried (with the exception of four sources queried). However, the order of this increase does not appear to be squared or even exponential, but rather linear. Search Times The four distinct features in the histogram of search times described in Figure 6 are due to the nature of the data sources and the value of the time-outs. The first small peak at 1 s is from search profiles that use exclusively local data sources. The second feature is a peak around the mean value and is caused by the six Internet resources. The small peak at 15 s is due to the large number of search profiles that have this value as a time-out. And finally, the tiny peak at 30 s is where the remaining searches time out. It was to be expected that local sources would be more reliable and have a shorter latency in response time. This is due to the controlled environment, compared to the uncontrolled Internet environment of the external sources. It is interesting to note the difference between the six external data sources. While some sources are very popular (eg, PubMed) and therefore are expected to be busy, others might lack the resources to keep up with demand. The time-out value of individual data sources is a trade-off between speed and quality of results and is determined by the intended usage of the system. However, under certain circumstances there are optimizations that can be carried out without affecting quality of results. For example, the search duration histogram for HealthInsite (Figure 8; top right) reveals that if a search has not completed within 10 s it is highly unlikely it will complete within 15 s. Therefore, a time-out value of 15 s can safely be reduced to 10 s without significantly compromising search quality. Future Work The current QC architecture has demonstrated in trials that it meets the technical design goals set for it, and it provides good evidence that our general approach to federated searching is sustainable and maintainable. We intend to pursue research and development in areas of current interest to meta-search engines, information retrieval systems, and artificial intelligence. These include automatic wrapper generation [17,18] so that new data sources can be easily integrated into QC. Using this approach, a component could automatically generate a wrapper from knowledge of the data source query inputs and results. Another area of continued research will be automated data source consistency checking. Data sources often change in their formats, and this needs to be monitored with either automated or human intervention in order to modify wrappers accordingly. A third area will be intelligent search agents [19]. We envisage incorporating an intelligent agent that will guide users through the search process, using domain knowledge to help frame clinical questions and choose search parameters. This agent could learn to work with its user. An area of continued development will be semantic understanding of result sets. We would like QC to combine search results into a meaningful coherent story that presents a concise, relevant, and digestible response to the user [20]. These approaches, coupled with user support, will allow us to develop and improve the system with a view to it becoming an integral part of a clinician’s daily practice. Even without these enhancements, we have demonstrated that the QC framework is a functional and useful approach for the delivery of online, just-in-time clinical evidence.

J_Urban_Health-2-2-1705501

Recruiting Injection Drug Users: A Three-Site Comparison of Results and Experiences with Respondent-Driven and Targeted Sampling Procedures

Several recent studies have utilized respondent-driven sampling (RDS) methods to survey hidden populations such as commercial sex-workers, men who have sex with men (MSM) and injection drug users (IDU). Few studies, however, have provided a direct comparison between RDS and other more traditional sampling methods such as venue-based, targeted or time/space sampling. The current study sampled injection drug users in three U.S. cities using RDS and targeted sampling (TS) methods and compared their effectiveness in terms of recruitment efficiency, logistics, and sample demographics. Both methods performed satisfactorily. The targeted method required more staff time per-recruited respondent and had a lower proportion of screened respondents who were eligible than RDS, while RDS respondents were offered higher incentives for participation. Introduction Injection drug users (IDU) remain an important population in the study of HIV/AIDS. The Centers for Disease Control and Prevention (CDC) estimates that 31.8% of cumulative male U.S. AIDS cases through 2004 and 40.7% of female cases were exposed through injection drug-use or male-to-male sexual contact and injection drug use.1 Unfortunately traditional sampling methods are inadequate when the population of interest consists of individuals who perform illegal or stigmatized behaviors. Injection drug users are one such population. Traditional probability-based sampling methods require the development of a sampling frame enumerating the entire population. This is prohibitive for these ‘hidden’ populations because their size is unknown, and their members are often reluctant to participate in studies due to legal issues or social stigmatization.2,3 Because of these difficulties, a number of alternative sampling methods have historically been employed to study hidden populations, including injection drug users.4 These methods have included convenience sampling, time/space or venue-based sampling and chain-referral methods. Chain-referral or “snow-ball” sampling5,6 methods involve selection of an initial set of members of the target population, or ‘seeds’, who provide information on, or referrals for, subsequent waves of respondents. The number of waves and potential referrals, as well as the seed selection methods, may vary. Versions of these methods have proven successful at quickly and easily identifying members of hidden populations.7–9 However, results obtained are thought to be biased by the initial seed selection method as well as characteristics of the respondents' social network and have generally been classified as convenience samples.6,10,11 Another method proposed by Watters and Biernacki3 known as targeted sampling (TS) has had some success in recruiting injection drug users.12 Targeted sampling requires extensive ethnographic and formative research to describe the area and population of interest and to identify appropriate locations within that area for inclusion in a sampling plan.13 Existing secondary data sources are also reviewed to further describe the target population and geographic areas of interest. Qualitative data in the form of focus groups and in-depth interviews with key informants may be conducted to gain a more complete understanding of the population of interest as well as to identify possible specific locations for sampling. These locations are then systematically mapped and ethnographic observations are conducted. Based on all of this information a sampling frame of locations is developed and weighted by the density of observed indicators. Locations are randomly selected and recruitment is conducted within the selected locations. Targeted sampling, as well as other time–space or venue-based sampling methods, may be biased to the extent that members of the population of interest do not attend the venues or areas that have been identified. Heckathorn10,14,15 has recently proposed a modified chain-referral method called respondent-driven sampling (RDS) as a promising strategy in surveying hidden populations. This approach retains the advantages of chain-referral sampling in terms of ease of respondent identification and recruitment while allowing population estimates and inferences to be made.10,15 Initial seeds are identified from known members of the population or through key informants. Information about the respondent's social network is collected, and seeds are provided with a set number of coupons for distribution to recruits within their own network. Eligible recruits are interviewed and provided coupons for distribution to the next ‘wave’. Recruitment continues in this way until the desired sample size is reached. Network information is then used to derive asymptotically unbiased population estimates for variables of survey interest. RDS has been shown to be a useful strategy for the recruitment of drug users.16–18 To date, however, no direct comparison of RDS with other sampling methods has been made in terms of the ability to recruit hidden populations such as IDUs, the “representativeness” of sample characteristics, and the practicality of administration.2,4 The objective of the current study is to compare and contrast RDS and TS methods on demographics, recruitment efficiency, and logistic concerns. Materials and Methods Three sites in large Metropolitan Statistical Areas (MSA) (Detroit, Houston and New Orleans) that participated in the CDC-funded National HIV Behavioral Surveillance among injecting drug users (NHBS-IDU) conducted a pilot investigation comparing RDS and TS. Each site assessed the feasibility of recruiting at least 100 eligible IDUs utilizing RDS and TS methods over an 8-week period in late 2004. What follows is a brief presentation of the strategies used by each site to locate and recruit potential respondents using RDS and TS methods. Targeted Sampling Formative Research Extensive ethnographic and formative research was conducted at all sites prior to sampling. Each site completed a thorough review of secondary data sources dealing with the IDU population. Data were compiled and synthesized from several sources including number of persons living with HIV/AIDS who have IDU as a reported mode of exposure, archival census data and findings from previous published and unpublished research. A total of 81 in-depth interviews were conducted with individuals who had knowledge of local injection drug-use communities. Those interviewed included local HIV prevention personnel and community planning group members as well as law enforcement representatives and current or former injectors. Ten focus groups with a total of 78 injection drug users were also conducted in order to provide specific knowledge about local injection habits and behaviors. These structured interviews were designed to provide a fuller understanding of the injection community at large as well as to solicit locations for possible sampling areas. Sites also utilized information on areas that had been previously identified by ethnographers with knowledge of the local IDU community or had been used in other local IDU studies. Area Identification and Recruitment Field observations of the possible sampling areas were systematically conducted at varying times of day and week over the course of 5 months (March–July 2004). Field staff conducted ethnographic observations within each area and recorded any IDU indicators that were present. IDU indicators included physical objects such as used syringes, baggies, balloons or injection works/equipment as well as behavioral indicators such as copping activity, loitering or commercial sex work. Sites used a combination of ethnographic research, secondary data analysis, key informant interviews, and field observations to form expected yield ratings to identify areas where injection drug use was likely to occur and where potential survey respondents could be recruited. Based on highest expected yield rating, Detroit and Houston identified four and ten sampling areas, respectively, within seven ZIP codes. For these sites line based enumeration was then used to approach possible respondents. New Orleans identified 35 sampling areas within 16 ZIP codes of high, medium and low expected yield. These ZIP codes were stratified by the expected yield, and order of ZIP codes was then randomly selected without replacement at the beginning of each 2-week period. Interviewers approached all individuals within each of the prescribed three block sampling areas within each ZIP code. Respondent-Driven Sampling Seed Selection and Recruitment Possible seeds were identified through key informant referrals and street outreach. Seeds were purposefully selected to maximize diversity on race, sex and drug of choice. Each seed was screened, and eligible seeds were interviewed using the NHBS-IDU instrument and provided with a $20 cash value incentive. Each initial seed was provided with three recruitment coupons and instructed to give them to individuals they knew injected drugs. Seeds were also informed that they would be compensated with $10 cash value for each person that they recruited. Peer-recruited respondents returning these coupons were then screened for eligibility, interviewed and provided with three coupons they were instructed to give to someone who they knew injected drugs. They were told that they would be compensated $10 for each person they recruited. A specialized coupon tracking system database was used to track referrals and facilitate incentive provision. Analysis All RDS participants were asked to provide information about the size and characteristics of their social networks from which they would draw potential recruits. Specifically, each participant was asked to report the number, sex and race/ethnicity of injectors they knew and had seen in the past 6 months. Relationship of the respondent to the recruiter (e.g., friend, injection partner, sex partner etc.) was also assessed. This information along with data from the coupon tracking system was analyzed using Respondent-Driven Sampling Analysis Tool (RDSAT) software (RDSAT (computer version). Version 5.4.0. Ithaca, New York: Volz E, Heckathorn DD; 2005) to provide estimated population proportions and confidence intervals for age, sex and race/ethnicity variables. Participants All participants provided informed consent and were carefully screened for eligibility criteria. Eligibility criteria for study participation were that the respondent was at least 18 years of age, was resident of the study MSA and had injected illicit drugs within the past 12 months. Targeted Sampling In Detroit 273 individuals were approached during TS. Two hundred twenty one individuals agreed to participate and were screened for eligibility. Of TS screened participants, 54% (n=120) were found to be eligible and completed the survey. In Houston 174 individuals were approached during TS. One hundred forty eight respondents agreed to participate and were screened for eligibility. Of TS screened participants, 65% (n=97) were found to be eligible and completed the survey. In New Orleans 560 individuals were approached during TS. Three hundred eighty-nine respondents agreed to participate and were screened for eligibility. Of TS screened participants, 35% (n=137) were found to be eligible and completed the survey. Respondent-Driven Sampling Six seeds were approached in Detroit for participation, and three of those individuals completed the survey and agreed to be seeds. One hundred sixty-nine recruitment coupons were distributed by Detroit during RDS over nine waves of recruitment; 106 of those individuals returned and were screened for eligibility. Of RDS screened participants, 96% (n=102) were found to be eligible and completed the survey. Thirteen seeds were approached in Houston for participation, and one additional seed was recruited after being interviewed in the targeted sampling component. Of the 14 seeds recruited, 1 was found to be ineligible. One hundred sixty-eight recruitment coupons were distributed during RDS over seven waves of recruitment; 62 of those individuals returned and were screened for eligibility. Of RDS screened participants, 77% (n=48) were found to be eligible and completed the survey. Ten seeds were approached in New Orleans for participation, and two of those individuals completed the survey and agreed to be seeds. Three hundred sixteen recruitment coupons were distributed during RDS over 19 waves of recruitment; 133 of those individuals returned and were screened for eligibility. Of RDS screened participants, 88% (n=118) were found to be eligible and completed the survey. Materials The standardized NHBS-IDU questionnaire was administered to all eligible participants. This 68-item questionnaire on HIV risk behaviors, requiring approximately 40 min, includes items on demographic characteristics, drug use, sexual risk behaviors, access to and use of health care and utilization of HIV prevention services. Variables of interest for this study included respondent demographic information, time required for survey completion, and cost of each survey. Results Sample Characteristics Demographic characteristics of the samples obtained for each site by method are presented in Tables 1, 2, and 3. Local HIV surveillance data on persons living with HIV/AIDS who have injection drug use as a reported mode of exposure are presented for comparison purposes only. Chi-square tests indicated no significant association of sampling method and gender for any site (Detroit: , P=0.33; Houston: , P=0.38; New Orleans: , P=0.23). No significant association between age and sampling method was found (Detroit: , P=0.52; Houston: , P=0.14; New Orleans: , P=0.23). Two sites showed significant association between race/ethnicity and sampling method (Detroit: , P=0.42; Houston: , P=0.01; New Orleans: , P<0.0001). Table 1Demographic characteristics of Detroit IDUs obtained through RDS and TS, and persons living with HIV/AIDS who are injection drug users (PLWH/A-IDU) TSRDSRDS pop. estimates, % (95% CI)PLWH/A-IDU, (%)Race/ethnicityN%N%African American101949696–91White 0011–5Hispanic1111–3Other6622–0Sex/genderMale7469717163 (52–75)61Female3431242437 (25–48)39TransgenderAge18–3421220.1(0.2–0.7)435–442423202017 (7–27)2245–546560545451 (40–67)4755+1716242432 (16–43)27Table 2Demographic characteristics of Houston IDUs obtained through RDS and TS, and persons living with HIV/AIDS who are injection drug users (PLWH/A-IDU) TSRDSRDS pop. estimates, % (95% CI)PLWH/A-IDU, (%)Race/ethnicityN%N%African American42522745–69White22281423–20Hispanic10121932–10Other6700–1Sex/genderMale5367467776 (45–96)56Female2025122024 (4–55)44Transgendera6823Age18–342634122014 (0.1–31)1735–442228254141 (33–86)4045+3039244045 (5–60)43aFemale and transgender categories were combined for purposes of Houston's RDS analyses.Table 3Demographic characteristics of New Orleans IDUs obtained through RDS and TS, and persons living with HIV/AIDS who are injection drug users (PLWH/A-IDU) TSRDSRDS pop. estimates, % (95% CI)PLWH/A-IDU, (%)Race/ethnicityN%N%African American12589675877 (64–90)65White54342920 (9–34)33Hispanic11223 (0.1–3)2Othera9613110Sex/genderMale11985948174 (58–88)76Female1914221926 (12–42)24Transgender21Age18–343726342927 (15–43)1635–444431262227 (12–44)3545–544029443839 (21–57)3755+191412107 (2–13)12aHispanic and other race/ethnicities were combined for purposes of New Orleans' RDS analyses. Also presented are the population estimates and confidence intervals obtained by RDSAT. No RDS population estimates were calculated for race/ethnicity for Detroit or Houston due to lack of cross-group recruitment (e.g., only African Americans were recruiters of other race/ethnicities in Detroit). For each RDS estimate presented, equilibrium was achieved. Cost Effectiveness Across all sites 47% of TS-screened respondents were found to be eligible IDUs, while 89% of screened respondents in RDS were eligible IDUs. TS required an average of 214 h performing ethnographic observation and mapping plus an average of approximately 2 h and 9 min of staff time locating, screening and interviewing each recruit (total = 4 h and 6 min per recruit) while RDS required approximately 1 h and 13 min per recruit. TS methods provided each participant with an average of $20 in incentives, while RDS recruits received an average of $26.68. RDS incentives did not total $50 for every participant because some respondents did not produce the maximum of three recruits, and some recruiters did not return to receive the added incentives for recruitment efforts. Logistics Staffing requirements were similar for both methods, and sites were able to use the same staff to conduct RDS and TS concurrently. Some differences were evident as a result of method selection. Targeted sampling was conducted in the field while RDS was usually conducted from a single storefront. Interviewers reported that this led to a more controlled environment when using RDS. Increased privacy, comfort and perceptions of safety were reported, while distraction level decreased. Weather was also more of a concern with TS while interviewing outdoors. Discussion The use of multiple sources of ethnographic data was beneficial in identifying high-yield TS recruitment areas because areas with the highest number of eligible recruits were often not necessarily areas with obvious drug use indicators but areas identified through HIV surveillance or key-informant interviews. However, even with good ethnographic information, many areas that were expected to be high-yield were not, and less than half of all individuals approached were found to be eligible. This led to low interviewer morale in Detroit, where the interviewers were paid per interview completed. It also led to some bias in recruitment in Houston, where interviewers made the decision to recruit only individuals who looked like injection drug users on one particularly unsuccessful recruitment day. The sample characteristics of those recruited through TS may not reflect the general IDU population but rather the population found during the recruitment times. New Orleans included low density areas and randomized area selection in the hopes of assembling a diverse sample, reflective of the general IDU population. However, this did not happen. The sample characteristics were reflective of the population found during recruitment times, and the sample may have missed less visible injectors, such as those with steady day time employment or child care responsibilities and those who remain indoors during the day or only come out at night. Houston and Detroit had similar problems. One solution might be to extend recruitment hours into the night or start very early in the morning, but this would further increase security concerns. TS offered less control in the working environment, compared to RDS. Safety was a concern: The interviewers tended to spread out in the targeted area and the interviewing process drew the attention of others. Groups often gathered to inquire about the interview and the incentive, heightening security concerns. Weather was also an important factor in recruitment success, as fewer individuals were on the streets during hot, cold, or rainy days, and the spring and summer of 2005 was particularly noteworthy, weather-wise. Overall the RDS storefront was preferable to conducting TS outdoors. Participants often scheduled interview appointments and came to a central location for their interviews. Adequate staffing and office space were essential. It was found to be best to have a general waiting area, private interview spaces for each interviewer, and an area for obtaining participants' coupon information and providing incentives. In addition, all sites concurred that a single staff person to monitor the flow of participants entering, waiting, and exiting the facility would have provided even more control of the environment. RDS success depends largely on the effective recruitment of seeds, an inter-connectedness of injection drug use networks, and the willingness of seeds and their recruits to travel to an interview location. Community partnerships were also important in the recruitment of successful seeds. In Detroit, seeds were recruited from two needle exchange locations in the city at the recommendation of staff familiar with the individuals' ability to recruit others. This worked well in Detroit, where three of six recruited seeds led to the recruitment of 106 eligible IDUs. In Houston, the initial seeds were recruited from outreach workers who were conducting other research studies in drug-using populations. This was problematic, as some of these initial seeds were not actually linked to the injection drug using community but rather spent their days recruiting participants for these other research projects. These “research hustler” seeds were often very good at recruiting individuals (for money), just not at recruiting eligible individuals. This resulted in very short referral chains since few of their recruits were eligible. In New Orleans, staff were only able to successfully recruit two seeds. Eight others identified through key informants agreed to participate but never came to the field location to be interviewed. The two seeds that were interviewed represented the two subgroups of the overall sample, as described in the formative research. One seed was an older African American male, the other a younger white female. The race/ethnicity and age differences between the New Orleans RDS sample (29% white, 19% under 25) and the TS sample (4% white, 5% under 25) may have reflected the characteristics of the two seeds' networks rather than methodological differences. Based on lessons learned in this pilot, when NHBS-IDU shifted to full implementation, the sites made several adjustments to the RDS protocols. In New Orleans, prior to the discontinuation of the project due to Hurricane Katrina, seeds were recruited from among participants who were known to interviewers from the TS pilot and who encouraged others to be interviewed. Second, two field offices were established; one was close to downtown and easily accessible by public transportation and the other was near a large public housing project in an area that yielded high eligibility rates in TS. Finally, transportation tokens were purchased and made available to participants who came to the field office in an attempt to ease the burden of travel on respondents. In Houston, successful seeds were individuals the interviewers/outreach workers found during the formative work for the targeted pilot. These were influential individuals or gatekeepers within small communities of injection drug users, often the individuals who were first to approach staff and inquire why interviewers were entering their geographic space. Discontinuing RDS recruitment required some special considerations. While targeted sampling may easily stop on any given date or after a pre-determined number of participants have been recruited, the coupons distributed to potential RDS subjects represent a commitment on the part of the researcher. It was therefore important to carefully decrease the number of coupons distributed and shorten the coupon validity period so that recruitment would continue even as the end of the study date approached. Ending the distribution of coupons too early may result in not meeting the recruitment goal (in this case 100 participants), while ending it too late may lead to a disgruntled public. Overall, both RDS and TS proved effective in recruiting IDUs in the three pilot MSAs. Although no claim as to the actual representativeness of the obtained samples is made, both methods yielded samples that were similar in terms of many demographic characteristics (such as age, race and sex) as well as drug of choice (not presented here). Demographic results of the samples from both methods also compared favorably to each city's Census and HIV surveillance data from injection drug users. All three sites were able to reach the goal of 100 respondents in TS. Issues with initial seed selection and time constraints most likely resulted in the failure of one site to recruit 100 respondents with RDS. Total cost efficiency for the two methods was also comparable. Extensive formative research and low screened-to-eligible respondent ratios resulted in larger person-hour expenditures for TS, while RDS required higher incentive payouts to participants. Logistical concerns did differ considerably between the two methods. While RDS provided a more controlled interview environment, recruitment success seemed to be affected by the ability to identify quality seeds. Quality of ethnographic assessment and size and cultural qualities of the MSAs also affected targeted sampling success. While more research is needed in assessing the methodological impact of choice of sampling method in identifying hidden populations, these experiences should provide researchers some insight into the some of the practical strengths and weaknesses of these two methods.

Pediatr_Nephrol-4-1-2259258

A genome search for primary vesicoureteral reflux shows further evidence for genetic heterogeneity

Vesicoureteral reflux (VUR) is the most common disease of the urinary tract in children. In order to identify gene(s) involved in this complex disorder, we performed a genome-wide search in a selected sample of 31 patients with primary VUR from eight families originating from southern Italy. Sixteen additional families with 41 patients were included in a second stage. Nonparametric, affected-only linkage analysis identified four genomic areas on chromosomes 1, 3, and 4 (p < 0.05); the best result corresponded to the D3S3681-D3S1569 interval on chromosome 3 (nonparametric linkage score, NPL = 2.75, p = 0.008). This region was then saturated with 26 additional markers, tested in the complete group of 72 patients from 24 families (NPL = 2.01, p = 0.01). We identified a genomic area on 3q22.2–23, where 26 patients from six multiplex families shared overlapping haplotypes. However, we did not find evidence for a common ancestral haplotype. The region on chromosome 1 was delimited to 1p36.2–34.3 (D1S228-D1S255, max. NPL = 1.70, p = 0.03), after additional fine typing. Furthermore, on chromosome 22q11.22–12.3, patients from a single family showed excess allele sharing (NPL = 3.35, p = 0.015). Only the chromosome 3q region has been previously reported in the single genome-wide screening available for primary VUR. Our results suggest the presence of several novel loci for primary VUR, giving further evidence for the genetic heterogeneity of this disorder. Introduction Vesicoureteral reflux (VUR) (OMIM 193000) is the most common disease of the urinary tract in children and affects 1–2% of the Caucasian population [1]. VUR may be associated with both acquired postinfectious and congenital parenchymal damage, currently known as reflux nephropathy (RN) [2, 3]. The most serious consequence of RN is chronic renal insufficiency (CRI), leading to end-stage renal failure (ESRF), dialysis, and/or renal transplantation: 25.4% of children affected with CRI have RN [4]. As a consequence, the impact of VUR on public health is considerable and, despite medical and surgical interventions for the past decades, the incidence of VUR-related renal failure has not decreased [5]. The reflux may occur isolated or in association with other congenital abnormalities of kidney/urinary tract (CAKUT) or as part of syndromic entities, such as renal-coloboma or branchio-oto-renal syndromes [6–8]. The exact etiology of primary VUR is not known, but it is probably related to an abnormal morphogenesis of the ureteral bud, leading to a defect of the ureterovesical junction [9]. The initial evidence suggesting a genetic origin of primary VUR came from twin studies, showing an 80–100% concordance for VUR in monozygotic twins vs. a 35–50% concordance in dizygotic twins [10, 11]. Subsequent evidence included familial clustering of VUR [12], ethnic differences between affected and nonaffected individuals [13], and an increased risk (30–50%) of developing VUR in first-degree relatives of an index case [14–16]. From family studies, a range of inheritance patterns was reported, including autosomal dominant with incomplete penetrance [17–20], autosomal recessive [21], polygenic [22], and even X-linked [23]. Previous studies suggest a urinary tract malformation locus on chromosome 6p [24, 25]. Studies of humans with chromosomal abnormalities also suggest candidate loci or genes on chromosomes 10q26 [26], 19q13 (USF2 gene) [27], and 13q33–34 [28]. Because mutations in PAX2 on 10q24 cause renal-coloboma syndrome, a rare autosomal dominant disease with kidney anomalies that include VUR, this gene was also proposed as a candidate [6, 7]. However, none of these loci or genes has been shown causally related to primary VUR [19, 20]. Recently, Lu et al. [29] showed that mutations in the ROBO2 gene contribute to the pathogenesis of VUR/CAKUT in a small proportion of families. In the only genome-wide linkage study reported to date, Feather et al. [18] demonstrated linkage to chromosome 1p13 for primary VUR under a model of autosomal dominant inheritance with reduced penetrance. Here, we describe the results of the second genome-wide scan for primary VUR. Differently from previous studies and aiming to collect a homogeneous sample set, our patients were ascertained in a single geographic region. Our results suggest the presence of several novel loci for primary VUR, giving further evidence for the genetic heterogeneity of this disorder. Methods Patients and families Fifty-one pedigrees with multiple patients with VUR coming from Campania (southern Italy) were enrolled in the study (Fig. 1). All families were ascertained through an index case, with VUR documented by voiding cystourethrography (VCUG) in males and direct radionuclide cystography (RNC) in females and family members. Three pediatric nephrologists and one radiologist assessed the patients. RN was diagnosed by DMSA scintigraphy (dimercaptosuccinic acid labeled with Technetium-99 m) and defined as focal defects of radionuclide uptake and/or by one-kidney differential uptake below 43% [30]. VUR grading was made according to the International Grading System of Vesicoureteral Reflux [31]. Fig. 1Selection and distribution of families and patients included in the study. VUR vesicoureteral reflux Additional family members were considered as “affected” based on the presence of reflux documented by VCUG/RNC and/or the diagnosis of RN, or the detection of ESRF/renal replacement in absence of other known causes. As VUR may spontaneously disappear during childhood and adolescence [32], the finding of scintigraphic signs of RN in relatives of VUR patients strongly suggests the previous occurrence of reflux [33]. Individuals with renal symptoms indicative of VUR, such as previous urinary tract infections and/or hypertension and/or proteinuria, not supported by additional findings were classified as “diagnosis unknown”. Patients with secondary VUR, i.e., neurogenic bladder and posterior urethral valves, or other urinary tract abnormalities, i.e., ureterocele and obstructive hydronephrosis, were excluded. The study focused on primary familial VUR. Eight families with 31 patients with VUR were selected for study phase 1 (genome scan) according to the following criteria: diagnosis of primary VUR in absence of any other malformation, two or more affected individuals per family, and a pattern of inheritance compatible with an autosomal dominant model. The second sample (follow-up) consisted of five affected relative pairs (parent–child trios, ten patients) and 11 small families (31 patients) fulfilling the same criteria (Fig. 1). Informed consent from patients and family members (parents for their children) and approval from the Ethic Committee at Second University of Naples were obtained previously. Laboratory analysis Genomic DNA was isolated from peripheral blood leukocytes by standard techniques and was sent from the Paediatrics Department of Second University of Naples to the Department of Clinical Genetics, Erasmus Medical Centre in Rotterdam. A systematic genome scan was performed using the ABI Prism MD-10 set (Applied Biosystems) consisting of 382 short-tandem-repeat polymorphisms markers (STRPs), average spaced 10 cM. Additional markers for further characterization of candidate regions were selected from the gender-average Marshfield genetic map. Information about marker order and distances were obtained from the National Center for Biotechnology Information (NCBI) physical map and Marshfield integrated genetic map. Polymerase chain reaction (PCR) products were resolved on an ABI3100 automated sequencer, and genotypes were analyzed using the GeneMapper software v.2.0 (Applied Biosystems). Linkage analysis One thousand simulations were performed (SLINK, MSIM) [34] to investigate the statistical power of our sample set. An autosomal dominant mode of inheritance was assumed with a penetrance of 70%, a mutant allele frequency of 1%, and a phenocopy rate of 1%. The calculations were done under the assumption of genetic heterogeneity. Assuming 35% of unlinked families, a maximum total log of odds (LOD) score of 5.4 was obtained with a low average LOD of 1.58 [standard deviation (SD) = 1.26], after analyzing all eight pedigrees selected previously. Increasing the percentage of unlinked families to 50%, a maximum LOD score of 4.94 was observed (average 0.99, SD = 1.04). Families 1, 4, and 12 had the largest contribution to the total LOD score (LOD = 0.86, 1.04, and 1.62, respectively), followed by families 5, 7, and 13 (LOD = 0.57). Due to their small size, families 2 and 11 had less contribution to the final LOD (LOD = 0.29). Simulations were calculated in the replication group (11 families) after exclusion of five parent–child trios, giving a maximum LOD of 4.57 (average 1.49, SD = 1.07) and a maximum LOD of 4.85 (average 1.14, SD = 1.08), under assumption of 35% and 50% unlinked families, respectively. Family 25 with seven patients was the main contributor to the total LOD score (LOD = 1.47). Finally, simulations were performed in all 19 families (genome scan group and replication group) reaching a maximum LOD of 9.19 (average of 2.76, SD = 1.78) and a maximum LOD of 7.36 (average of 1.71, SD = 1.34), under assumption of 35% and 50% unlinked families, respectively. Mega2 [35] was used to process the genetic data, whereas the accuracy of allele segregation within the families was confirmed with Pedcheck. The program GENEHUNTER v.2.1 [36] was used to compute multipoint parametric and nonparametric (or model-free) linkage analysis. As VUR may often disappear with age, all individuals in whom VUR, RN, or both were not clinically proven were classified (and analyzed) as “diagnosis unknown”. Thus, a conservative “affected-only” analysis was performed based on these criteria. Due to the uncertain pattern of inheritance, nonparametric analysis was computed. The rationale of a nonparametric analysis is that, among affected relatives, excess sharing of haplotypes identical by descent would be expected, irrespective of the mode of inheritance. The nonparametric linkage statistics examining all individuals simultaneously (NPL-all), is reported. A parametric analysis was performed as well. An autosomal dominant mode of inheritance with reduced penetrance, as described above (power calculations), was used. Marker allele frequencies were calculated using all spouses (unrelated individuals) coming from the same geographic area. Due to the genetic heterogeneity of the disease, the genome scan data was analyzed, maximizing the heterogeneity LOD score (HLOD) with respect to the proportion of linked families (α). Nonparametric (NPL) and parametric (LOD) scores were calculated for each of the families, and then total NPL and HLOD scores were obtained. Results Patients Fifty-one pedigrees with multiple patients with VUR, originating from the same region of southern Italy (Campania), were available for the study. All persons in the study were ascertained through an index case documented by VCUG/RNC. A total of 143 patients were detected. According to the phenotype, three groups of families were identified: primary VUR; VUR associated with additional abnormalities of kidney/urinary tract such as duplicated collecting system, renal agenesis, or hypospadia; and VUR occurring in syndromes, such as renal-coloboma, branchio-oto-renal syndromes, and reflux associated with congenital ichthyosis (Fig. 1). A total of 78 primary VUR patients belonging to 25 pedigrees were identified (33 based on a positive VCUG/RNC, 18 based on detection of RN, 21 diagnosed as having both reflux and RN, and six with ESRF). During the course of the study, a new patient affected with RN was detected in family 1 and included in the second stage. Twenty-eight patients were males and 51 were females (ratio M/F = 0.57). The median age at diagnosis was 3.5 years (range 1 month–68 years). Among the 79 patients, 19 were treated surgically, and four out of six who developed ESRF underwent renal transplantation. The most informative pedigrees are shown in Fig. 2a,b. The pedigrees showed patients in several generations, and male-to-male transmission was observed in some families, supporting an autosomal dominant mode of inheritance. As reported before for primary VUR, we observed several obligate carriers in our families, i.e., families 4, 6, 12, and 13. Family 6 is especially interesting: when assessing the parents (asymptomatic) of the four symptomatic children, both individuals were found to have RN. No consanguinity was reported, but history of ESRF in both branches was described. We are currently recruiting clinical information of those patients (family 6, second generation, Fig. 2). Due to this bilinear transmission, this family was excluded from the linkage analysis. Fig. 2A selection of pedigrees enrolled in the study is shown. Squares indicate males and circles indicate females. Family members with unknown phenotype are in grey, whereas those unaffected (normal voiding cystourethrography or direct radionuclide cystography before age of 5 years and unrelated spouses) are in white. An asterisk highlights the individuals genotyped in the study. A number following the asterisk indicates the patients/obligate carriers reported in Fig. 3. Individuals with urinary tract infections by history and “obligate carriers” are considered as unknown in all analyses. a Nine multiplex pedigrees included in the genome-wide scan are shown. An additional five parent–child trios are not displayed. Family 6 was excluded from the linkage analysis due to the bilineal inheritance. b All 11 multiplex pedigrees included in the fine-typing stage are shown. VUR vesicoureteral reflux, RN reflux nephropathy, ESRF end-stage renal failure, UTIs urinary tract infections Genome search We performed a systematic genome-wide scan using 382 microsatellite markers. Results from both parametric and nonparametric linkage analysis excluded most of the genome (data not shown). Furthermore, negative scores were found on both VUR loci, 1p13 [18] (NPL = −0.86, p = 0.86) and 13q33–34 [28] (NPL = −0.21, p = 0.58) previously reported. We used a nominal p value of  < 0.05 to decide whether a region was promising for further study. Table 1 summarizes the genome-wide scan results, showing all regions that yielded a total NPL corresponding to p < 0.05. These regions were located on chromosome 1 (D1S468-D1S255, D1S213-D1S2785), chromosome 3 (D3S3681-D3S1569), and chromosome 4 (D4S402-D4S1597). Furthermore, on chromosome 22q11.22–12.3, patients from a single family showed excess allele sharing (D22S539-D22S280). The best evidence of linkage was observed on chromosome 3p12.3–3q24, which yielded the highest NPL score (2.75, p = 0.008, HLOD = 1.52, α = 0.76) and on chromosome 1p36.32–1p34.3 (NPL = 2.22, p = 0.02, HLOD = 1.13, α = 0.62). Recently, mutations in ROBO2 were described in two VUR/CAKUT patients [29]. ROBO2, which is an ideal functional candidate gene for VUR/CAKUT, maps on chromosome 3p12.3 at the border of our region. Thus, we retrospectively sequenced all gene exons in our index patients, and no mutations were found. Table 1Summary of the genome-wide scan results. Total nonparametric linkage (NPL) scores with p values <0.05PedigreesD1S468-D1S255D1S213-D1S2785D3S3681-D3S1569D4S402-D4S15971p36.32–1p34.31q41–1q433p12.3–3q244q26–4q32.34–65 cM242–266 cM109–158 cM117–169 cM1−0.80 (−1.09)0.59 (0.19)2.48 (0.87)−0.37 (0.07)21.34 (0.27)−0.42 (−0.76)1.34 (0.28)0.44 (0.27)41.14 (0.72)0.26 (−0.36)1.04 (−0.31)3.05 (1.3)51.9 (0.55)1.78 (0.55)1.89 (0.58)1.69 (0.57)71.59 (0.54)1.07 (0.42)1.75 (0.58)−0.33 (−1.37)111.34 (0.27)1.34 (0.27)1.34 (0.28)0.44 (0.28)121.47 (0.19)1.58 (0.35)0.65 (0.10)−0.04 (−1.66)131.25 (0.54)1.97 (0.54)1.25 (0.57)1.25 (0.57)Total NPL (p)2.22 (p = 0.02)2.35 (p = 0.02)2.75 (p = 0.008)2.15 (p = 0.02)Total HLOD (α)1.13 (0.62)0.61 (0.57)1.52 (0.76)1.31 (0.51)NPL and (LOD) scores per family are shown at each locus. Total NPL (p value) and HLOD (α) obtained from the analysis of all eight pedigrees are displayed. HLOD was obtained maximizing the “heterogeneity” LOD score with respect to the proportion of linked families (α). Negative NPL and LOD scores indicate no linkage or inconclusive resultNPL nonparametric LOD score, HLOD heterogeneity LOD score, α proportion of “linked” families The NPL score of 2.15 (p = 0.02) on chromosome 4q26–32.3 was mainly due to family 4 (NPL = 3.05, p = 0.062, LOD = 1.3). Yet, allele sharing among the patients of this family was observed also on chromosome 2p23.2–2p15 (D2S165-D2S337, NPL = 3.05, p = 0.062, LOD = 1.3). The only region in which patients from family 12 (Fig. 2) showed excess allele sharing was on chromosome 22q11.22–12.3 (NPL = 3.65, p = 0.015, LOD = 1.49). We then closely inspected the haplotypes from the positive regions in all genotyped families. Families 1, 2, 5, 7, 11, and 13 displayed haplotype sharing among each family’s patients on chromosome 3p12.3–3q24, supporting the linkage to this area. Thus, in six out of eight informative families, we observed allele/haplotype sharing in an overlapping area on chromosome 3q. We noted also on chromosome 1p haplotype sharing in four out of eight multiplex families (families 2, 5, 7, and 11) extending from D1S2667 until D1S255. Therefore, we decided to explore these genomic areas. Refinement of the chromosome 3 locus The chromosome 3q region between D3S3681 and D3S1569 was saturated with 26 additional microsatellites markers (average spaced approximately 2 cM). A maximum HLOD = 2.69 (α = 0.95) was reached with an NPL = 2.96, p = 0.001. In a second stage, the fine mapping was extended with 16 additional families with 41 patients. The expansion of pedigrees introduced additional unlinked families, which lowered the maximum HLOD to 1.24 (α = 0.36), giving a total NPL = 2.01 (p = 0.01) (Table 2). Pedigree 25 was the main contributor to the total score, with an NPL = 4.63, p = 0.03, (LOD = 1.45). We then performed a detailed haplotype analysis in all 24 families. Patients from families 2, 5, 7, 13, 20, and 25 shared a common region delimited by markers D3S3641 and D3S1764, containing 36 genes according with the NCBI map, built 36.2. Instead, families 1 and 11 were sharing a more centromeric area not overlapping with the other families (Fig. 3). We did not observe a common “ancestral” haplotype shared across the families. Fig. 3Haplotype analysis in families supporting the chromosome 3 region after fine typing. A selection of microsatellite markers is shown. The shared genomic region is shown in grey; recombinants are shown in white blocksTable 2Summary of fine-typing results in chromosome 1 and 3 (total NPL scores with p values <0.05)PedigreesD1S468-D1S255D3S3681-D3S15691p36.32–1p34.33p12.3–3q244–65 cM109–158 cM1−0.79 (−2.19)1.43 (0.74)21.34 (0.28)1.34 (0.28)41.59 (1.17)0.46 (−0.43)51.89 (0.56)1.79 (0.58)71.6 (0.57)1.75 (0.57)111.34 (0.28)1.34 (0.28)121.51 (0.05)0.81(−0.73)131.26 (0.57)1.98 (0.57)141.41 (0.27)0.004 (0.001)15−0.24 (−0.07)1.41 (0.28)16−0.07 (−0.02)0.0004 (−0.008)171.41 (0.28)1.41 (0.28)18−0.37 (−0.32)−0.57 (−0.48)190.9 (0.28)−0.81 (−0.9)20−0.37 (−0.29)1.73 (0.48)21−0.48 (−0.45)−0.58(−0.48)221.37 (0.27)−0.57 (−0.48)232.43 (0.86)−1.41 (−0.96)250.22 (0.28)4.63 (1.45)Total NPL (p)1.70 (p = 0.03)2.01 (p = 0.01)Total HLOD (α)1.65 (0.55)1.24 (0.36)NPL and (LOD) scores per family are shown at each locus. Total NPL (p value) and HLOD (α) obtained from the analysis of all eight pedigrees are displayed. HLOD was obtained maximizing the “heterogeneity” LOD score with respect to the proportion of linked families (α). Negative NPL and LOD scores indicate no linkage or inconclusive resultNPL nonparametric LOD score, HLOD heterogeneity LOD score, α proportion of “linked” families Refinement of chromosome 1 locus The 1p36.22-p34.3 region between D1S468 (4.2 cM) and D1S255 (65.5 cM) was saturated with seven extra microsatellite markers (total 12, spaced 4–5 cM) in the complete group of 24 families (Table 2). We observed a maximum HLOD of 1.65 (α = 0.55) and NPL of 1.7 (p = 0.03). The haplotype analysis showed allele sharing in families 2, 5, 7, 11, 19, and 23 between D1S228 (29.8 cM) and D1S255 (65.5 cM). Patients from families 13, 14, and 22 displayed haplotype sharing, but it was limited to the upper or lower part of the region, thus showing no overlapping with the rest of the families. Candidate genes There were several interesting genes map on the chromosome 3q region. The PPP2R3A gene encodes the protein phosphatase 2A, one of four major protein phosphatases identified in eukaryotic cells, implicated in the regulation of most major metabolic pathways. It has been shown to be expressed ubiquitously in 15-day-old kidneys, regulating the mitogenic activity in the early embryonic kidney [37]. The FNDC6 gene encodes a type of fibronectin, expressed mainly in fetal kidney [38], implicated in the development of renal basement membrane [39]. RBP1 and RBP2 genes (Retinol-binding-proteins type 1 and 2) are involved in the metabolism of vitamin A, which in its active form of retinoic acid plays a critical role during kidney development, even connecting ureters to bladder [40]. The AGTR1 gene encodes the angiotensin receptor II type 1 (AT1), shown to be related to gross abnormalities in renal morphogenesis in mutant mice (hydronephrosis) as well as a poor proliferation of ureteral smooth cells [41]. Discussion This study reports the second genome-wide search for primary VUR. Our results suggest the existence of several loci mapping to chromosomes 1, 3, 4, and 22, further supporting the hypothesis that primary VUR is genetically heterogeneous. We, as others, encountered several pitfalls when studying VUR. We observed clinical variability among and within families and the presence of obligate carriers (individuals who carry and transmit the disease allele but do not manifest any disease sign or symptom). Although the appropriate clinical investigations were performed in several apparently healthy individuals (carriers), no evidence of disease was found, indicating a reduced or age-dependent penetrance. As most of these individuals were recruited during their adulthood, we could not exclude an earlier disease condition that evolved to a spontaneous resolution. To overcome these problems, we first performed a careful clinical evaluation of patients and available relatives. All individuals older than 5 years of age with insufficient or no evidence of VUR were classified as diagnosis unknown, despite the consequent loss of power for the statistical analysis. We performed the first genome search in VUR that includes families with the same ethnic origin. In order to strengthen the genetic homogeneity of the patients, all families included in this study originated from the same geographic area in the southern part of Italy. Yet, our results strongly support that primary VUR is a (highly) genetic heterogeneous condition. Whereas a large number of our patients and families supported the chromosome 3q locus, one relatively large pedigree (family 12) showed evidence of linkage on chromosome 22. Six of our families supported a locus on chromosome 3q22.2–23 (149.8–153.2 cM). This region is fully overlapping with one of the loci reported by Feather et al. [18]. In their study, the interval on chromosome 3q (from GATA128C02 to D3S1763, 112–176 cM) showed a high α (0.98) with an NPL = 3, p = 0.008. This region was supported by one of their largest pedigrees (with seven patients) that was clearly not linked to the chromosome 1p13 locus. It is interesting that two independent studies have found the same genomic region in distinct groups of patients. Besides the number of patients included in our genome scan, the results of the statistical analysis are quite modest and do not reach the criteria suggested by Lander and Kruglyak [42] to declare significant linkage. Therefore, some of the identified loci may represent false positive hits and should be interpreted with caution. The complex disease etiology of primary VUR has shown to be difficult to disentangle. Genetic heterogeneity and lack of knowledge of the true genetic model for VUR are probably the main difficulties in the identification of the genetic etiology of VUR. Although genetic studies in VUR are still in an early phase, we can presume that primary VUR is likely a complex disorder, with a number of not fully penetrant genes causing most of the familiar cases. Finally, primary VUR could be caused by simultaneous gene-environment interactions. In conclusion, our results show further evidence for the genetic heterogeneity in primary VUR. We will next focus on the refinement of the identified genomic regions and the sequence analysis of the candidate genes according to their tissutal expression and biological function. Replication of the results in additional families will be essential, first to confirm and eventually to evaluate the contribution of these loci to the pathogenesis of primary, nonsyndromic VUR.

Int_J_Cardiovasc_Imaging-4-1-2373860

Calcium scoring using 64-slice MDCT, dual source CT and EBT: a comparative phantom study

Purpose Assessment of calcium scoring (Ca-scoring) on a 64-slice multi-detector computed tomography (MDCT) scanner, a dual-source computed tomography (DSCT) scanner and an electron beam tomography (EBT) scanner with a moving cardiac phantom as a function of heart rate, slice thickness and calcium density. Methods and materials Three artificial arteries with inserted calcifications of different sizes and densities were scanned at rest (0 beats per minute) and at 50–110 beats per minute (bpm) with an interval of 10 bpm using 64-slice MDCT, DSCT and EBT. Images were reconstructed with a slice thickness of 0.6 and 3.0 mm. Agatston score, volume score and equivalent mass score were determined for each artery. A cardiac motion susceptibility (CMS) index was introduced to assess the susceptibility of Ca-scoring to heart rate. In addition, a difference (Δ) index was introduced to assess the difference of absolute Ca-scoring on MDCT and DSCT with EBT. Results Ca-score is relatively constant up to 60 bpm and starts to decrease or increase above 70 bpm, depending on scoring method, calcification density and slice thickness. EBT showed the least susceptibility to cardiac motion with the smallest average CMS-index (2.5). The average CMS-index of 64-slice MDCT (9.0) is approximately 2.5 times the average CMS-index of DSCT (3.6). The use of a smaller slice thickness decreases the CMS-index for both CT-modalities. The Δ-index for DSCT at 0.6 mm (53.2) is approximately 30% lower than the Δ-index for 64-slice MDCT at 0.6 mm (72.0). The Δ-indexes at 3.0 mm are approximately equal for both modalities (96.9 and 102.0 for 64-slice MDCT and DSCT respectively). Conclusion Ca-scoring is influenced by heart rate, slice thickness and modality used. Ca-scoring on DSCT is approximately 50% less susceptible to cardiac motion as 64-slice MDCT. DSCT offers a better approximation of absolute calcium score on EBT than 64-slice MDCT when using a smaller slice thickness. A smaller slice thickness reduces the susceptibility to cardiac motion and reduces the difference between CT-data and EBT-data. The best approximation of EBT on CT is found for DSCT with a slice thickness of 0.6 mm. Introduction The presence of calcium in coronary arteries is known to be a strong indicator for coronary artery disease (CAD) [1]. It has been shown that quantification of coronary calcium enables the assessment of cardiac event risk stratification [1]. In 1990, Agatston et al. described a method which determines the amount of coronary calcium from tomographic images [2]. This method, known as the Agatston score (AS), depends on the area and the maximum CT density of the calcification detected by electron beam tomography (EBT). Since then, EBT is generally accepted as the gold standard for determining the amount of coronary calcium. Alternative scoring methods have been proposed, such as volume scoring (VS), depending on the volume of the calcification, and equivalent mass (EM) scoring, which depends on the volume and the average density of the calcification [3–5]. Calcium scoring (Ca-scoring) on EBT is known to be less susceptible to cardiac motion compared to other CT-modalities, because of its relatively high temporal resolution. However, since the appearance of multi-detector computed tomography (MDCT), scanners of this type are also widely used for Ca-scoring as an alternative to EBT. Although the temporal resolution of MDCT is lower than EBT, the spatial resolution is much higher (0.4 vs. 1.0 mm), enabling the detection of smaller lesions. Whereas Ca-scoring on EBT can only be used in sequential scanning mode, MDCT facilitates Ca-scoring in sequential and spiral mode. Spiral mode scanning has shown to decrease the variability of Ca-scoring when compared to sequential mode scanning [6]. With the development of dual source computed tomography (DSCT) in 2006, CT is finally approaching the temporal resolution of EBT combined with a high spatial resolution [7]. In order to use Ca-scoring as a useful diagnostic test, it must be demonstrated as accurate, clinically relevant and reproducible. Monitoring of coronary atherosclerosis by repeated scans is advocated by Callister et al. to test the response to lipid-lowering pharmacologic therapy [8] and Budoff et al. [9] showed that statin therapy induced a 61% reduction in coronary calcium progression rate. Therefore a highly reproducible scan-method independent of in-vivo conditions to test the accuracy of Ca-scoring is desirable. In this study a cardiac phantom was used to investigate the influence of cardiac motion on the absolute Ca-score for different kinds of scanners. To our knowledge no previous study has systematically investigated the influence of the heart rate on the absolute Ca-score using EBT, 64-slice MDCT and DSCT. The purpose of this study was therefore to assess Ca-scoring on 64-slice MDCT and DSCT versus EBT on a moving cardiac phantom as a function of heart rate, slice thickness and calcification density using 3 different Ca-scoring methods. Methods and materials Cardiac phantom A moving cardiac phantom (QRM, Möhrendorf, Germany) was used to simulate the movement of the coronary arteries (Fig. 1, left) [7, 10]. The phantom consists of a robot arm which performs a pre-programmed motion (Fig. 2). The robot arm moves in a water container inside a thorax phantom (QRM, Möhrendorf, Germany) [11]. Different inserts can be attached to the robot arm. The motion curves used in this study were based on velocity curves for the LAD given in literature in order to simulate the human coronary motion as realistically as possible [12]. Three different artificial arteries were investigated which were custom built by QRM. The artificial arteries were made of hydroxyapatite (HA) with a diameter of 4 mm and a length of 55 mm (Fig. 1). Each artery contained three artificial calcifications with a length of 10 mm, a spacing of 5 mm and a thickness of 0.5, 1.0 and 2.0 mm, respectively. The density of the calcifications was different in each artery, one with high density calcifications (HDC), one with medium density calcifications (MDC) and one with low density calcifications (LDC). The concentration and density of the calcifications in the three artificial atereries is given in Table 1. The artificial artery had a density of 50 Houndsfield Units (HU), simulating human blood. Fig. 1Left: the cardiac phantom. Right: schematic figure of the artificial artery, the dimensions are given in millimetersFig. 2Motion curve of the phantom at 70 bpm. The curve is defined by the time-deflection points T1–T8 and the reconstruction intervals of the DSCT and 64-slice MDCT are indicated by the grey areas. Other heart rates are obtained by a time scaling of the data points. For higher heart rates (>90 bpm) the data point T5 was omitted to reflect the relative larger diminishing of the diastolic phaseTable 1The three artificial coronary arteries high, medium and low density calcification (HDC, MDC and LDC) with the properties of the inserted calcifications as specified by the manufacturerArtificial arteryConcentration (mgHA/cm3)Density (g/cm3)HDC7961.58MDC4011.30LDC1971.16 Data acquisition The phantom was positioned at an angle of 45 degrees relative to the center axis of the scanner. Every scan was repeated five times with a small random translational (approximately 2 mm) and small random rotational repositioning (approximately 2 degrees) of the phantom after each scan. The ECG signal from the phantom was recorded during scanning to enable synchronization with the scanner. The scan parameters on the 64-slice MDCT (Somatom Sensation 64, Siemens, Forchheim, Germany) were: tube voltage 120 kV, tube current 250 mAs effective, collimation 64 × 0.6 mm and rotation time 330 ms. A DSCT (Somatom Definition, Siemens, Forchheim, Germany) was used with similar scan parameters: tube voltage 120 kV, tube current 100 mAs/rot (equivalent to the tube current of 64-slice MDCT), collimation of 64 × 0.6 mm and rotation time 330 ms. A spiral scanning mode was used on both scanners for a better reproducibility. A standard hospital calcium scoring protocol was used on the EBT-scanner (e− Speed, GE Imatron, San Francisco, USA). This protocol uses a sequential mode with a tube voltage of 130 kV, a tube current of 44 mAs, a collimation of 3.0 mm and a scan speed of 50 ms. A standard calcium scoring kernel (B35f) was used for reconstruction of the CT-data. Images were retrospectively reconstructed with a slice thickness of 0.6 mm (increment 0.4 mm) and 3.0 mm (increment 3.0 mm) for both CT scanners. The phases with minimal motion were selected from the motion curves of the coronary arteries (Fig. 2) and used for reconstruction of the raw data (Table 2). The data from the EBT-scanner were reconstructed with a slice thickness of 3.0 mm (increment 3.0 mm) at 40% of the RR-interval with a standard calcium kernel according to the standard calcium scoring protocol used in our hospital. Table 2Phases used for reconstruction of the images in percentage of the beat time at different heart rates used in beats per minute (bpm)Heart rates (bpm)506070809010011064-Slice MDCT-phase (%)76746058565351DSCT-phase (%)83827069696766 Ca-scoring was performed on the reconstructed image sets with commercially available software (Syngo CaScore, Siemens, Forchheim, Germany). Three different scoring methods were used: Agatston scoring, volume scoring and equivalent mass scoring. A standard scoring threshold of 130 HU was used during the procedure. Detailed descriptions of these scoring methods can be found extensively elsewhere [4, 11, 13–15]. The three calcifications of the arteries could not be detected individually at higher heart rates (at heart rates larger than 60 bpm for 64-slice MDCT and larger than 90 bpm for DSCT) combined with thin slices in some of the scans. Therefore the Ca-score of the total artery was used instead of the Ca-scores of the individual calcifications. Data analysis Two root mean square measures were used to analyze the scoring results. The first measure quantifies the susceptibility of the calcium score to cardiac motion. The second measure quantifies the deviation of the calcium score from the reference value. We defined a cardiac motion susceptibility (CMS) index in order to assess the susceptibility to cardiac motion of the Ca-scoring methods: in which x0 is the Ca-scoring result at 0 bpm, xi is the scoring result at heart rate i and N is the total number of heart rates used. In the equation for the CMS-index a factor 1/x0 is introduced to make the index independent of the absolute score which enables comparison of Ca-scores obtained at different slice thicknesses and with different scoring methods as a function of cardiac motion. A small CMS-index is equivalent to a low susceptibility of Ca-scores to cardiac motion. A second measure was introduced to compare the calcium score results of the two CT scanners to the results of the EBT scanner. The deviation of the calcium score on CT versus the reference value on EBT is defined using a Δ-index: in which yi is the EBT-score at heart rate i, zi is the CT-score at heart rate i and yav is the average EBT-score over all heart rates. The normalization factor yav was inserted to make the Δ-index independent of the absolute score and to enable comparison of Δ-indexes obtained with different Ca-scoring methods and slice thicknesses. A low Δ-index is equivalent to a small difference between Ca-scores on CT and EBT. The delta-index, as defined in Eq. 2, is used to quantify the difference in Agatston and volume scores on CT and EBT. For these scoring methods EBT provides the reference value. For the equivalent mass score, however, the reference value is given by the physical mass. The use of a phantom enables the possibility of calculating the true amount of calcium. Therefore the equivalent mass scoring results have been compared to the true values instead of the EBT-values, thus yi is the true value and zi is the CT/EBT-score at heart rate i. Noise levels were measured using a standard Region of Interest (ROI) technique. The ROI was placed in a section of a slice containing only water. The standard deviation of the measured HU-values within the selected ROI was considered to be a measure for the noise level. All measurements are considered to be normally distributed. Mean and standard deviation (sd) are given for each measurement. Results The Ca-scoring results of the different arteries obtained with 64-slice MDCT, DSCT and EBT are shown in Fig. 3 as a function of slice thickness and heart rate using the three different scoring methods. The scoring results are relatively constant at low heart rates (50–60 bpm). At heart rates higher than 60 bpm, however, the scores deviate from the values at lower heart rates and an increase or decrease of scoring results is observed depending on modality, slice thickness, calcification density and scoring method. Fig. 3Calcium scores as a function of heart rate in beats per minute using 64-slice MDCT at 0.6 mm (dotted line with circles), 64-slice MDCT at 3.0 mm (solid line with triangles), DSCT at 0.6 mm (dotted line with circles), DSCT at 3.0 mm (solid line with triangles), EBT (solid line with squares). Agatston score (AS), volume score (VS) and equivalent mass (EM) score from top to bottom; artificial arteries high density calcification (HDC), medium density calcification (MDC) and low density calcification (LDC) from left to right. The thick dotted black lines in the figures in the bottom row represent the physical amount of calcium The results show a general underestimation of the Ca-score for Ca-scoring obtained at 3.0 mm slice thickness when comparing CT-data and EBT at all heart rates except for the Agatston and volume score of the high density calcifications at 70 and 80 bpm. In general, the Ca-scores obtained with 0.6 mm slice thickness on CT are overestimated compared to the EBT-data or are similar to the EBT-data at all heart rates. The scores obtained with EBT (squares) increased at heart rates above 90 bpm for the artery containing the high density calcifications (Fig. 3, left column), whereas the artery containing the medium density calcifications remained relatively constant throughout the whole range of heart rates (Fig. 3, middle column). The artery containing the low density calcifications showed decreased scoring results at higher heart rates (Fig. 3, right column). The 64-slice MDCT with a slice thickness of 3.0 mm (solid lines with triangles) showed increased Ca-scores for the Agatston score at 70–90 bpm and for the volumes score at all heart rates for the high density calcification, whereas the equivalent mass score showed a slight decrease. The medium and low density calcification also showed a decrease in scoring results at higher heart rates. The 64-slice MDCT with a slice thickness of 0.6 mm (dotted lines with circles) showed highly increased Ca-scores above 70 bpm for the high density calcification for all scoring methods. This is also seen for the medium density calcification for the volume score, whereas the equivelnt mass and Agatston score showed a peak in Ca-scores at 80 bpm. The low density calcification showed diminished results at higher heart rates for all scoring methods. The Ca-scores of the medium and low density calcification obtained with DSCT with a slice thickness of 3.0 mm (solid lines with triangles) were decreased at elevated heart rates. The results of the high density calcification were relatively constant over the whole range of heart rates. Finally DSCT at 0.6 mm (dotted lines with circles) showed increased results for Agatston and volume score of the high density calcification. The Agatston score of the medium density calcification showed a small decrease and relatively constant results were observed for the equivalent mass score of the high density calcification and volume and equivalent mass score of the medium density calcification. Diminishing results with increasing heart rate were observed for all methods for the low density calcification. The influence of cardiac motion on the Ca-score (CMS-index) using the different scoring methods is calculated using Eq. 1 and is summarized in Fig. 4a–c for all scanners and slice thicknesses. Looking at the results of the Agatston score, the average CMS-index for EBT was approximately similar to the CMS-index of DSCT at 0.6 mm, which was for its part approximately 60% smaller than the CMS-index of 64-slice MDCT at 0.6 mm. The CMS-index of DSCT at 3.0 mm was approximately 50% higher than the CMS-index at 0.6 mm. The CMS-index of 64-slice MDCT at 3.0 mm was approximately twice as large as the index of DSCT at 3.0 mm (Fig. 4a). The results of the susceptibility to cardiac motion using volume and equivalent mass score were similar to the results obtained with the Agatston score, except for the relatively small CMS-index for 64-slice MDCT at 0.6 mm using for equivalent mass score. The absolute CMS-indexes using equivalent mass were approximately 10% lower compared to the other two methods. The CMS-indexes averaged over scoring method, slice thickness and calcification density were 2.5 for EBT, 3.6 for DSCT and 9.0 for 64-slice MDCT. Fig. 4Cardiac motion susceptibility-index (see text) determined with Agatston score (AS) (a), volume score (VS) (b) and equivalent mass (EM) (c) score for the high, medium, low density lesions and the average using EBT (with slice thickness of 3.0 mm), 64-slice MDCT (with slice thickness of 3.0 and 0.6 mm) and DSCT (with slice thickness of 3.0 and 0.6 mm). A small CMS-index represents a low susceptibility to cardiac motion. The standard deviations of the CSM-index are indicated by error bars. 64S = 64-slice MDCT; DS = Dual Source CT The difference between the scoring results using Agatston and volume score of 64-slice MDCT and DSCT compared to EBT are calculated using Eq. 2 and are shown in Fig. 5a–b. For Agatston score (Fig. 5a), the best Δ-index was observed for DSCT with a slice thickness of 0.6 mm (35.9 ± 10.0 averaged over all densities). A Δ-index approximately twice as large was observed for 64-slice MDCT at 0.6 mm (65.7 ± 9.0 averaged over all densities). Both CT-modalities at 3.0 mm had a Δ-index approximately two times the Δ-index of DSCT at 0.6 mm (91.0 ± 10.1 and 88.4 ± 9.1 for DSCT and 64-slice MDCT respectively averaged over all densities). Comparable results were observed for the volume score measurement (Fig. 5b), although the Δ-indexes for the measurements at 0.6 mm were higher with the highest Δ-index for 64-slice MDCT at 0.6 mm. Fig. 5Δ-index (see text) determined with Agatston score (AS) (a), volume score (VS) (b) and equivalent mass (EM) (c) score for the high, medium, low density lesions and the average using 64-slice MDCT and DSCT, both with slice thicknesses of 3.0 and 0.6 mm. For Agatston and volume score, EBT has been used as a reference value (a and b, respectively), whereas for equivalent mass score the physical mass has been used as a reference value (c). The equivalent mass measurement includes the EBT as well. A small Δ-index represents a good correspondence with the EBT results. 64S = 64-slice MDCT; DS = Dual Source CT A Δ-index was calculated for all scanners comparing the equivalent mass results to the theoretical true values. The results are shown in Fig. 5c. The smallest Δ-index was observed for 64-slice MDCT (55.9 ± 6.8) followed by higher indexes for DSCT (68.3 ± 8.3) and EBT (71.3 ± 7.9) both with a slice thickness of 0.6 mm, however the indexes of 64-slice MDCT and DSCT and the indexes of EBT and DSCT are within each margins of error shown by the error bars. Both CT-modalities at 3.0 mm showed Δ-indexes approximately twice as large compared to the results at 0.6 mm (140.1 ± 7.8 and 131.1 ± 8.5 for DSCT and 64-slice MDCT respectively averaged over all densities). The Δ-indexes were 53.2 for DSCT and 72.0 for 64-slice MDCT both with a slice thickness of 0.6 mm averaged over the scoring methods and densities. The Δ-indexes at 3.0 mm were 102.0 for DSCT and 96.9 for 64-slice MDCT averaged over the scoring methods and densities. Noise levels were as follows: 64-slice MDCT showed 36.1 ± 2.9 HU and 13.2 ± 1.2 HU for 0.6 and 3.0 mm slice thickness, respectively. DSCT showed 43.0 ± 1.6 HU and 16.1 ± 1.0 HU for 0.6 and 3.0 mm slice thickness, respectively. EBT with a slice thickness of 3.0 mm showed a noise level of 20.5 ± 0.8 HU. The noise did not vary at different heart rates. Discussion An assessment was made of Ca-scoring on 64-slice multi-detector computed tomography and dual-source computed tomography versus electron beam tomography on a moving cardiac phantom as a function of heart rate, slice thickness and calcification density using 3 different Ca-scoring methods. From the results it can be concluded that the Agatston, volume and equivalent mass scores depend on heart rate, slice thickness and the CT-system used. Furthermore DSCT is approximately 50% less susceptible to cardiac motion as 64-slice MDCT in Ca-scoring. It has been shown in previous studies that the amount of calcium in coronary arteries is generally underestimated in MDCT with respect to the gold standard EBT. Stanford et al. showed an underestimation of coronary calcium with 4-slice MDCT compared to EBT [16] and the same effect was reported by Horiguchi et al. using 16-slice MDCT [14, 17]. Our results showed underestimation as well, but only for 3.0 mm slice thickness, whereas 0.6 mm showed an overestimation at all heart rates. Surprisingly the Agatston scores of the medium density calcification at rest using 3.0 mm slices are different for the 64-MDCT and DSCT, while similar scores are expected (approximately 165 for 64-slice MDCT and 135 for DSCT). The same effect is observed for heart rates of 50 and 60 bpm. A possible explanation for this phenomenon lies within the scoring algorithm of the Agatston score. For each calcification the maximum HU value within the calcification is obtained. Based on this maximum value the area of the calcification is multiplied by a weighting factor. For a maximum of more than 400 HU this factor is 4, for a maximum between 300 HU and 400 HU this factor is 3 [2]. The medium density calcification has a CT density of 400 HU. The difference in scoring results can be explained by a small difference in HU between the two scanners. Where the maximum CT density within the medium density calcification could be over 400 HU using the 64-slice MDCT, the maximum CT might have been below 400 using the DSCT. If this explanation is applied, the score obtained using 64-slice MDCT is more similar to the score obtained using DSCT (165*3/4 = 124). Although the difference in HU is very small, the weighting factor of the Agatston algorithm can cause a large difference in scoring result. At heart rates above 70 bpm Agatston, volume and equivalent mass score differ from the results at rest and at low heart rates. This difference depends on the density of the calcification as can be seen from Fig. 3: calcifications with a high density show elevated scoring results, whereas low density calcifications are associated with diminished scoring results. A decrease of Agatston and equivalent mass score on increased heart rates using a calcification of 400 HU has also been reported by Ulzheimer et al. using a 4-slice MDCT in accordance with our results [11]. We considered the influence of image blurring on the Ca-score as a function of the calcification density in Fig. 6 for a possible explanation for this effect. Two calcifications of identical size are shown by black lines, one with a high density (X) and one with a low density (Y). The corresponding apparent images at a relatively low and high heart rate are given by the solid grey and dotted grey line, respectively. In addition, the default Ca-scoring threshold of 130 HU is shown by the dotted black line. At the level of the threshold the apparent width at high heart rates is larger than the apparent width at low heart rates for the high density object. The reverse effect is observed for the low density object; at high heart rates the apparent size is reduced compared to the apparent size at low heart rates. From this analysis it can be concluded that at high heart rates the apparent volume of high density objects is increased and the apparent volume of low density objects is decreased. With this model we can explain the increase of calcium score on increasing heart rate for high density calcifications, and a decrease of calcium score on increasing heart rate for low density calcifications, as observed in Fig. 3. Decreasing scoring results with increasing movement have previously been reported on 4-slice CT [15]. Fig. 6Theoretical estimated CT profiles for two objects (black) with high (X) and low density (Y) exhibiting a relatively low (solid grey) and high (dotted grey) movement. The dotted black line represents the standard Ca-scoring threshold of 130 HU The susceptibility of calcium score on heart rate has been assessed by the CMS-index using the 3 different scoring methods available. The results show that the CMS-index of EBT is the lowest for all methods. Therefore it can be concluded that EBT is the least susceptible to cardiac motion. The CMS-index of DSCT is approximately half the CMS-index of 64-slice MDCT, showing a reduction of 50% of the influence of cardiac motion on Ca-scoring on DSCT with respect to 64-slice MDCT. These results can be explained with the improved temporal resolution of DSCT compared to 64-slice MDCT (83 vs. 165 ms). A reduction of the slice thickness also results in a lower CMS-index. Therefore we conclude that the use of a small slice thickness reduces the susceptibility to cardiac motion for both 64-slice MDCT and DSCT. The difference between CT-data and EBT-data has been assessed by the Δ-index using the Agatston and volume score, the equivalent mass results have been compared to the physical amount of calcium. The results show the lowest Δ-index for DSCT with a slice thickness of 0.6 mm for Agatston and volume score. The CT modalities at 0.6 mm and EBT showed similar Δ-indexes for the approximation to the physical mass. A reduction of the Δ-index was observed comparing the two CT-modalities at 0.6 mm and 3.0 mm. The best resemblance between EBT and CT was observed for DSCT with a slice thickness of 0.6 mm. The use of a smaller slice thickness has some disadvantages although it was beneficial to the scoring results in this phantom study. The noise measurements showed increased noise levels for the 0.6 mm slices compared to 3.0 mm slices. It is expected that for patient scanning the noise levels at 0.6 mm are too high to guarantee a reliable outcome of the Ca-scoring. To overcome these increased noise levels the tube current can be increased. However, this increases the patient dose as well. Although dose-reduction techniques have been investigated leading to dose-reductions up to 57% [18–21], a good balance between patient dose and accuracy of calcium scoring needs to be found. Limitations The EBT-data acquisition of this study was performed with a standard hospital protocol using a tube voltage of 130 kV, whereas CT scanning was performed with a tube voltage of 120 kV. Although higher energies tend to show less density, Nelson et al. reported very small differences between EBT at 130 kV and CT at 120 kV [22]. Therefore we expect that the influence of the difference in tube voltage can be neglected. The pre-programmed movement of the calcified coronary arteries was 1-dimensional in contrast with the in vivo situation where the motion of human coronary arteries is 3-dimensional and the direction and orientation of the human coronary arteries can vary. In our study the movement of the calcified coronary arteries was in the (x,z) plane with a 45o angle relative to the z-direction of the scanner. We expect that movement more perpendicular to the z-direction of the scanner will cause more blurring in the (x,y) plane and reduce blurring in the z-direction. In addition, we expect that movement more parallel to the z-direction of the scanner will be more subject to partial volume effects when using thick slices. Thin slices will be less subject to the PVE due to the isotropic resolution of 0.6 mm. The motion of the robot arm was programmed according to patient data [11] and therefore we expect that our analysis shows a good correspondence with a clinical situation, but a clinical validation is advocated. The coronary artery we used for our simulation, the LAD, exhibits lesser motion than the LCX and especially the RCA, which exhibits very large motion swings especially in systole. In our study we, however, wanted to show the influence of motion on the coronary calcium score independent of a specific major coronary artery. We therefore have used motion curves with velocities similar to the LAD to simulate the motion, because if a dependency of calcium score on coronary motion could be proven for the lowest velocity of the LAD, we expect an even stronger dependence for the higher velocities of the LCX and RCA. In our study we have shown that for higher heart rates the under- or overestimation of the calcium score increases as a function of calcification density, independently of the absolute velocity of the artery, but depending on the relative heart rate difference from 0 bpm. Because this motion dependent effect is pronounced visible for the relative low velocity of the LAD, we expect that the results can also be applied to the vaster moving other major arteries. Conclusion The results of Ca-scoring are influenced by heart rate, slice thickness and modality used. DSCT is approximately 50% less susceptible to cardiac motion than 64-slice MDCT using a robot phantom. Susceptibility is further reduced with a smaller slice thickness. DSCT gives a better approximation of the absolute calcium score on EBT than results obtained with 64-slice MDCT when using a smaller slice thickness (0.6 mm). The two modalities show similar results when using larger slice thicknesses (3.0 mm). In general, the use of a smaller slice thickness further reduces the difference between CT-data and EBT-data. The best approximation to the physical amount of calcium was found using a small slice thickness, where 64-slice MDCT and DSCT show similar results. The best approximation of Ca-scoring on EBT is observed for DSCT with a slice thickness of 0.6 mm.

Bioinformation-2-1-2139988

A database for medicinal plants used in the treatment of diabetes and its secondary complications

Effective treatment of diabetes is increasingly dependent on active constituents of medicinal plants capable of controlling hyperglycemia as well as its secondary complications. Sensing the importance of documenting such medicinal plants, here we describe a web database containing information (name, literature citation, active compounds and few related full text articles) of the diabetes medicinal plants exhibiting hypoglycemic, antioxidant and antimicrobial effects. Background Diabetes mellitus is possibly the world's largest growing metabolic disorder and around 30 million people around the world suffer from this disorder.[1] Diabetes can be defined as a group of syndromes characterized by hyperglycemia, altered metabolism of lipids, carbohydrates and proteins.[2] The complications of diabetes include vascular diseases, eye disorders, renal disorders and a host of secondary infections. [3] The treatment of diabetes with synthetic drugs is generally not preferred because of its high cost and the range of side effects caused. Hence development of traditional or alternative medicine is needed. Herbal drugs constitute an important part of traditional medicine and literature shows that there are more than 400 plant species showing antidiabetic activity. [4,5] Our interest is to study these anti diabetic medicinal plants. Research findings suggest that many of these plants control diabetes by exhibiting hypoglycemic and antioxidant effects. The secondary infections associated with diabetes are also restricted to antimicrobial effect by plant products. Sensing the opportunities provided and in an effort to translate research into technology, this database ‘DIAB’ was created to document the research literature available on these medicinal plants and their active compounds. Methodology Step 1: Data collection Data of antidiabetic plants were collected from various literature sources such as PubMed [6], ScienceDirect [7], Biomed Central [8] Springerlink [9], Scirus [10], Wiley journals [11] and also from folklore medicinal usage. The current dataset contains information for about 258 genus and 287 species of plants exhibiting antidiabetic effect. Of these 287 antidiabetic plants, 129 species show proven antioxidant effect and 53 species exhibit antimicrobial effect besides their hypoglycemic activity. Step 2: Database Design The database is developed using MS Access as back-end and ASP.NET as front-end on a Windows platform and updated regularly. Step 3: Features of DIAB Three separate links (antidiabetic, antioxidant, and antimicrobial) are provided to access the literature citations of antidiabetic plants showing hypoglycemic, antioxidant and antimicrobial properties respectively. The record entry consists of the following literature: plant name, part investigated, authors and abstract. The ‘Active principles’ link provides information (plant name, part investigated, active compounds and properties) on the 46 compounds isolated from the antidiabetic medicinal plants. The ‘Articles’ link provides few free text articles for further reading. A screen shot of the database is given in figure 1. Utility In the current scenario there are several other databases which give information on medicinal plant name, distribution, drugs formulated and usage in treatment of diabetes. We believe that the freely available database called DIAB will give supplementary information on the antidiabetic plants capable of controlling some secondary complications. This database finds utility to the scientific community for a quick review on plants for diabetes medicinal plant research and provides enormous scope for development of drugs. Future development We plan to refine and keep updating this database and hopefully a link solely for indigenous plants will be made available in the nearest future. The database will also be modified to develop provisions to search the database to identify plants of interest using keywords.

Liver_Int-1-_-2156150

Interferon and lamivudine vs. interferon for hepatitis B e antigen-positive hepatitis B treatment: meta-analysis of randomized controlled trials

Aims To compare interferon monotherapy with its combination with lamivudine for hepatitis B e antigen (HBeAg)-positive hepatitis B treatment. Chronic hepatitis B is a common medical condition, affecting more than 400 million individuals worldwide, leading to hepatic inflammation and injury (1–4). This viral-triggered, immune-mediated condition predisposes those affected to cirrhosis and hepatocellular carcinoma, thus necessitating treatment (1–3). The treatment consists of individualized, single-agent therapy with interferon-α or nucleoside analogues. Unfortunately, this treatment fails to yield long-lasting outcomes in majority of the treated population, prompting the notion of their use in combination to enhance the therapeutic efficacy (4–8). The notion of combination therapy for chronic hepatitis B treatment has been previously examined, yielding inconclusive results (9–16). In our study, we aim to elucidate this topic comparing interferon monotherapy to its combination with the best-studied antiviral agent for that purpose, lamivudine. Furthermore, the focus of our analysis is hepatitis B e antigen (HBeAg)-positive patients, a subset of the patient population in which disease activity, risk of complications and the subsequent need of efficacious therapy are more pronounced. Methods Literature search and study design Two independent researchers conducted the literature search, study selection and data extraction, with any disagreements resolved by consensus among them. The researchers conducted a systemic literature search using the electronic databases MEDLINE (1966 to January 2006), EMBASE (1980 to June 2006), OVID (1966 to January, week 3, 2006) and the Cochrane library clinical trials registry (issue 1, 2007). The following keywords were used: ‘Hepatitis B’, ‘Interferon’, ‘Lamivudine’ and ‘combination therapy’. In addition, a manual search using citations in previous publications was preformed. The following inclusion criteria were used: (i) study design: randomized controlled trials; (ii) study population: HBeAg-positive patients; (iii) intervention: interferon vs. interferon and lamivudine therapy. Our search was not restricted by language. The following exclusion criteria were used: (i) examining the nonadult population; (ii) not reporting any of the primary efficacy measures as defined by the authors. When several publications pertaining to a single study were identified, the most recent and complete publication was used. The included studies were divided into two groups according to their use of conventional (CON) or pegylated (PEG) interferon-α, with patients within each group given interferon monotherapy, or interferon and lamivudine combination therapy. Data were extracted for study methodology and for the defined efficacy measures. Only data pertaining to the regimens in question were extracted, while data concerning other regimens were reviewed, and if found to be of significance to our study, were noted and discussed. Separate meta-analyses examining the defined efficacy measures were preformed. In addition, we compared the rates of sustained responses across groups aiming at the identification of a preferable regimen. Intention to treat analysis was used throughout this study, excluding histological response analysis, because of its low outcomes reporting rates. Efficacy measures and definitions End-of-follow-up (sustained) virological and biochemical response rates, and sustained HBeAg clearance and seroconversion rates were used as primary efficacy measures. Histological response, emergence of YMDD mutations, liver-related and all-cause mortality, and treatment safety were used as secondary efficacy measures. Virological response was defined as attainment of undetectable (or below 400 copies/mL) levels of hepatitis B virus DNA, as determined by polymerase chain reaction, which was previously found to be the most accurate measure of virological response monitoring (2). Biochemical response was defined as normalization of alanine aminotransferase levels, HBeAg clearance as HBeAg disappearance and seroconversion as HBeAg antibodies appearance. Histological response was defined as a two-point reduction or increase in the histologic activity index score, signifying histological improvement and worsening respectively. Treatment safety was assessed using the occurrence rate of adverse effects necessitating treatment discontinuation. Study quality and homogeneity The included studies methodological quality was assessed using the Jadad quality scale (15), an established composite score evaluating randomization, concealment and reporting of patient withdrawal and dropout rates, with scores ≥3 signifying high-quality studies. Heterogeneity was assessed for each analysis. Statistical analysis Quantitative meta-analyses were performed to assess differences between monotherapy and combination groups. Statistical analysis was performed and the Forest plots were generated using the comprehensive meta analysis® software application, Version 2.0 (Biostat, Englewood, NJ, USA). The odds ratios (OR) were calculated along with their, respective, 95% confidence intervals (CI) and presented for each individual study as well as interferon type and across all studies. Subgroup analyses were presented using OR and their corresponding 95% CI. Heterogeneity was assessed for each of the meta-analyses by means of Q-statistics and their corresponding P-values. Results Study selection and characteristics The literature search yielded 13 studies. Five studies were excluded on account of the following: (i) examining the nonadult population (n=1); and (ii) not examining or reporting the sustained response rates (n=4). The eight remaining trials, involving 1321 patients, were included. Six trials used conventional interferon-α (n=503) (16–21) and two trials used pegylated interferon-α (n=808) (22, 23). Two of the CON group trials exclusively studied treatment-naïve patients (16, 19), whereas the others studied a mixture of treatment-naïve and previously treated patients (17, 18, 20–23). The majority of patients were treated for a period of 1 year (n=6) (16–23), with some treated for 6 months (n=2) (17, 18). Sustained response rates were obtained at 6 months following treatment completion in most studies (n=6) (17, 19–23), and at 40 or 54 weeks following treatment completion in some (n=2) (16, 18). The studies were of heterogeneous methodological quality (Jadad scores of 2–5). All studies were published as full publications, with six studies published in English (16–19, 22, 23) and two in Chinese (20, 21). One study used sequential therapy (18), six used concurrent therapy (16, 17, 19,21–23) and one used both (20) (see Tables 1 and 2). Table 2 Patient selection criteria of studies included in the meta-analysis Study Inclusion criteria Exclusion criteria Ayaz (2006) 1. HBsAg positive for >6 m and anti-HBeAg and HBsAg negative 2. Presence of HBV DNA 3. Evidence of inflammation on biopsy within 6 m of enrolment and ALT>1.5 NL 1. Previous treatment with INF, antiviral or immunosuppressive agents 2. HIV, hepatitis C or D 3. Other aetiologies of liver disease, alcohol intake >40 g/day, decompensated liver disease or cancer 4. No informed consent 5. Pregnancy 6. Any contraindications for INF use 7. Leucocytes, neutrophil or platelet count of <2500, <1000 and <100 000/mL, respectively, or haemoglobin <10 g/dL Song (2004) 1. 19–65 years old 2. HBsAg positive for >6 m and HBeAg positive 3. HBV DNA>500 000 copies/mL 4. Evidence of inflammation by 2 NL <ALT <500 Not reported Deng (2003) 1. 15–60 years old 2. HBeAg and HBV DNA positive for >6 m 3. HBV DNA>103 000 copies/mL 4. Evidence of inflammation by ALT>2 NL 1. Immunosuppressive or antiviral therapy within 6 m 2. Hepatitis of other aetiologies 3. Decompensated liver disease 4. Pregnancy or breast feeding Yalcin (2002) 1. 16–80 years old 2. HBeAg and HBsAg positive 3. HBV DNA positive 4. Evidence of inflammation by histology and 1.5<ALT<10 NL, on three occasions within 6 m 1. Previous INF therapy, antiviral or immunosuppressive therapy, or contraindication for INF therapy 2. HIV, hepatitis C or D 3. Decompensated liver disease or carcinoma 4. Alcohol consumption >40 g/day or other liver disease causes 5. Pregnancy 6. Leucocytes <2500/mm3, neutrophils <1000/mm3, platelets <100 000/mm3, or haemoglobin <10g/dL 7. Unable to obtain consent Cindoruk (2002) 1. Adults 2. HBeAg positive 3. HBV DNA positive 4. Evidence of inflammation by histology and by abnormal ALT levels for >6 m 1. Previous INF therapy 2. HIV, hepatitis C or D 3. Decompensated liver disease 4. Diabetes, autoimmune, or other psychiatric or serious medical illness 5. High alcohol intake or current drug abuse 6. Pregnancy Schalm (2000) 1. 16–70 years old 2. HBsAg and HBeAg positive at screening and at >6 and >3 m prior respectively 3. HBV DNA>500 000 copies/mL 4. Evidence of inflammation by histology or persistently elevated ALT for >3 m 1. Contraindication to or previous INF therapy, or antiviral therapy within 6 m 2. HIV, hepatitis C or D 3. Decompensated liver disease 4. Liver disease of other aetiology Lau (2005) 1. Adults 2. HBsAg positive for >6 m and HBeAg positive 3. HBV DNA>500 000 copies/mL 4. Evidence of inflammation on biopsy and 1<ALT<10 NL 1. Treatment within 6 m 2. HIV, hepatitis C or D 3. Decompensated liver disease 4. Serious medical or psychiatric illness 5. Alcohol or drug use within 1 y 4. Neutrophils <1500 g/dL, platelets <90 000/mm3, or creatinine >1.5 NL Janssen (2005) 1. >16 years old 2. HBsAg positive for >6 m and HBeAg positive on two occasions within 8 w of randomization 3. Evidence of inflammation by two measurements of ALT>2 NL within 8 w of randomization 1. Antiviral or immunosuppressive therapy within 6 m 2. HIV, hepatitis C or D 3. Advanced liver disease or carcinoma 4. Serious medical or psychiatric illness, or uncontrolled thyroid disease 5. Substance abuse within 2 y 6. Pregnancy or inadequate contraception 7. Leucocytes <3000/mm3, neutrophils <1800/mm3, or platelets <100 000/mm3 ALT, alanine aminotransferase; HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; INF, interferon; m, months; NEG, HBeAg negative; NL, upper limit of normal; POS, HBeAg positive; w, weeks; y, years. Table 1 Characteristics of studies included in the meta-analysis Study n Study design Jadad score Therapy period Follow-up period Therapy regimen Conventional interferon-α  Ayaz (2006)  68 RCT 2 12 m  6 m INF-α-2a 9 MU × 3/w with or without LMV 100 mg/day  Song (2004)  90 RCT 2 12 m  6 m INF-α 3 MU × 3/w with or without LMV 100 mg/day  Deng (2003)  62 RCT 2 48 w 24 w INF-α-1b 5 MU × 3/w with or without LMV 100 mg/day  Yalcin (2002)  49 RCT 2 52 w 52 w INF-α-2b 10 MU × 3/w with or without LMV 100 mg/day  Cindoruk (2002) 100 RCT 2 6 m  6 m INF-α 9 MU × 3/w with or without LMV 100 mg/day  Schalm (2000) 144 RCT, DB 4 24 w 40 w INF-α 10 MU × 3/w with or without LMV 100 mg/day Pegylated interferon-α  Lau (2005) 542 RCT, DB 5 48 w 24 w PegINF-α-2a 180 μg × 1/w with or without LMV 100 mg/day  Janssen (2005) 266 RCT, DB 4 52 w 26 w PegINF-α-2b 100 μg × 1/w with or without LMV 100 mg/day DB, double blind; INF, conventional interferon; LMV, lamivudine; m, months; PegINF, pegylated interferon; RCT, randomized controlled; w, weeks. Sustained virological response Greater sustained virological response rates were observed for patients given combination as compared with monotherapy in the CON group [61.1 vs. 35.4%, OR=11.7, 95% CI (7.8–17.6), P<0.0001], and overall [28.9 vs. 18.5%, OR=2.1, 95% CI (1.3–3.3), P=0.002], although not in the PEG group [12.2 vs. 11.8%, OR=1.1, 95% CI (0.5–2.3), P=0.8]. Heterogeneity was assessed and not found to be a concern (Q=3.5, P=0.06) (see Fig. 1). Fig. 1 Sustained virological response. CON, conventional interferon monotherapy vs. its combination with lamivudine; PEG, pegylated interferon monotherapy vs. its combination with lamivudine. *Concurrent and sequential administration. Sustained biochemical response Greater sustained biochemical response rates were observed for patients given combination as compared with monotherapy in the CON group [46.2 vs. 34.0%, OR=1.8, 95% CI (1.2–2.7), P=0.007], although not in the PEG group [37.9 vs. 38.1%, OR=1.0, 95% CI (0.7–1.3), P=0.94], or overall [36.1 vs. 36.7%, OR=1.2, 95% CI (0.9–1.5), P=0.15]. Heterogeneity was assessed and not found to be a concern (Q=3.3, P=0.07) (see Fig. 2). Fig. 2 Sustained biochemical response. CON, conventional interferon monotherapy vs. its combination with lamivudine; PEG, pegylated interferon monotherapy vs. its combination with lamivudine. Sustained hepatitis B e antigen clearance No significant differences in sustained HBeAg clearance rates were observed between patients given combination and monotherapy in the CON group [33.5 vs. 24.0%, OR=1.6, 95% CI (0.9–2.7), P=0.09], PEG group [30.6 vs. 34.4%, OR=0.8, 95% CI (0.6–1.1), P=0.26] and overall [31.5 vs. 31.9%, OR=1.0, 95% CI (0.7–1.3), P=0.88]. Heterogeneity was assessed and not found to be a concern (Q=2.6, P=0.11) (see Fig. 3). Fig. 3 Sustained hepatitis B e antigen (HBeAg) clearance. CON, conventional interferon monotherapy vs. its combination with lamivudine; PEG, pegylated interferon monotherapy vs. its combination with lamivudine. *Concurrent and sequential administration. Sustained seroconversion Greater sustained seroconversion rates were observed for patients given combination as compared with monotherapy in the CON group [30.0 vs. 18.9%, OR=1.8, 95% CI (1.1–2.8), P=0.01], although not in the PEG group [27.9 vs. 31.0%, OR=0.9, 95% CI (0.6–1.2), P=0.34], or overall [28.7 vs. 27.1%, OR=1.1, 95% CI (0.8–1.4), P=0.59]. Heterogeneity was assessed and not found to be a concern (Q=3.5, P=0.06) (see Fig. 4). Fig. 4 Sustained seroconversion. CON, conventional interferon monotherapy vs. its combination with lamivudine; PEG, pegylated interferon monotherapy vs. its combination with lamivudine. *Concurrent and sequential administration. Histological response Greater histological improvement rates were observed for patients given combination as compared with monotherapy in the CON group [83.8 vs. 26.6%, respectively, n=1, OR=14.3, 95% CI (3.3–61.3), P<0.001], although not in the PEG group [48.1 vs. 53.4%, respectively, n=1, OR=0.8, 95% CI (0.4–1.7), P=0.70], or overall [61.4 vs. 47.9%, respectively, n=2, OR=1.7, 95% CI (0.9–3.3), P=0.11]. No significant differences in histological worsening rates were observed between patients given combination and monotherapy in the CON group [0 vs. 6.7%, respectively, n=1, OR=0.23, 95% CI (0.02–3.3), P=0.16], PEG group [9.6 vs. 10.3%, respectively, n=1, OR=0.9, 95% CI (0.3–3.1), P=0.9] and overall [6.0 vs. 9.6%, respectively, n=2, OR=0.6, 95% CI (0.2–1.9), P=0.55]. All-cause and liver-related mortality There were no reported deaths of any aetiology for either group (0% for both). Safety No significant differences in safety rates were observed between patients given combination and monotherapy in the CON group [1.6 vs. 0.9%, OR=1.7, 95% CI (0.3–9.0), P=0.55, n=6], PEG group [6.0 vs. 4.2%, OR=1.7, 95% CI (0.9–3.2), P=0.12, n=2] and overall [4.1 vs. 2.7.%, OR=1.7, 95% CI (0.9–3.1), P=0.10]. Heterogeneity was assessed and not found to be a concern (Q=0.001, P=0.98). YMDD mutation emergence Greater YMDD mutation emergence rates were observed for patients given combination as compared with monotherapy in the PEG group [4.0 vs. 0%, OR=18.1, 95% CI (2.4–136.6), P=0.005, n=2] and overall [4.5 vs. 0%, OR=14.8, 95% CI (2.8–77.6), P=0.001], although not in the CON group [5.9 vs. 0%, OR=9.9, 95% CI (0.5–177.1), P=0.12, n=1]. Heterogeneity was assessed and not found to be a concern (Q=0.11, P=0.74). Conventional interferon combination therapy vs. pegylated interferon monotherapy Excluding virological response [61.1 vs. 11.8%, OR=11.7, 95% CI (7.8–17.6), P<0.0001], no significant differences in rates of biochemical response [46.2 vs. 38.1%, OR=1.4, 95% CI (1.0–1.9), P=0.052], HBeAg clearance [33.5 vs. 34.9%, OR=0.9, 95% CI (0.6–1.4), P=0.77] or seroconversion [30.0 vs. 31.4%, OR=0.9, 95% CI (0.7–1.3), P=0.73] were observed between patients given combination therapy in the CON group and those given monotherapy in the PEG group. Significantly greater virological response rates were observed with monotherapy in the CON as compared with the PEG group [35.4 vs. 11.8%, OR=4.1, 95% CI (2.6–6.4), P<0.0001]. Discussion The suboptimal outcomes of current hepatitis B therapies have prompted the notion of their use in combination to achieve a synergistic effect and decreased mutagenicity (2). Furthermore, it has been suggested that the enhanced efficacy of the combination will allow for the dose reduction of its components, thus decreasing the risk of potential adverse effects (2). In our study, we explored this notion in the subset of HBeAg-positive patients. Our study is the first to examine the combination of interferon and lamivudine for chronic hepatitis B treatment, pooling data from all pertinent randomized-controlled trials into meta-analysis. This analysis will aid in achieving evidence-based conclusions on the matter, resolving the controversy in its regard and directing future investigational efforts. In our analysis, we found pegylated interferon monotherapy to be of comparable efficacy to its combination with lamivudine, providing similar rates of sustained virological and biochemical responses, and HBeAg clearance and seroconversion (P=0.66, 0.94, 0.26 and 0.34 respectively). Furthermore, while the pegylated interferon trials predominantly involved treatment-naïve patients, analysis of previously treated patients within one of those studies yielded similar outcomes (24). In contrast, the addition of lamivudine to conventional interferon resulted in superior sustained virological, biochemical and seroconversion rates (P<0.001, P=0.007, 0.01 and 0.09 respectively), similarly observed with sequential and concurrent administration (20). A similar trend was observed with HBeAg clearance rates, although the sample size was insufficient to detect this effect (P=0.09). As with pegylated interferon, treatment-naïve patients comprised the majority of the studied population and to a greater extent. Nonetheless, a controlled, nonrandomized trial of previously treated patients reported similar outcomes (25). These outcomes are corroborated by those of our histological analysis (P<0.001 and P=0.70 for histological improvement in the CON and PEG groups respectively) and by those of others (26). Importantly, our analysis provides an explanation to the discordance between the combinations' effectiveness with conventional and not with pegylated interferon, with lamivudine-induced mutagenicity suppressed with the former, while not with the latter (P=0.12, and 0.05 respectively). Accordingly, two possible regimens emerged from our analysis: pegylated interferon monotherapy, and conventional interferon and lamivudine combination therapy. A comparison between the two found them to be of comparable efficacy (P>0.05), with the exception of virological response (P<0.001). That said, it is the authors opinion that this combinations' favourable virological response should not prompt its use as the regimen of choice, as a greater portion of treatment-naïve and thus easier to treat patients, comprised the CON as compared with the PEG group, with three CON group studies exclusively examining this patient population (16, 17, 19). Our hypothesis is further supported by the superior virological outcomes of conventional as compared with pegylated interferon monotherapy (P<0.0001), which is in conflict with current knowledge (27), and is easily explained by this hypothesis. Accordingly, we suggest that in comparable populations, pegylated interferon monotherapy is likely to be similarly or more efficacious than lamivudine and conventional interferon combination therapy. More so, the thrice-weekly injection therapy required with conventional interferon poses a risk of low-patient compliance rates (1–3, 28), with the risk further exacerbated by the addition of a second agent. The weekly administration of pegylated interferon monotherapy is likely to alleviate this concern, while carrying similar economic costs (29). Consequently, we conclude that pegylated interferon monotherapy is likely to be the treatment of choice for HBeAg-positive chronic hepatitis B, with this conclusion being supported by others (27). That said, when conventional interferon therapy is considered, particularly in highly compliant patients, its combination with lamivudine should be entertained. Similarly, studies examining the HBeAg-negative hepatitis B population did not find the addition of lamivudine to pegylated (30), or conventional (31), interferon to be advantageous. In addition, while the superiority of the combination over lamivudine monotherapy was suggested in previous studies, this effect is likely to represent interferon's greater inherent efficacy as compared with lamivudine, rather than the enhanced properties of the combination, as demonstrated in those very studies (22, 30). Our study contains several limitations. Firstly, our use of intention to treat analysis, the methodological heterogeneity of the included studies, and the heterogeneity of their treatment and follow-up protocols, may have introduced some inaccuracies in our analysis. Notably, while the PEG group comprised large, carefully planned, well-executed studies, the CON group involved smaller, lower quality ones, thus weakening our conclusions in its regard. Secondly, the absence of adequate controls precluded the authors from studying the subsets of treatment-naïve and previously treated populations. Those concerns, however, were alleviated by the low patient lost for follow-up rates, the lack of statistically significant heterogeneity across studies, the beneficial effects of the combination with conventional interferon across the measured indicators and the agreement between our conclusions and those of other studies. While the focus of our study was lamivudine and interferon combination therapy, a plethora of other combinations have been explored as well. Among those studied were combinations of interferon and various antiviral agents (32, 33), interleukin-12 (34) and prednisone (35). All yielded disappointing results. Additionally, studies investigating various antiviral combinations resulted in conflicting outcomes (36–40). Those results indicate the need for further study, as the goal of a safe and efficacious therapy is yet to be attained. Conclusion Pegylated interferon-α monotherapy is the treatment of choice for HBeAg-positive chronic hepatitis B, with no added benefit with lamivudine addition. However, when conventional interferon therapy is considered, its combination with lamivudine should be entertained.

J_Behav_Med-4-1-2413087

Parental problem drinking, parenting, and adolescent alcohol use

The present study examined whether parental problem drinking affected parenting (i.e., behavioral control, support, rule-setting, alcohol-specific behavioral control), and whether parental problem drinking and parenting affected subsequent adolescent alcohol use over time. A total of 428 families, consisting of both parents and two adolescents (mean age 13.4 and 15.2 years at Time 1) participated in a three-wave longitudinal study with annual waves. A series of path analyses were conducted using a structural equation modeling program (Mplus). Results demonstrated that, unexpectedly, parental problem drinking was in general not associated with parenting. For the younger adolescents, higher levels of both parenting and parental problem drinking were related to lower engagement in drinking over time. This implies that shared environment factors (parenting and modeling effects) influence the development of alcohol use in young adolescents. When adolescents grow older, and move out of the initiation phase, their drinking behavior may be more affected by other factors, such as genetic susceptibility, and peer drinking. Introduction Time trends in Dutch epidemiological research show a significant increase in frequency and intensity of alcohol consumption among 12–15 year olds (Poelen et al. 2005). Alarming high numbers of Dutch adolescents (75%) also report problem drinking behaviors such as binge drinking (consuming more than 5 amounts of alcohol on one occasion), when compared to their American counterparts (19%; Newes-Adeyi et al. 2005; Van Dorsselaer et al. 2007). In addition, previous studies show that high levels of alcohol-related problems such as social consequences of alcohol use (e.g., family problems) and dependence symptoms (e.g., loss of control) occur frequently in Western societies, with approximately 10% of both American and Dutch populations reporting 3 or more alcohol-related problems (Cornel et al. 1994; NIAAA 1997; Van Dijck and Knibbe 2005; Wallitzer and Connors 1999). Parental problem drinking increases risk for alcohol use in children (e.g., Chassin et al. 1996; Hawkins et al. 1992; Sher et al. 1991). Children of alcoholics are not only at a higher risk for early alcohol initiation (Hill et al. 2000), they also show a greater increase in alcohol consumption over time than adolescents without alcoholic parents (Chassin and Barrera 1993). In addition, children with a family history of alcoholism show more escalation of alcohol use (Lieb et al. 2002), and more often develop alcohol disorders and dependence (Hill et al. 2000) than children without a family history of alcoholic parents. In an attempt to explain these associations, social theorists suggested a modeling effect (Bandura 1977) that causes youngsters to imitate their parents. Others have proposed that parental substance abuse may impair parenting (Sher 1991; Van der Vorst et al. 2006; Van Zundert et al. 2006), which subsequently may affect adolescent alcohol consumption. As Mayes and Truman (2002) pointed out, personality characteristics, disabilities, or impairments accompanying an addiction may affect the ability to raise a child. In addition, substance use alters the state of consciousness, memory, affect, and impulse control, each of which may impair the adult’s parenting capacities. Indeed, empirical studies have shown that children of alcoholic parents receive less discipline (King and Chassin 2004) and less emotional support from their parents (Rutherford et al. 1997). In addition, Chassin et al. (1993) found that parental alcoholism decreased the amount of parental monitoring. This is all the more problematic, since discipline and rule setting, in turn, reduce the likelihood of youngsters’ drunkenness (Engels and Van der Vorst 2003), and more parental monitoring is related to less heavy drinking in adolescents (Kerr and Stattin 2000; Van der Vorst et al. 2006). In addition, parental support appears to prevent early onset of alcohol use, as well as frequent and heavy alcohol use among adolescents (Barnes et al. 1994). Thus, numerous cross-sectional studies have demonstrated associations between parental alcohol use, parenting, and adolescent alcohol consumption (e.g., Chassin et al. 1993; Kerr and Stattin 2000). However, with the exception of two prospective studies that showed that monitoring by fathers and parental discipline mediated between parental alcoholism and adolescents’ alcohol use (Chassin et al. 1996; King and Chassin 2004), longitudinal studies are lacking. In addition, to allow generalization of findings and to examine effects in potentially less severe cases, it is necessary to investigate community-based samples (Russell et al. 1990). Accordingly, the central aim of the present study was to longitudinally examine the nature of the relations between parental problem drinking, parenting, and adolescent alcohol use in a three-wave community-based sample. Alcohol-specific parenting Although studies on parenting and adolescent alcohol use have been informative, two important issues have hardly been addressed. First, most studies on the link between parenting and adolescent alcohol use have focused on general parenting. However, alcohol-specific socialization, which refers to the actions parents undertake to discourage or prevent their offspring from drinking (Jackson et al. 1999; Van der Vorst et al. 2005), has received less attention in relation to parental drinking and adolescent alcohol use. Wood et al. (2004) found that late adolescents drank less alcohol when their parents disapproved of drinking. In addition, imposing strict rules prevented youngsters from heavy drinking (Jackson et al. 1999; Van der Vorst et al. 2005; Yu 2003). However, whether parental problem drinking affects alcohol-specific parenting has not yet been examined. From studies on smoking we know that parents who smoke are less frequently engaged in anti-smoking socialization practices than parents who do not smoke (Harakeh et al. 2005). A similar process might be at work regarding alcohol-specific socialization, suggesting that parents with alcohol problems may engage less frequently in alcohol-specific socialization, and as such provide fewer alcohol-specific rules, are more permissive towards alcohol use and exert less alcohol-specific control. Second, it is crucial to acknowledge that the association between parenting and adolescent problem behavior may be bidirectional: Parents do not only influence their children, but children’s behavior also exerts an effect on parents. Indeed, recent longitudinal studies showed a bidirectional relation between parenting and adolescent substance use. Adolescent drinking, smoking or deviant behavior decreased the level of parental monitoring and rule setting (Huver et al. 2006; Stice and Barrera 1995; Van der Vorst et al. 2006). This implies that when these child effects are not taken into account, this may lead to an overestimation of parental influences (Kerr and Stattin 2003; Van der Vorst et al. 2006). Current study and expectancies We longitudinally investigated the direct effect of parental problem drinking on adolescent alcohol use, the role of alcohol-specific and general parenting practices in this relationship, and the reciprocal effects of adolescent alcohol use on parenting (see Fig. 1). It was expected that parental problem drinking would have a direct positive effect on adolescent alcohol use, with more parental alcohol-related problems leading to more adolescent alcohol use. In addition, an indirect relationship was expected via parenting; more specifically, higher levels of parental problem drinking were thought to have a negative effect on both general and alcohol-specific parenting practices, which in turn would lead to more adolescent alcohol use. Moreover, the drinking behaviors of the adolescents were expected to influence parenting, with more adolescent drinking resulting in less parental discipline and monitoring. Fig 1Longitudinal Model of Parental Problem Drinking, Parenting, and Adolescent Alcohol Use Methods Participants and recruitment The data were derived from an ongoing Dutch longitudinal survey called ‘Family and Health’, which examines different socialization processes in relation to various health behaviors in adolescence (see Harakeh et al. 2005; Van der Vorst et al. 2005). A total of 428 Dutch families, consisting of mother, father, and two adolescent children, participated in our study in the first wave (2002–2003). Families were included when the parents were married or living together, and when all family members were biologically related. Families with twins, or with mentally or physically disabled offspring were excluded. Numbers of drop-outs were extremely low in the second (2003–2004) and third wave (2004–2005), with 416 (97%) and 404 (94%) participating families, respectively. The majority of the families were of Dutch origin (>95%). The mean age of the participants at Time 1 was 15.2 years (SD = .60) for the older adolescents, 13.4 years (SD = .50) for the younger adolescents, 46.2 years (SD = 4.00) for the fathers, and 43.8 years (SD = 3.57) for the mothers. Of the older adolescents, 47% were girls, compared to 52% in the younger group. Concerning educational level, an equal distribution was realized, with about one third of the adolescents following low education, one third following intermediate general education, and one third following the highest level of secondary school. The different levels of the Dutch secondary school system are comparable with the different tracks within a middle class public high school in the USA, although they may not be completely interchangeable. In our sample, when compared to national Dutch figures, the intermediate general education is slightly underrepresented, while the low and high levels are slightly overrepresented (CBS 2007). Procedure The families were visited at home by a trained interviewer. In his or her presence all four family members individually filled out an extensive questionnaire, which took about 2 h to complete. The participants were not allowed to consult each other or to discuss the answers. When all family members had completed the questionnaire, each family received 30 € (39 $). In addition, after completion of the first three waves of the project, 5 traveler cheques of 1,000 € (1,300 $) each were raffled among all participating families. Approval was obtained from the Central Committee on Research Involving Human Subjects on collecting the data. Measure Self-reports were used to measure parental problem drinking and adolescent alcohol use. The four parenting practices were based on adolescents’ reports, reflecting how they perceived their parents’ behaviors. The questions regarding the parenting variables were asked in such a way that the adolescents were able to discriminate between the parenting practices of their mothers and fathers. Problem drinking To measure the severity of fathers’ and mothers’ alcohol-related problems, both parents completed the problem drinking list of Cornel et al. (1994). The original scale was based on three commonly used instruments to measure problem drinking: CAGE (Cut down, Annoyed, Guilty, Eye-opener, CAGE is an acronym formed by taking the first letter of key words from each of the following questions; Mayfield et al. 1974), Short Michigan Alcohol Screening Test (SMAST; Selzer et al. 1975), and a shortened version of the Self-Administered Alcohol Screening Test (SAAST; Davis et al. 1987). Seriousness of problem drinking was developed as a Rasch scale with items arranged in order of increasing severity. The more severe the items, the less frequently they are scored positively. Since all requirements of the Rasch model were met, the items form a reliable and unidimensional scale (Cornel et al. 1994). Examples of items were ‘Do you ever drink alcohol to forget your concerns?’ (item 2) and ‘Have you ever lost your job because of your drinking’ (item 18). Respondents could respond 0 ‘no’, or 1, ‘yes’. Severity of problem drinking was reflected by the aggregated score with a maximum score of 18. Because of the skewness of the summed variable’s distribution, scores were categorized into 3 meaningful groups: 1 = never had problems due to alcohol; 2 = has had problems due to alcohol a couple of times, 3 = problem drinkers (see Cornel et al. 1994). General parenting To measure parental behavioral control, we used a Dutch translation of the scale developed by Kerr and Stattin (2000). The scale consisted of 5 items with response categories ranging from 1 ‘no, never’ to 5 ‘yes, always’. Examples of items were: ‘Do you need to have your mother’s permission to stay out late on a weekday evening?’ and ‘Before you go out on a Saturday night, does your father require you to tell them where you are going and with whom?’. Internal consistencies as measured with Cronbach’s alphas ranged from .71 to .90 for the reports of both adolescents, about their mothers and fathers over the three waves. To measure parental support, we used the Relationship Support Inventory (RSI; Scholte et al. 2001) tapping several aspects of emotional and instrumental support. Examples of items were ‘My mother shows me that she loves me’ and ‘My father supports me in what I do’. The adolescents had to answer 12 items on a scale from 1 ‘absolutely untrue’ to 5 ‘absolutely true’. The amount of support was the mean score on 12 items. Cronbach’s alpha coefficients were between .76 and .88 across the three waves. Alcohol-specific parenting Van der Vorst et al. (2005) developed a 10-item scale to measure the degree to which parents permit their children to consume alcohol. Examples of items were: ‘I am allowed to drink alcoholic consumptions when my mother/father is at home’ and ‘I am allowed to drink alcohol on weekdays’. Participants had to respond on a 5-point scale that ranged from 1 ‘completely applicable’ to 5 ‘not applicable at all’. The internal consistency was high, with Cronbach’s alphas between .89 and .92 over the three waves. In addition, the general behavioral control scale of Kerr and Stattin (2000) was adapted to measure behavioral control aimed at affecting adolescents’ alcohol consumption. Examples of the 5 items were ‘Do you need your mother’s permission to drink alcohol on weekdays?’ and ‘Does your mother want to know whether your friends drink alcohol?’. As in the original scale, the response categories ranged from 1 ‘never’ to 5 ‘always’. Cronbach’s alpha coefficients were between .74 and .88 across the three waves. Adolescent alcohol use Intensity of drinking was assessed by questions that asked about the number of glasses consumed in the previous week, during weekdays and weekends, both outside and inside the house (Engels et al. 1999). The aggregated score on these four questions was used as an indication of the adolescents’ intensity of alcohol use (Van der Vorst et al. 2005). Because of the skewness in the distribution of this variable, total scores were categorized into 7 groups (0 = 0 glasses, 1 = 1–2 glasses, 2 = 3–5 glasses, 3 = 6–10 glasses, 4 = 11–20 glasses, 5 = 21–30 glasses, 6 = 31 glasses and above). Strategy of analyses For the descriptive part of the analyses we applied t tests, Pearson correlations and general linear modeling with repeated measures (the latter to test changes over time in alcohol-related problems and alcohol use). We performed cross-lagged path analyses (see Fig. 1), using version 4.1 of the Mplus statistical package (Muthén and Muthén 1998–2006), to test (a) to which degree parental alcohol-related problems, parental practices and alcohol use of adolescents were stable over time, (b) whether parental problem drinking was related to parental practices and alcohol use of the adolescent over time and (c) whether parental practices and alcohol use of the adolescent were cross-related over time (Finkel 1995). Cross relations over time allow to test causal predominance: Are specific parenting practices the ‘cause’ of adolescent alcohol use, or does adolescent alcohol use provoke specific parenting practices (Byrne 1998)? Structural regression models are generally somewhat more sophisticated than the path models used in our study because they correct for measurement error (Kline 1998, p. 211). This controlling for error variance by means of latent variables that are measured by multiple manifest indicators plus their error variance, however, also increases the number of parameters to be estimated. In addition, more complex models, i.e., models with more parameters, require larger sample sizes than do more parsimonious models in order for the estimates to be comparably stable (Kline 1998, p. 111). Kline (1998) recommends a parameter—subjects ratio of 1:10. As such, we used path models in which one manifest parameter represented all the individual items of one scale by means of the mean or sum score. The model depicted in Fig. 1 was tested for each of the four parenting variables separately. A total of 4 (parenting variables) × 2 (fathers and mothers) = 8 models were tested. The variables at T1 and the disturbance terms of the variables at T2 and T3 were free to correlate. Because adolescent alcohol consumption and parental problem drinking were relatively skewed and the measurement level was ordered more categorical (ordinal) than interval, maximum likelihood estimation methods (demanding multivariate normal distributed variables) were less suited. We used the weighted least square method with adjusted mean- and variance chi-square (WLSMV) estimator, an estimation method specifically developed for ordered categorical dependent variables (Muthén and Muthén 1998–2006). To test model fit, standard chi-square tests as well as the number of degrees of freedom (df) were replaced by robust chi-square tests (mean- and variance-adjusted chi-squares) and estimates of df (Muthén and Muthén 1998–2004, pp. 19–20). The latter estimates are dependent on sample information and this explains why df with identical models can vary across different groups. Together with the robust chi-square tests we used two fit measures: the Root Mean Square Error of Approximation (RMSEA; Byrne 1998; Steiger and Lind 1980), and the Comparative Fit Index (CFI) of Bentler (Bentler 1990). RMSEA is utilized to assess approximate fit preferably with values less than or equal to .05, but values between .05 and .08 are indicative of fair fit (Browne and Cudeck 1993). CFI is a comparative fit index, values above .95 are preferred (Kaplan 2000), but should not be lower than .90 (Kline 1998, see also Hu and Bentler 1999, and, for commentary on existing guidelines Marsh et al. 2004). Mplus has several possibilities to handle missing values depending on the estimation method used. In our case (using the WLSMV-estimator) all available information in the data was used by means of pair-wise information of each combination of two variables. Results Descriptives on alcohol consumption and problem drinking Table 1 shows the means, standard deviations, and percentages of parental problem drinking. Fathers reported higher levels of problem drinking than mothers at all three waves, as was tested with separate t tests. (T1: t(424) = 8.11, p < .001; T2: t(426) = 8.13, p < .001; T3: t(424) = 7.93, p < .001). With general linear modeling repeated measures we tested whether maternal and paternal problem drinking differed over time (within factor). Both paternal and maternal problem drinking showed significant differences over time (for fathers: F(2, 421) = 23.18, p < .001, partial eta squared (PES) = .10, and for mothers: F(2, 424) = 59.10, p < .001, and PES = .22).1 Subsequently carried out repeated contrasts revealed significant differences over time only from T1 to T2, for both parents (p < .001), but not from T2 to T3. At T1, the older adolescents consumed on average 4.36 glasses in the past week (SD = 6.81; T2: M = 7.78, SD = 10.86; T3: M = 9.75, SD = 12.35), compared to 1.23 glasses (SD = 3.41) consumed in the past week by the younger siblings (T2: M = 3.70, SD = 8.99; T3: M = 6.22, SD = 10.32). Older adolescents reported significantly higher levels of alcohol consumption than younger adolescents at all three waves (T1: t(417) = 9.30, p < .001; T2: t(414) = 6.85, p < .001; T3: t(405) = 5.09, p < .001). Repeated measures showed a significant increase in alcohol consumption over time for both adolescents, for T1–T2, and T2–T3 (for older adolescents: F(2, 394) = 34.15, p < .001, PES = .15, and for younger adolescents: F(2, 414) = 42.64, p < .001, PES = .17). Table 1Means, standard deviations and percentages of parental problem drinking (PD) and adolescent alcohol use (A) at Time 1 (T1), Time 2 (T2), and Time 3 (T3)T1T2T3M*SD%**M*SD%**M*SD%**PD father1.84a2.1819.42.33b1.9925.52.21b2.0022.7PD mother.87a1.575.61.44b1.518.41.42b1.49 9.1A younger adolescent1.22a3.41n.a.3.11b8.35n.a.5.27c9.76n.a.A older adolescent4.37a6.80n.a.7.15b10.62n.a.8.79c12.08n.a.Note: *M represents the mean score calculated from the aggregated scores of all 18 items (maximum score = 18) of which the problem drinking scale consists (Cornel and Knibbe 1994). **Percentages of problem drinkers are computed with a cut-off score > 3. n.a. = not applicable. Values for adolescent alcohol use (A) represent the intensity of alcohol use, i.e., the number of glasses of alcohol consumed in the past week. Fathers had significantly more alcohol-related problems than mothers at all three time points, with p < .01. Older adolescents reported significantly more alcohol than younger adolescents at all three time points. Means in the same row that do not share superscripts (a, b, c) are significantly different (p < .001) Correlations between cross-sectional and longitudinal variables Maternal and paternal problem drinking correlated positively, but marginally with adolescents’ alcohol consumption (.02 ≤ r ≤ .19). Parental problem drinking correlated negatively with support (−.18 ≤ r ≤ −.02), and alcohol-specific behavioral control (−.22 ≤ r ≤ −.01), and positively with permissiveness (.03 ≤ r ≤ .24), while both positive and negative correlations were found between parental problem drinking and general behavioral control (−.13 ≤ r ≤ .15). Adolescent alcohol use correlated low to moderately with general behavioral control (−.24 ≤ r ≤ −.05), support (−.15 ≤ r ≤ −.02) and alcohol-specific behavioral control (−.29 ≤ r ≤ −.03), and positively with permissiveness (.18 ≤ r ≤ .46). General behavioral control correlated positively with support (.11 ≤ r ≤ .40) and negatively with permissiveness (−.29 ≤ r ≤ −.02). Moderate correlations existed between the general behavioral control scale and the alcohol-specific behavioral control scale (.19 ≤ r ≤ .55), indicating that they share the same basis, but can be seen as separate constructs. Correlation tables are available upon request. Structural equation models All models showed an acceptable fit (Table 2), with all Comparative Fit Indices (CFI) at least above .90 and all Root Mean Square Errors of Approximation (RMSEAs) below .08. Table 2Fit indices for all modelsFatherMotherPMASBCSUPMASBCSUdf2630302627302928χ²36.1358.7376.0541.7847.9256.8078.4942.95p0.090.000.000.030.010.000.000.04CFI0.980.960.930.980.960.960.920.98RMSEA0.030.050.060.040.040.050.060.04Note: PM = Permissiveness, AS = Alcohol-specific behavioral control, BC = Behavioral control, SU = Support. Each column represents one model with a specific parenting variable, separately for mothers and fathers Alcohol-specific parenting: permissiveness and alcohol-specific behavioral control Standardized regression weights (β) of parental problem drinking, permissiveness, alcohol-specific behavioral control, and adolescent alcohol consumption showed a strong stability over time, with values between .36 and .88 (see Table 3). Table 3Structural parameters estimates of the alcohol-specific parenting practices, parental problem drinking (PD) and adolescent alcohol use (standardized beta weight)PermissivenessAlcohol-specific behavioral controlFathersMothersFathersMothersStability paths1. PD T1–PD T2.79.76.80.762. PD T2–PD T3.82.88.82.823. Parenting OA T1–Parenting OA T2 .68.67.55.554. Parenting OA T2–Parenting OA T3.73.73.64.645. Alcohol use OA T1–Alcohol use OA T2.46.47.55.546. Alcohol use OA T2–Alcohol OA T3.66.66.67.677. Parenting YA T1–Parenting YA T2.75.76.50.498. Parenting YA T2–Parenting YA T3 .76.76.49.489. Alcohol use YA T1–Alcohol use YA T2.36.37.42.4210. Alcohol use YA T2–Alcohol use YA T3.55.54.59.57Paths from PD11. PD T1–Parenting OA T2.11**.12**.07−.0812. PD T2–Parenting OA T3.05.03−.11**−.0713. PD T1–Alcohol use OA T2.08.16**.09.18***14. PD T2–Alcohol use OA T3.02.02.03.0315. PD T1–Parenting YA T2.07.03.05−.0216. PD T2–Parenting YA T3.05.04−.14**−.15**17. PD T1–Alcohol use YA T2.00−.02.00−.0318. PD T2–Alcohol use YA T3.19***.14**.20***.15**Cross-lagged paths19. Parenting OA T1–Alcohol use OA T2.15**.12*.05.0420. Parenting OA T2–Alcohol use OA T3.02.03−.02−.0721. Alcohol use OA T1–Parenting OA T2−.01.01−.07−.0522. Alcohol use OA T2–Parenting OA T3−.04−.04−.10*−.0923. Parenting YA T1–Alcohol use YA T2.17**.17**−.23***−.24***24. Parenting YA T2–Alcohol use YA T3.04.05.05−.0325. Alcohol use YA T1–Parenting YA T2.01.02−.02−.10*26. Alcohol use YA T2–Parenting YA T3.01.01−.12*−.10*27. Alcohol use OA T1–Alcohol use YA T2.08.09.09.0928. Alcohol use OA T2–Alcohol use YA T3.11*.12*.09.11*Note: PD = Parental Problem Drinking, OA = Older Adolescent, YA = Younger Adolescent. All stability paths are significant at p < .001. The numbered paths in the table correspond to the arrowed paths depicted in Fig. 1* p < .05, ** p < .01, *** p < .001 Regarding both the older and younger adolescents, no significant associations were found between parental problem drinking and parental permissiveness, with the exception of problem drinking of both fathers and mothers at T1 which was significantly and positively related to permissiveness towards the older adolescent at T2 (for fathers: β = .11, p < .01, for mothers: β = .12, p < .01). More problem drinking of the mother at T1 and T2 was significantly associated with more alcohol use of the older adolescent at T2 and drinking of the younger adolescent at T3, respectively (β = .16, p < .01; β = .14, p < .01). Problem drinking of the father at T2 was significantly and positively associated to alcohol use of the youngest adolescent at T3 (β = .19, p < .001). More parental permissiveness at T1 led to more alcohol use at T2 in both younger and older adolescents (.12 ≤ β ≤ .17, p < .05). This significant relation was not found between T2 and T3. We did not find that parents adapt their levels of permissiveness in response to adolescent alcohol use. Paths from parental problem drinking at T2 on alcohol-specific behavioral control at T3 were significant for the father regarding both the older and younger adolescents (respectively β = −.11 and β = −.14, p < .01), and for the mother regarding the younger adolescent (β = −.15, p < .01). Problem drinking of the father at T2 related substantially to alcohol use of the younger adolescent at T3 (β = .20, p < .001). Problem drinking of the mothers affected both the older (β = .18, p < .001) and the younger adolescents’ alcohol consumption (β = .15, p < .01). For the younger, but not for the older adolescents, more alcohol-specific behavioral control at T1 was related to lower levels of adolescent alcohol use at T2 (β = −.23, p < .001 for fathers, β = −.24, p < .001 for mothers). Adolescent alcohol use negatively affected alcohol-specific behavioral control of both parents (.10 ≤ β ≤ .12, p < .05) General parenting: behavioral control and support The standardized regression weights of parental problem drinking, support, behavioral control, and adolescent alcohol consumption showed a strong stability over time, with values between .42 and .85 (see Table 4). Table 4Structural parameters estimates of general parenting practices, parental problem drinking (PD) and adolescent alcohol use (standardized beta weight)Behavioral controlSupportFathersMothersFathersMothersStability paths1. PD T1–PD T2.80.73.81.772. PD T2–PD T3.83.82.85.803. Parenting OA T1–Parenting OA T2 .61.64.76.734. Parenting OA T2–Parenting OA T3.70.70.76.725. Alcohol use OA T1–Alcohol use OA T2.54.53.53.536. Alcohol use OA T2–Alcohol OA T3.66.66.67.677. Parenting YA T1–Parenting YA T2.66.63.70.678. Parenting YA T2–Parenting YA T3 .63.73.69.709. Alcohol use YA T1–Alcohol use YA T2.43.42.43.4210. Alcohol use YA T2–Alcohol use YA T3.58.57.59.57Paths from PD11. PD T1–Parenting OA T2.03−.06.00.0712. PD T2–Parenting OA T3−.05−.04−.06−.0113. PD T1–Alcohol use OA T2.09.18***.10.18***14. PD T2–Alcohol use OA T3.03.00.03.0215. PD T1–Parenting YA T2.06.00.08.0316. PD T2–Parenting YA T3−.04−.12**−.03−.0617. PD T1–Alcohol use YA T2.00.00.00−.0118. PD T2–Alcohol use YA T3.20***.15**.20***.14**Cross-lagged paths19. Parenting OA T1–Alcohol use OA T2.04.03−.04−.0720. Parenting OA T2–Alcohol use OA T3−.11*−.18***−.02−.0221. Alcohol use OA T1–Parenting OA T2−.07−.05−.01−.0222. Alcohol use OA T2–Parenting OA T3−.12**−.14**−.06.0123. Parenting YA T1–Alcohol use YA T2−.15**−.14**−.10*−.15**24. Parenting YA T2–Alcohol use YA T3−.05−.08.07−.0125. Alcohol use YA T1–Parenting YA T2−.04−.09*−.11**−.08*26. Alcohol use YA T2–Parenting YA T3−.05−.07.02−.0227. Alcohol use OA T1–Alcohol use YA T2.10*.09.09.1028. Alcohol use OA T2–Alcohol use YA T3.10*.10*.11*.11*Note: PD = Parental Problem Drinking, OA = Older Adolescent, YA = Younger Adolescent. All stability paths are significant at p < .001. The numbered paths in the table correspond to the arrowed paths depicted in Fig. 1* p < .05, ** p < .01, *** p < .001 Regarding both the older and younger adolescents, paths of both maternal and paternal problem drinking with behavioral control were generally not significant. Only maternal problem drinking at T2 showed a significant association with behavioral control towards the youngest adolescent at T3 (β = −.12, p < .01). Problem drinking of the mother at T1 and T2 was significantly associated with alcohol use of the older adolescent at T2 and drinking of the younger adolescent at T3, respectively (β = .18, p < .001, β = .15, p < .01). Problem drinking of the father at T2 related substantially to alcohol use of the younger adolescent at T3 (β = .20, p < .001). Considering the cross-lagged paths, more parental behavioral control at T2 was associated with less alcohol consumption of the older adolescents at T3 (β = −.11, p < .05 for fathers, β = −.18, p < .001 for mothers), but not from T1 to T2. Younger adolescents also consumed less alcohol at T2 when their parents exerted more behavioral control at T1 (β = −.15, p < .01 for fathers, β = −.14, p < .01 for mothers). In addition, older adolescents’ drinking at T2 negatively affected parental behavioral control at T3 (β = −.12, p < .01 for fathers, β = −.14, p < .01 for mothers), indicating that when older adolescents drank more, parents exerted less general behavioral control. This result was not found for the younger adolescent. No significant associations were found between parental problem drinking and the support parents provide to both the younger and older adolescent. Paternal problem drinking at T2 directly affected alcohol use of the younger adolescent at T3 (β = .20, p < .001). Mothers’ problem drinking at T1 and T2 affected alcohol use of the older adolescent at T2, and drinking of the younger adolescent at T3, respectively (β = .18, p < .001; β = .14, p < .01). More parental support at T1 was related to less alcohol use of the younger adolescents at T2 (β = −.10, p < .05 for fathers, β = −.15, p < .01 for mothers). These associations were not found between T2 and T3, nor for the older adolescents. In addition, more alcohol use of the older adolescents at T1 was associated with less parental support at T2 (β = −.11, p < .01 for father, β = −.08, p < .05 for mothers). Additional analyses We also tested whether older adolescents influenced their younger siblings in drinking behavior. Results showed that alcohol consumption of the older adolescents tended to directly affect alcohol use of the younger adolescent (.10 ≤ β ≤ .12, p < .05). Discussion The aim of the present study was to gain insight into the associations between parental problem drinking, parenting, and adolescent alcohol use in a sample of Dutch families. The first main finding shows that, except for alcohol-specific behavioral control, parental problem drinking does not structurally affect parenting over time. More alcohol-related problems did not result in less behavioral control, less general support, or higher permissiveness. Our results differ from those of other studies in which significant relations between parental problem drinking and parenting were found (Chassin et al. 1993; King and Chassin 2004; Rutherford et al. 1997). These differences could be due to methodological issues, as problem drinking or alcoholism in the latter studies were often diagnosed in conformity with the DSM-IV criteria, whereas we concentrated on a broader range of alcohol-related problems in a community sample. Thus, because of our assessment of problem drinking,2 instead of alcohol dependence or abuse, and because of our focus on a community sample instead of a clinical sample, the present study reflects the situation in the general population, and as such enhances the generalizability of the findings. Another explanation for the non-significant relation between parental problem drinking and general parenting comes from the buffering hypothesis, which states that “support protects persons from the potentially pathogenic influence of stressful events” (Cohen and Wills 1985, p. 310). Accordingly, children of one problem-drinking parent may be protected from the parent’s inadequate parenting by the support and adequate parenting of the other (non-problem drinking) parent. A buffering mechanism has been reported in the literature. For example, Van Aken and Asendorpf (1997) found that low support from one parent could be compensated by support from the other parent in affecting adolescent self-esteem. In addition, peer friendships, positive peer relations and family cohesion have each shown to be a protective factor against children’s externalizing problems in family conflict situations (Criss et al. 2002; Farrell et al. 1995). With respect to problem drinking, the possibility of enhancing resiliency in children and adolescents, by protecting against possible harmful influences from one parent by a strong relationship with the other parent, siblings, or peers, should be a topic of examination in future research. In addition, with regard to the persons in our sample, being part of a stable, nuclear family that consists of two biological parents with two or more children living together may be protective in itself. Our findings do not imply that problem drinking has no effect on personal cognition or functioning, but suggest that parents are able to regulate their problem behavior with regard to their children and parenting practices. However, parental problem drinking may affect the way in which parents handle alcohol use within the family. Parents with more alcohol-related problems are not more permissive than parents who do not have these problems. Maintaining the set rules by means of alcohol-specific behavioral control, however, does appear to be a problem. Our second main finding is that higher levels of behavioral control, support, rules, and alcohol-specific behavioral control account for less alcohol consumption in mainly the younger adolescents, which corresponds with the literature on this topic (e.g., Kerr and Stattin 2000; Van der Vorst et al. 2005, 2006). Alcohol-specific rule setting played an important role in drinking of both the younger and older adolescents. When parents were more permissive toward alcohol use, adolescents reported higher levels of drinking, which is in accordance with other studies on rule enforcement and adolescent alcohol use (Jackson et al. 1999; Van der Vorst et al. 2005; Van Zundert et al. 2006; Yu 2003). However, support and both general and alcohol-specific behavioral control were only associated with less alcohol use of the younger adolescent, up to the age of about 14 years. For older adolescents the effect of parenting disappeared, and parental problem drinking was found to directly affect adolescent alcohol use. In explaining this pattern, it might be that parenting exerts influence before and during the initiation phase of alcohol use, which in Dutch adolescents takes place around the age of 14 years (Poelen et al. 2005), but that parenting is no longer important once the habitual drinking pattern has been established (DeCourville 1995). Further, it has been suggested that genetic effects increase in importance over time during late adolescence, whereas environmental factors decrease in importance. Twin studies have indeed shown that shared environmental factors, such as parenting, play a profound role in the initiation of alcohol use, but that genetic factors are more important in frequency of alcohol use and problem drinking in young adulthood (see Hopfer et al. 2003; Pagan et al. 2006). Moreover, during adolescence, parental factors decrease in importance, whereas the influence of peers increases, making the latter a strong predictor for adolescent drinking (Fergusson et al. 1995). As such, shared environmental factors, such as parenting and parental modeling, affect alcohol consumption in young adolescence. Drinking in later adolescence may be related to other, non-shared factors, such as genes and peer drinking. Accordingly, future research should apply a longitudinal, behavioral genetic design, preferably examining the different stages of adolescent drinking (initiation, frequency of consumption, drinking to intoxication) in order to partial out the different effects of genetic and environmental factors. Our third main finding considers the reciprocal associations between adolescent alcohol use and parental behaviors. More adolescent alcohol use made parents decrease their levels of general and alcohol-specific behavioral control over time. Levels of support were also negatively adjusted in response to adolescent alcohol use, but only in the younger adolescents. Our findings concur with recent studies that also reported bidirectional findings between parenting practices and adolescent substance use (Huver et al. 2006; Stice and Barrera 1995; Van der Vorst et al. 2006). However, parents did not adjust their levels of permissiveness in response to adolescent alcohol use. Perhaps rule-setting is more stable over time, and is not affected by fluctuations in adolescent alcohol use. The application of those rules, however, by means of exerting control, was influenced by the levels of alcohol that adolescents consumed. Since our study is one of the first to examine reciprocal effects between adolescent drinking and parental factors, more research on this topic is warranted. Regarding differences between fathers and mothers, we found that maternal, but not paternal problem drinking was directly associated with alcohol use of the oldest adolescent. This is a remarkable finding, considering the fact that in most parenting studies with a focus on alcoholism or problem drinking, women are underrepresented (e.g., Chassin et al. 1993). Future research should specifically include mothers in studies regarding alcoholic or problem-drinking parents. Limitations Despite the advantages of our study, such as multi-informant data, longitudinal design and the testing of reciprocal associations in path analyses, some limitations should be addressed. First, we did not further examine relationships in subgroups (for example, sex differences) because of a lack of statistical power and, subsequently, the risk of making Type II errors. Nonetheless it should be stressed that in this type of longitudinal study with a full-family design, the sample size was substantial and the low attrition rates over the three waves were remarkable. Second, parental factors explained only a small part of the variance in adolescent drinking. However, finding small effects does not imply that parental factors are unimportant. The finding that parenting influences adolescent alcohol consumption can have large practical implications (see Abelson 1985). Third, parents may have under-reported their alcohol-related problems because of social desirability and adolescents may have under-reported their alcohol use because of the presence of their parents at home while filling in the questionnaires. In an attempt to anticipate these biases, and to ensure confidentiality, the questionnaires had to be completed individually and separately, without the possibility for family members to discuss the answers. In addition, studies have shown that self-reports concerning alcohol use are a reliable source of information (Engels et al. 2007). Fourth, although the sample was carefully selected, the results cannot be generalized to the whole Dutch population, because of the lack of for example, single-parent families and step-families. Fifth, in the Netherlands the legal age to drink beer and wine is 16, and the legal age to drink liquor is 18. This may make it difficult to compare previous research from the USA, where the legal age to drink is 21, with our results. Sixth, it might be that the initial measurements triggered follow-up discussions about alcohol use within families, which might have acted as an intervention. However, since our study is longitudinal and ongoing, we did not want to encourage any speculations or thoughts on the content of the questionnaires which might affect the following measurements, and as such we did not consult the families on these matters. Seventh, in our questionnaires, the definition of “glasses” of alcohol was left up to the respondents to interpret. This may have lead to a reporter bias in the exact amount of alcohol consumed because of different (non-standard) glass sizes. However, measurement of the precise amount of alcohol consumed is rather difficult to realize in our current study design. Experimental designs or diary studies will be able to more accurately measure and control the precise quantity of consumed alcohol. See Kerr et al. (2005) for an elaborated discussion on this topic. Taking these limitations into account, this study is the first to disentangle the prospective relations between parental alcohol-related problems, parenting, and adolescent alcohol use in a community sample using multi-informant data. The results show that parental problem drinking does not substantially and systematically affect parenting, and that parenting influences adolescent alcohol use, but only up to the age of about 14 years. This implies that shared environment factors (e.g., parenting and modeling effects) influence the development of alcohol use in young adolescents. When adolescents grow older, and move out of the initiation phase, their drinking behavior may be more affected by other factors, such as genetic susceptibility and peer drinking.

Bioinformation-2-4-2255066

Database of neurodegenerative disorders

A neurological disorder is a disorder caused by the deterioration of certain nerve cells called neurons. Changes in these cells cause them to function abnormally, eventually bringing about their death. In this paper we present a comprehensive database for neurodegenerative diseases, a first-of-its kind covering all known or suspected genes, proteins, pathways related to neurodegenerative diseases. This dynamically compiled database allows researchers to link neurological disorders to the candidate genes & proteins. It serves as a tool to navigate potential gene-protein-pathway relationships in the context of neurodegenerative diseases. The neurodegenerative disorder database covers more then 100 disease concepts including synonyms and research topics. The current version of the database provides links to 728 abstracts and over 203 unique genes/proteins with 137 drugs. Also it is integrated well with other related databases. The aim of this database is to provide the researcher with a quick overview of potential links between genes and proteins with related neurodegenerative diseases. Thus DND providing a user-friendly interface is designed as a source to enhance research on neurodegenerative disorders. Background Neurodegenerative disease leads to neurodegeneration and disabilities as a result of deterioration of neurons. [1] Progressive loss of motor, sensory neurons and the ability of the mind to refer sensory information to an external object is affected in different kinds of neurological disorders. [2] Mutations in the genes identified for these disorders leads to accumulation of misfolded protein resulting in protein aggregation and intracellular inclusions. [3] The knowledge gained from the genetic studies of many neurodegenerative disorders explains the disease mechanism that includes few pathways leading to neuronal death of cells. [4] Amyloid precursor proteins (APP), SNCA, Parkin, UCHL1, NR4A2, DJ1, PINK1 and LRRK2 are the genetic causes of some of the primary neurodegenerative disorders like Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and prion diseases. [5] The treatment strategies for these disorders include therapies and drugs. Because of the complexity involved in the neurodegenerative disease mechanism and treatment strategies, it is of interest to collate different Neurodegenerative Disorders in the form of a database. Hence we developed DND (Database of Neurodegenerative Disorders), an on-line web based database that contains more than 100 neuro related disease concepts and provides with a covering of all related genes, proteins, pathways and drug information. Methodology Database model and content Database of Neurodegenerative Disorders (DND) is developed as an open source software system using Mysql - 5.0.18 - Win32 [6] and PHP - 5.2.0. [7] DND uses the relational data model. A schema diagram describing the DND is shown in the [figure 1]. MySQL tables are shown as rectangles. Mandatory attributes are in bold, optional are in italics. The relationships between tables in the database are shown as connecting lines. The public databases NCBI, SwissProt, Kegg and DrugBank are used for populating the database. The gene sequences related to various neurological disorders are obtained from NCBI. Both gene sequence and coding sequence can be obtained in Fasta format. Also HGNC links for each entry is provided. All protein sequences are extracted from UniProt, and fields that mapped to PDB, Pfam, Interpro, Prints and Smart are parsed and stored. Links from DND to these source databases are also provided for each entry. Fasta format of protein sequences too can be obtained. Pathway information for Neurodegenerative diseases as well as known neuro related drug compounds in KEGG is searched and the results obtained are incorporated in to the database. The articles are chosen to represent a diverse selection of reports on major Neurodegenerative diseases and corresponding Pubmed ID is provided for all the entries. Neurological Associations and Organizations interested in neuroscience-related fields are tabulated with links to their corresponding web pages. Searching DND There are three ways by which the user can query the database. The first one is the keyword search in home page that can be performed by giving keywords like Disease name, Gene name and Drug name to retrieve the required data. The second one is the advanced search option for specific requirements with two main entry points namely gene/protein and drug/drug properties. Gene/protein include search options like Gene name, Gene id, Omim, Hgnc, Pubmed, Swissprot id, Pdb, Interpro, Pfam, Smart and Prints. The second entry point Drug/drug properties include Drug name, Kegg id, DrugBank id, Pubchem, and IUPAC name and Molecular formula. Alternatively, the users can also browse the database via the seven entry points namely browse by Diseases, Genes, Proteins, Drugs, Pathways, Related Articles and Organizations. Utility The current version of the DND is comprehensive with enormous data related to every aspect of neurological disorders, providing public access to sequence, genetics, structural, and bibliographic information. Structures of drugs can be viewed using the browser plug in Chime [8] that allows chemical structures to rotate, reformat, and save in various file formats. A glossary describing the terms used in database is also provided to help the end users. We believe that DND will assist the intended neurological researchers in understanding of fundamental molecular and genetic processes that control various neurodegenerative diseases.

Pediatr_Nephrol-3-1-1805050

Ambulatory blood pressure monitoring and renal functions in children with a solitary kidney

The aim of this study is to investigate the blood pressure (BP) profile, microalbuminuria, renal functions, and relations with remaining normal kidney size in children with unilateral functioning solitary kidney (UFSK). Sixty-six children with UFSK were equally divided into three groups: unilateral renal agenesis (URA), unilateral atrophic kidney (UAK), and unilateral nephrectomy (UNP). Twenty-two age-, weight-, and height-matched healthy children were considered as a control group. The serum creatinine level and first-morning urine microalbumin and creatinine concentrations were determined by the standard methods. Also, the BP profile was determined by ambulatory blood pressure monitoring (ABPM). We found that the serum creatinine level was higher and creatinine clearance was lower in each patient groups compared to those of the control group (p < 0.05). Compared with the controls, each group of patients had mean office, 24-h, daytime, and night-time systolic and diastolic BP values similar to those of the controls (p > 0.05). An inverse correlation was found between the renal size standard deviation scores (SDS) of normal kidneys and 24-h systolic and diastolic BP load SDS in all of the patients (p < 0.05; r = −0.372, r = −0.295, respectively). The observed relationship between renal size SDS and 24-h mean arterial pressure (MAP), systolic and diastolic BP load SDS suggests that children with UFSK should be evaluated by using ABPM for the risk of hypertension. Introduction Arterial hypertension, proteinuria, and impaired renal functions are potential complications of unilateral functioning solitary kidney (UFSK), including unilateral renal agenesis (URA), unilateral atrophic kidney (UAK), and unilateral nephrectomy (UNP). The reduction of renal mass leads to compensatory hypertrophy of the remaining renal tissue in these patients [1–3]. Functionally, the remnant kidney will partly compensate for the lost function by increasing its workload, which is accompanied by increased glomerular blood flow and blood pressure (BP), so-called hyperfiltration [4]. It has been shown in animal models of unilateral renal ablation that the remaining kidney undergoes accelerated structural renal damage, generally in the form of glomerulosclerosis, with varying degrees of renal insufficiency, partly amenable to a variety of therapeutic interventions [5, 6]. A relatively small number of long-term follow-up studies in humans has documented that the surgical loss of renal functional mass in the presence of a normal remnant kidney rarely leads to renal insufficiency, although the incidence of mild to moderate proteinuria and hypertension has increased [7–10]. Kasiske et al. [11] reported that UNP does not cause progressive renal dysfunction, but it may be associated with a small increase in BP. On the other hand, Wikstad et al. [8] showed that adults born with URA or UNP in childhood did not have a marked increase in arterial BP or renal insufficiency. Some reports, however, indicate that patients with URA or with an UFSK left after nephrectomy may have proteinuria and focal glomerular sclerosis [5, 12]. Janda et al. [13] recorded marginal diastolic hypertension in one third of their subjects in a study of 40 children and/or adolescents (23 URA, 17 UNP). Although some investigators have reported an increase in the prevalence of hypertension and/or proteinuria, others have failed to document these abnormalities [11]. The discrepancy among the results of these studies may be caused due to a difference in the amount of renal mass removed, the age at the time of renal mass reduction, or unsuspected damage in the remaining kidney. In the past few years, ABPM has become an accepted method for investigating the BP profile in children with renal disorders [14–16]. Over the years, ambulatory blood pressure monitoring (ABPM) has been increasingly used to investigate hypertension in different pediatric populations [17–20]. A careful review of the literature shows that there are few reports on ABPM, microalbuminuria, and renal functions in children with UFSK [21]. Furthermore, the utility of BP in children with UFSK is not well established. Therefore, the aim of this study was to investigate the BP profile by ABPM, microalbuminuria, renal functions, and relations with remaining normal kidney size in children with UFSK. Materials and methods Sixty-six children with a mean age of 8.32 ± 4.23 and range 0.5 and 18 years with UFSK were equally divided into three groups: URA group (n = 22), UAK group (n = 22), and UNP group (n = 22). Twenty-two Age-, weight-, and height-matched healthy children were considered as a control group (Table 1). Renal ultrasound was normal in all controls. Informed consent was obtained from the children and their parents prior to the testing, and the study was approved by the local ethics committee of the School of Medicine, Çukurova University, Adana, Turkey. Table 1Demographic data, anthropometric data, and renal functional parameters of the patients and control groupsParametersURA (n = 22)UAK (n = 22)UNP (n = 22)Controls (n = 22)Gender (M/F)11/1112/1010/1210/12Age (years)8.63 ± 4.508.03 ± 4.308.31 ± 4.048.78 ± 3.49Weight (kg)28.44 ± 18.0727.01 ± 17.1426.57 ± 10.4829.19 ± 10.92Height (cm)124.55 ± 27.62126.05 ± 25.22123.96 ± 21.21128.36 ± 26.44BMI (kg/m2)16.60 ± 2.9517.96 ± 3.1416.77 ± 2.0516.96 ± 1.91UMA (mg/L)5.46 ± 3.914.80 ± 2.034.56 ± 8.115.18 ± 4.09UMA/UCr (mg/mg)0.23 ± 0.720.17 ± 0.300.07 ± 0.130.06 ± 0.06GFR (ml/dk/1.73 m2)1121.95 ± 34.19123.66 ± 43.67108.57 ± 26.71155.85 ± 41.27Serum Cr (mg/dl)20.59 ± 0.130.60 ± 0.150.65 ± 0.100.47 ± 0.13Renal size (mm)100.32 ± 18.4291.32 ± 17.5697.23 ± 17.4992.36 ± 16.00Values are expressed as mean±SD, M: male, F: female, BMI: body mass index, UMA: urine microalbumin, UCr: urine creatinine, GFR: glomerular filtration rate1p < 0.05 URA vs. controls, UAK vs. controls, and UNP vs. controls2p < 0.05 URA vs. controls, UAK vs. controls, and UNP vs. controls Both renal agenesis and atrophic kidney were established through renal ultrasound and scintigraphy. A non-functioning kidney is caused due to unilateral vesicoureteral reflux (in seven cases), ureteropelvic junction obstruction (in 10 cases), and multicystic dysplastic kidney (in five cases) in the UAK group. In contrast, indications for nephrectomy contained unilateral Wilms’ tumor (in six cases), a non-functioning kidney is caused due to unilateral vesicoureteral reflux (in 10 cases) and ureteropelvic junction stenosis (in two cases), multicystic dysplastic kidney (in two cases), and nephrolithiasis (in two cases) in the UNP group. Patients with Wilms’ tumor have favorable type and they did not receive chemotherapy. The mean time due to nephrectomy was 4.06 ± 3.87 years (range 0.5–17 years) in this group. All patients had a normal renal function defined as a glomerular filtration rate (GFR) of over 90 ml/min 1.73 m2 (as determined by Schwartz et al.’s formula [22]) and normal urinary sediment. The remaining kidney has findings of normal ultrasound and normal scintigraphy in all patient groups. Only children without any anatomical abnormalities of the remaining kidney and with normal GFR were included in the study. No patient was treated with antihypertensive drugs or drugs interfering with BP. Neither the study patients nor children in the control groups had a positive (first-degree-relative) family history of hypertension. Patients with a renal scar in their solitary kidney or who have other urinary tract anomaly were withdrawn from the study. In all of the study group participants, urinalysis, serum uric acid, creatinine, cholesterol, protein and albumin levels, and first-morning urine microalbumin and creatinine concentrations were determined by the standard methods. In all subjects, ABPM was performed over 24 h using the SpaceLabs 90217 oscillometric device (Redmond, Washington). The appropriately sized cuff, chosen based on arm width, was placed on the non-dominant arm. The BP was automatically recorded in every 15 min during the day and every 30 min at night. Only ABPM profiles with at least 30 recordings, including at least eight readings between midnight and 06.00 h, were accepted. Daytime was defined as 08.00–20.00 h and night-time as 00.00–06.00 h, according to the study of Soergel et al. [23]. For each subject, the BP values corresponding to the 95th percentile, according to sex and height, was determined [23, 24]. Hypertension was defined as a systolic BP and/or diastolic BP>95th percentile. The percentage of systolic BP and diastolic BP readings above this value constituted a measurement of 24-h systolic and diastolic BP load, respectively. The dipping status was calculated by subtracting the mean night-time BP from the mean daytime BP, and then dividing this value by the mean daytime BP. Dipping was defined as a ≥10% drop in mean systolic BP or diastolic BP between daytime and night-time. Renal size and BP standard deviation scores (SDS) was calculated according the left main stem (LMS) method [24, 25]. The renal size of the normal kidney was evaluated by performing abdominal ultrasound and measuring the lower-to-upper-pole length in the supine longitudinal view by the same observer. Renal size SDS values were correlated to the BP SDS values for each patient. Renal size percentiles were determined and evaluated according to standard length-against-height-nomogram defined by Dinkel et al. [26]. Compensatory hypertrophy was accepted when the measured renal size was found to be greater than the 95th percentile, and its effect on the BP parameters was evaluated. All data were stored and analyzed using the SPSS statistical package (SPSS Inc., Chicago, Illinois). The normality Kolmogorov-Smirnov test was performed to determine whether continuous variables were normally distributed or not. Differences between groups were analyzed using the non-parametric Kruskal-Wallis test and the Mann-Whitney U test. All values are expressed as mean±SD. The prevalence of hypertension was determined using Fisher’s exact test. The relationships between variables were analyzed with the non-parametric Spearman’s correlation test in each group. A value of p < 0.05 was considered to be statistically significant. Results The demographic data and renal function parameters of all of the study groups and controls are shown in Table 1. Age, height, weight, BMI, urine microalbumin, urine microalbumin to urine creatinine ratio, and renal size values are not different between each patient group and the controls (p > 0.05). The mean serum creatinine level was higher in all patient groups compared to that of the control group (p = 0.006 between URA and control, p = 0.004 between UAK and control, p < 0.001 between UNP and control) and the GFR was lower in all patient groups compared to that of the control group (p = 0.005 between URA and control, p = 0.016 between UAK and control, p < 0.001 between UNP and control). Mean heart rates (24 h, daytime, night-time), mean systolic and diastolic BP parameters (office BP, 24 h, daytime, night-time), mean systolic and diastolic BP dipping, and 24-h systolic and diastolic BP loads are all given in Table 2. When compared to the controls, each group with UFSK had mean office, 24-h, daytime, and night-time systolic and diastolic BP, and heart rate values similar to those of the controls (p > 0.05). Only the mean 24-h diastolic BP was higher in the URA group than that of the control group (p = 0.039). In addition, the diastolic load was higher in the UAK group than that of the control group (p = 0.012). There was no significant difference in the mean office, 24-h, daytime, and night-time systolic and diastolic BP, and heart rate between all of the patient groups (p > 0.05). The mean office systolic and diastolic BP, respectively, were higher than the mean daytime systolic and diastolic BP in all groups. The prevalence of hypertension in the URA group was 23% (5/22), in the UAK group was 23% (5/22), in the UNP group was 32% (7/22) [in all patients, it was 26% (17/66)], and in the controls it was 5% (1/22). The prevalence of hypertension in patients was higher compared to that in the controls (p = 0.035). The prevalence of non-dipping phenomenon in the URA group was 23% (5/22), in the UAK group was 36% (8/22), in the UNP group was 32% (7/22) [in all patients, it was 30% (20/66)], in the controls it was 9% (2/22). There was a statistically significant difference between patients and the controls (p = 0.049). Table 2Average pulse rate and blood pressure (BP) of the patients and the control groupsParametersURA (n = 22)UAK (n = 22)UNP (n = 22)Controls (n = 22)Av. 24-h HR (beats/min)89.36 ± 15.7994.64 ± 17.7488.18 ± 12.9590.82 ± 14.79Av. daytime HR (beats/min)92.64 ± 15.5197.14 ± 17.2793.09 ± 12.9697.41 ± 15.55Av. night-time HR (beats/min)80.23 ± 15.9986.46 ± 18.0177.86 ± 12.4382.50 ± 16.65Av. office SBP (mmHg)113.96 ± 16.00116.96 ± 11.24114.55 ± 14.83114.68 ± 12.66Av. office DBP (mmHg)74.73 ± 10.4871.50 ± 11.0671.05 ± 13.0072.00 ± 9.67Av. 24-h MAP (mmHg)80.00 ± 7.9278.82 ± 7.1678.41 ± 7.0678.27 ± 3.89Av. 24-h SBP (mmHg)106.64 ± 10.87105.59 ± 9.31104.22 ± 8.04105.05 ± 6.90Av. 24-h DBP (mmHg)165.91 ± 6.8264.55 ± 6.5964.09 ± 6.8763.41 ± 3.63Av. daytime MAP (mmHg)81.14 ± 9.1480.00 ± 8.0479.68 ± 7.3380.68 ± 4.86Av. daytime SBP (mmHg)109.59 ± 11.75107.18 ± 10.01106.55 ± 7.95108.14 ± 6.83Av. daytime DBP (mmHg)68.36 ± 8.3666.46 ± 7.1766.18 ± 7.2867.41 ± 4.36Av. night-time MAP (mmHg)72.86 ± 6.5571.86 ± 7.7072.09 ± 6.7673.64 ± 4.99Av. night-time SBP (mmHg)99.82 ± 9.3699.32 ± 9.9999.00 ± 7.7898.55 ± 6.60Av. night-time DBP (mmHg)59.00 ± 5.4658.23 ± 7.1858.09 ± 6.7357.59 ± 3.75Av. SBP dipping (%)8.26 ± 4.297.31 ± 4.686.58 ± 3.428.83 ± 3.74Av. DBP dipping (%)13.12 ± 6.5412.24 ± 7.6412.03 ± 7.1914.87 ± 6.32Av. 24-h SBP load (%)19.71 ± 25.7526.31 ± 25.1421.88 ± 20.0814.45 ± 13.81Av. 24-h DBP load (%)221.22 ± 17.1233.0.2 ± 28.9926.24 ± 24.5613.54 ± 13.75Values are expressed as mean±SD, Av.: average, HR: heart rate, MAP: mean arterial pressure, SBP: systolic blood pressure, DBP: diastolic blood pressure1p = 0.039 between URA and control groups2p = 0.012 between UAK and control groups Compensatory hypertrophy (renal size percentile >95%) was seen in 59% (39/66) of patients. Age, height, weight, renal size, and GFR were statistically significantly higher in patients with compensatory hypertrophy than in patients without compensatory hypertrophy. In contrast, the mean heart rates were lower in patients with >95th percentile of renal size (Table 3). Table 3Some characteristics of the patients with unilateral functioning solitary kidney (USFK) according the percentile of renal sizeParametersRenal size <95th percentile (n = 27)Renal size >95th percentile (n = 39)pAge (year)5.91 ± 3.169.99 ± 4.09<0.001Height (cm)113.19 ± 21.24132.92 ± 23.47<0.001Weight (kg)19.80 ± 9.8932.56 ± 16.41<0.001Renal size (mm)80.37 ± 11.13107.31 ± 12.71<0.001GFR (ml/dk/1.73 m2)107.82 ± 37.31125.15 ± 33.09<0.05Av. 24-h HR (beat/min)99.19 ± 15.4984.87 ± 12.96<0.001Av. daytime HR (beat/min)102.00 ± 13.8188.95 ± 13.99<0.001Av. night-time HR (beat/min)88.59 ± 17.0576.62 ± 13.02<0.05Values are expressed as mean±SD, Av.: average, GFR: glomerular filtration rate, HR: heart rate Renal size SDS was correlated with some BP SDS parameters in the patients and the controls. Statistically significant correlations are shown in Table 4. There was no correlation between the renal size SDS and BP parameters, but an inverse correlation was found between the renal size SDS and 24-h MAP SDS, 24-h systolic and diastolic BP load SDS in all of the patients, but these correlations were not found in the controls. Table 4Correlation between renal size standard deviation scores (SDS) and some parameters SDS in patients with UFSK and the controlsGroupsParametersrpPatients (n = 66)Av. 24-h MAP SDS−0.2840.021Av. 24-h SBP SDS−0.1750.159Av. 24-h DBP SDS−0.0340.788Av. 24-h SBP load SDS−0.3720.002Av. 24-h DBP load SDS−0.2950.016Controls (n = 22)Av. 24-h MAP SDS−0.0110.962Av. 24-h SBP SDS−0.0670.767Av. 24-h DBP SDS−0.3120.158Av. 24-h SBP load SDS−0.1480.510Av. 24-h DBP load SDS−0.0190.934SDS: standard deviation scores, Av.: average, MAP: mean arterial pressure, SBP: systolic blood pressure, DBP: diastolic blood pressure Discussion The UFSK is usually considerably enlarged, this constituting a compensatory diffuse but not partial, lobar enlargement, usually with a larger than normal pelvis [27]. The reduction of renal mass in the rat is associated with hyperfiltration and hypertrophy of the remaining nephrons, ultimately resulting in proteinuria, renal failure, and hypertension. Nagata et al. [28] reported that unilateral nephrectomy in young rats leads to a higher incidence of glomerular sclerosis than is observed in adult rats. A study conducted by Regazzoni et al. [29] in a follow-up of UNP in children has reported a decreased renal reserve capacity, but detected no evidence of hypertension or proteinuria. In our study, subjects were screened with urine microalbumin, urine and serum creatinine, GFR, and urine microalbumin-to-creatinine ratios in first-morning urine for the detection of proteinuria and renal functions. No statistically significant difference was found between the urine microalbumin and microalbumin-to-creatinine ratios in the first-morning urine in all of the patient and control groups. However, the GFR was lower and the serum creatinine level was higher in the subgroups of patients than those of the control group. These results show that a mild renal dysfunction may be detected in patients with UFSK. Although some investigators have reported an increased prevalence of hypertension, others have failed to document these abnormalities in children with UFSK [11]. Janda et al. [13] recorded marginal diastolic hypertension in one third of their subjects in a study of 40 children and/or adolescents (23 URA, 17 UNP). As we mentioned before, there are few reports which have utilized 24-h ABPM in UFSK children. Mei-Zahav et al. [21] showed that the presence of an UFSK, from whatever cause, leads to an increase in BP. This is particularly manifested in systolic BP and even more so in daytime systolic BP. Kasiske et al. [11] reported that UNP does not cause progressive renal dysfunction, but may be associated with a small increase in BP. On the other hand, Wikstad et al. [8] concluded that adults born with URA or UNP in childhood did not have a marked increase in arterial BP or renal insufficiency. We measured the ABPM in three groups of patient with UFSK and found that they have the same characteristics as in healthy controls. However, in our study, the mean office, 24-h systolic, daytime, and night-time systolic and diastolic BP, mean 24-h, daytime, and night-time heart rate, 24-h systolic BP load, systolic and diastolic BP dipping in all groups of patients were similar to those of the controls. In contrast, mean 24-h diastolic BP was higher in the URA group than those of the control group, but this finding showed no difference between the other patient groups and controls. The 24-h diastolic BP load was increased in the UAK group. In addition, an increased prevalence of hypertension was found in all of the patients groups. According to these results, we suggest that patients with a single kidney have potential risk factors for hypertension in the early childhood period. Compensatory hypertrophy of the remaining kidney may be seen in patients with UFSK. In adult animals, a compensatory increase in size following UNP is, for the most part, attributable to hypertrophy (an increase in cell size), which is predominantly of proximal tubular origin. In young animals, renal growth is achieved primarily by cell multiplication, that is, hyperplasia [3]. In the present study, an inverse correlation was found between renal size SDS and 24-h MAP SDS, 24-h systolic and diastolic BP load SDS in all of the patients, but was not seen in the controls. Therefore, we think that there is an increased risk of hypertension in children without compensatory hypertrophy. To this end, we conclude that subtle alterations of renal function might be already present in children with UFSK, while no obvious pathological findings were made for BP or urinary protein excretion in the early period. However, an increased BP load was seen in these patients (particularly in patients without compensatory hypertrophy). Nevertheless, this might not exclude an increased long-term risk for the development of hypertension or impaired renal function in this patient group. Because patients in the present study have a small sample and a shorter follow-up period, further prospective studies with a larger sample size and of longer term are suggested to find the relationship between these parameters.

Neurochem_Res-4-1-2413118

Silencing α-Synuclein Gene Expression Enhances Tyrosine Hydroxylase Activity in MN9D Cells

α-Synuclein has been implicated in the pathogenesis of Parkinson’s disease (PD). Previous studies have shown that α-synuclein is involved in the regulation of dopamine (DA) metabolism, possibly by down-regulating the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA biosynthesis. In this study, we constructed α-synuclein stably silenced MN9D/α-SYN− cells by vector mediated RNA interference and examined its effects on DA metabolism. We found that there were no significant differences in TH protein and mRNA levels between MN9D, MN9D/α-SYN− and MN9D/CON cells, suggesting that silencing α-synuclein expression does not affect TH gene expression. However, significant increases in phosphorylated TH, cytosolic 3, 4-dihydroxyphenylalanine (l-DOPA) and DA levels were observed in MN9D/α-SYN− cells. Our data show that TH activity and DA biosynthesis were enhanced by down-regulation of α-synuclein, suggesting that α-synuclein may act as a negative regulator of cytosolic DA. With respect to PD pathology, a loss of functional α-synuclein may result in increased DA levels in neurons that may lead to cell injury or even death. Introduction α-Synuclein is a 140-amino acid protein, which has been implicated in the pathogenesis of Parkinson’s disease (PD) [20, 30]. Previous studies have shown that α-synuclein mutations (A53T, A30P, E46K) are associated with some autosomal-dominant PD [14, 15, 27] and that aggregated α-synuclein is the major component of Lewy bodies and Lewy neurites, pathological hallmarks of PD [2, 21, 34]. Although the normal function of α-synuclein and its role in the pathogenesis of PD remain unclear, several hypotheses have been proposed based on its physical properties or interacting partners [17–19, 25, 28, 31, 36]. One theory suggested that α-synuclein might be linked to PD via the regulation of dopamine (DA) homeostasis [1, 18, 25, 31, 41]. Several studies have shown that α-synuclein may be involved in regulating the biosynthesis, vesicular storage and release, as well as reuptake of DA [3, 9, 26, 32, 33, 38, 40, 42]. However, some of these findings remain controversial. It has also been reported that α-synuclein may down-regulate the gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme involved in DA biosynthesis [3, 42]. Others report that α-synuclein regulates DA biosynthesis by reducing TH activity [26]. Another study showed that the tyrosine-mediated enhancement of phosphorylated TH in nigrostriatal DA neurons was inhibited in α-synuclein knockout mice, suggesting that α-synuclein may either enhance TH phosphorylation or hinder TH inactivation during accelerated neuronal activity [9]. Although it has been established that α-synuclein plays an important role in enhancing TH activity, conflicting reports indicate that further studies are required to examine the role of α-synuclein in regulating DA metabolism. RNA interference (RNAi) is a new strategy for silencing gene expression that uses short double-stranded (ds) RNA to mediate the degradation of sequence specific mRNA [10–12]. It has been reported that introducing small interfering RNAs (siRNAs) or short hairpin RNA (shRNA) into mammalian cells could specifically silence the expression of target genes [7, 23]. The high specificity, efficiency and convenience of this technique means that it is a powerful tool for studying gene function. In the present study, we silenced the expression of α-synuclein using vector mediated RNAi in dopaminergic MN9D cells and examined the effect of down-regulation of α-synuclein on TH and DA biosynthesis. Materials and Methods Construction of shRNA Expression Plasmids Short hairpin RNA were designed to target specific regions of mouse α-synuclein mRNA (GenBank accession no. AF179273). We identified two 19-nucleotide stretches within the coding region of the α-synuclein gene that were 40–50% GC rich, located within one exon and unique within the mouse genome. A nine-nucleotide loop structure was designed to form the hairpin. We designed three sets of oligonucleotides as instructed by the BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit (Invitrogen, Carlsbad, CA, USA): SH1/α-SYN: 5′-CACCGAGCAAGTGACAAATTTGTTCAAGAGACAACATTTGTCACTTGCTC-3′ (forward), 5′-AAAAGAGCAAGTGACAAATGTTGTCTCTTGAACAACATTTGTCACTTG CTC-3′ (reverse); SH2/α-SYN: 5′-CACCAGGCTACCAAGACTATGAGTTCAAGAG ACTCATAGTCTTGGTAGCC-3′ (forward), 5′-AAAAGGCTACCAAGACTATGA GTCTCTTGAACTCATAGTCTTGGTAGCCT-3′ (reverse); and SH/CON: 5′-CACCGGATCGCCAGAACAAGTATTTCAAGAGAATACTTGTTCTGGCGATCC-3′ (forward), 5′-AAAAGGATCGCCAGAACAAGTATTCTCTTGAAATACTTGTTCTGG CGATCC-3′ (reverse). Single-stranded (ss) DNA oligos (BioSia, Co., Ltd, Shanghai, China) were synthesized. Equal amounts of each pair of ss oligos were annealed to generate ds oligos. The ds oligos were inserted into the pENTRTM/H1/TO vector (Invitrogen) using T4 DNA ligase and transformed into One Shot® TOP10 chemically competent Escherichiacoli (Invitrogen). The transformants were verified by PCR and sequencing. The positive plasmids were named pSH1/α-SYN, pSH2/α-SYN and pSH/CON, respectively. Cell Culture The MN9D dopaminergic cell line was generated by fusion of rostral mesencephalic neurons from embryonic C57BL/6J (embryonic day 14) mice with N18TG2 neuroblastoma cells [8] (gift from Dr Bastian Hengerer, Novartis AG). Cells were cultured in DMEM/F12 media supplemented with 10% neonatal calf serum (GIBCO BRL, Grand Island, NY, USA; pH 7.2) and incubated at 37°C in an atmosphere of 5% CO2. MN9D cells endogenously express α-synuclein and TH, and produce measurable levels of DA. Transfection and Selection MN9D cells were transfected with pSH1/α-SYN, pSH2/α-SYN, pSH/CON or pcDNA3.1 containing human wild-type (WT) α-synuclein cDNA (constructed in our laboratory). For stable transfection of pSH1/α-SYN, pSH2/α-SYN and pSH/CON, cells were seeded in 6-well tissue culture plates pre-coated with poly-l-lysine (Sigma, St Louis, MO, USA) at 80–85% confluency. Transfections were performed using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s instructions. Four to six hours after transfection, the media was replaced with fresh growth media. The following day, cells were trypsinized and replated into a larger sized tissue culture format. Stably transfected MN9D cells were selected using 400 μg/ml Zeocin™ (Invitrogen) for 12 days. The Zeocin™ resistant clones were then selected and expanded. Cells were maintained with media containing 50 μg/ml Zeocin™. Seventy-two hours post-transfection, cells transfected with pcDNA3.1 containing human WT α-synuclein cDNA were collected and named MN9D/α-SYN+. RNA Isolation, RT-PCR and Real-time RT-PCR Total RNA was extracted from pSH1/α-SYN, pSH2/α-SYN and pSH/CON stably transfected MN9D cells and MN9D/α-SYN+ cells using Trizol reagent. First-strand cDNA was synthesized from 5 μg of total RNA, using the SuperScript™ First-strand synthesis System for RT-PCR (Invitrogen). The reaction mixture for PCR consisted of 1 μl of cDNA template, 17 μl of sterile ddH2O and 10 pmol (1 μl) of each specific primer. The following primers were used: α-synuclein: 5′-ATAAGAATGCGGCCGCATGGATGTATTCATGAAAG-3′ (forward) and 5′-CCGCTCGAGGCTTCAGGTTCGTAGTCTTGA-3′ (reverse). As an internal control, β-actin cDNA was co-amplified using the following primer sequences: 5′-CCCATCTACGAGGGCTACGC-3′ (forward) and 5′-AGGAAGGAGGGCTGGAACA-3′ (reverse). Each PCR was started by predenaturation at 96°C for 3 min. Each PCR cycle consisted of 94°C for 30 s, 50°C for 55 s and 72°C for 55 s, followed by a final 10 min extension at 72°C. PCR amplification was carried out for 35 cycles for α-synuclein and 22 cycles for β-actin using a Perkin–Elmer DNA thermal cycler 480 (Applied Biosystems, Foster City, CA, USA). The reaction mixture (20 μl) for real-time PCR consisted of 10 μl SYBR GREEN PCR Master Mix (Applied Biosystems), 0.5 μl of cDNA template, 0.4 μl of each specific primer (10 μM) and 9.7 μl of sterile ddH2O. The following primers were used: α-synuclein: 5′-TGACGGGTGTGACAGCAGTAG-3′ (forward) and 5′-CAGTGGCTGCTGCAATGC-3′ (reverse); TH: 5′-CAGCCCTACCAAGATCAAAC-3′ (forward) and 5′-TACGGGTC AAACTTCACAGA-3′ (reverse); and β-actin: 5′-ACCACCATGTACCCAGGCATT-3′ (forward) and 5′-CCACACAGAGTACTTGCGCTCA-3′ (reverse). Quantitative PCR was performed in duplicate or triplicate using a 7300 real-time PCR thermal cycler (Applied Biosystems). Each reaction was started by 50°C for 2 min, 95°C for 10 min and 96°C for 3 min, followed by 40 cycles of 94°C for 15 s, 59°C for 20 s and 72°C for 30 s. Immunofluorescence Staining Cells were washed with 0.01 M PBS, fixed in 4% paraformaldehyde (30 min), permealized with 0.5% Triton-X (20 min) and blocked in 5% normal goat serum (30 min). Samples were incubated with a specific monoclonal antibody against α-synuclein (2E3; 1:1000) (gift from Dr Shun Yu, Xuanwu Hospital, Capital Medical University, Beijing, China) overnight at 4°C. Cells were washed with 0.01 M PBS and incubated with goat anti-mouse IgG conjugated to cyanine 3 (Cy3TM; 1:400, Sigma) at 37°C for 30 min. After a final wash, cells were examined by fluorescence microscopy. Cell Viability Assay Cell viability was assessed using a MTT Kit (Promega, WI, USA). Briefly, the transfected MN9D cells were plated into 96-well plates (1.0 × 104 cells per well) and cultured for 24 h prior to the assay. Sixteen wells were measured for each stably transfected MN9D cell line. Western Blot Analysis Lysates of MN9D cells were prepared as previously described [39]. Protein concentrations were determined using BCA relative to BSA protein standards according to the manufacturer (Pierce, Rockford, IL, USA). Whole cell protein extracts were resolved by SDS-PAGE and electrophoretically transferred to nitrocellulose membrane. Prestained protein standards were used to determine the relative molecular mass of proteins. The membranes were blocked in 5% nonfat dry milk in Tris-buffered saline, then incubated with the TH specific monoclonal antibody (1:5000, Sigma) or α-synuclein specific monoclonal antibody (2E3; 1:1000) at room temperature for 3 h, followed by a 1-h incubation with a peroxidase-conjugated secondary antibody (1:5000, ZhongShan, Beijing, China) at room temperature. The blots were washed and immunodetection was carried out by chemiluminescence using SuperSignal (Pierce). Blots were then stripped and re-probed with an antibody against β-actin (1:500, Zhongshan). Blots were scanned using Kodak Image Station (440 CF; Kodak, Rochester, MN, USA) and the optical densities of the bands relative to β-actin within each lane were obtained. HPLC for Assaying DA and 3, 4-Dihydroxyphenylalanine (l-DOPA) Levels MN9D cells were collected and re-suspended in HPLC buffer consisting of 0.1 N perchloric acid, 0.3 mM EDTA and 0.1% l-lysine. Cells were freeze-thawed three times, then centrifugated at 15,000 g for 15 min at 4°C to remove particulates. The supernatants were collected and stored at −80°C until analysis. Briefly, the supernatant samples (20–50 μl) were injected onto a Phase II Column (ODS, 3.2 × 100 mm cartridge, Φ3.2 μm, Bioanalytical Systems, Inc, USA). The mobile phase consisted of 0.05 M CH3COONa, 0.05 M citric acid, 1 mM sodium octyl sulfate, 5 mM TEA, 0.075 mM Na2EDTA and 10% methanol (v/v), pH 2.7. The mobile phase was pumped through the system at 0.4 ml/min using a PM-80 pump (Bioanalytical Systems, Inc). Compounds were detected and quantified with an LC-4C detector (Bioanalytical Systems, Inc). The limit of detection for DA was 2 nmol/20 μl. Peaks were identified by retention times set to known standards. Tyrosine hydroxylase activity was assessed by measuring the accumulation of l-DOPA within cells treated with the aromatic l-amino acid decarboxylase (AADC) inhibitor n-hydroxybenzylhydrazine dihydrochloride (NSD-1015) and measured by HPLC as described above. Briefly, MN9D cells were cultured in 12-well tissue culture plates, washed twice in artificial CSF (ACSF containing 147 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl2 and 1.0 mM MgCl2) and equilibrated for 20 min at 37°C before addition of 200 μM NSD-1015 in ACSF for 30 min. ACSF samples were collected and assayed by HPLC as described above. Statistical Analysis ANOVA followed by Bonferroni post hoc multiple comparisons were used to examine the significance between differences among the experimental groups. A value of p < 0.05 was considered to be statistically significant. All data are expressed as mean ± SE. Results Construction of the shRNA Expression Plasmids After synthesis, each pair of the ss oligos was annealed to generate ds oligos. The annealing efficiency was analyzed by 4% agarose gel electrophoresis. Each ds oligo annealing reactant showed a clearly detectable molecular weight band around 50 bp, as expected for the length of the designed ds oligos. The 50 bp bands were brighter than the corresponding lower molecular weight band, which represented the unannealed ss oligos in the annealing reactant. These data indicate that each pair of ss oligos annealed effectively to form ds oligos. The ds oligos were ligated with linearized pENTR™/H1/TO vectors and the positive clones were expanded and verified by PCR. Bands near 350 bp were found in all PCR reactants, indicating that the ds oligos were ligated into the pENTR/H1/TO vectors. Further analysis by sequencing confirmed that the orientation and sequence of the inserted ds oligos were correct. α-Synuclein Expression in MN9D Transfected Cells The gene expression of α-synuclein in the transfected MN9D cells was assayed by RT-PCR, real-time RT-PCR, immunofluorescence and Western blot. RT-PCR analysis revealed that α-synuclein mRNA levels were decreased in pSH2/α-SYN-transfected MN9D cells compared with MN9D and pSH/CON-transfected MN9D cells (Fig. 1a). However, α-synuclein mRNA levels in pSH1/α-SYN-transfected cells were not significantly different to levels observed in control MN9D and pSH/CON-transfected MN9D cells (Fig. 1a). These data suggest that α-synuclein expression may be inhibited by transfection with pSH2/α-SYN. In addition, real-time RT-PCR revealed that α-synuclein mRNA levels in pSH2/α-SYN-transfected cells (9.8 ± 2.4%) were significantly lower than in MN9D and MN9D/CON-transfected (81.6 ± 4.9%) cells (Fig. 1b). The immunofluorescence data also indicated that α-synuclein gene expression was down-regulated in pSH2/α-SYN-transfected cells (Fig. 1c). Weak immunofluorescence staining was observed in pSH2/α-SYN-transfected cells compared to strong α-synuclein staining in control MN9D, pSH/CON-transfected and pSH1/α-SYN-transfected MN9D cells. Furthermore, western blot analysis revealed that α-synuclein protein levels were significantly lower in pSH2/α-SYN-transfected cells (0.150 ± 0.004) than in MN9D cells (0.401 ± 0.038) (Fig. 1d, e). Thus, α-synuclein expression was inhibited after transfection of MN9D cells with pSH2/α-SYN. These data suggest that SH2/α-SYN was an effective siRNA sequence and that α-synuclein expression was stably silenced in pSH2/α-SYN-transfected MN9D cells. The pSH2/α-SYN and pSH/CON stably transfected cells were then named MN9D/α-SYN- and MN9D/CON, respectively. Fig. 1Inhibition of α-synuclein expression in pSH2/α-SYN-transfected cells. (a) α-Synuclein mRNA levels were measured by RT-PCR. Lane 1 pSH1/α-SYN-transfected MN9D cells; Lane 2 pSH2/α-SYN-transfected MN9D cells; Lane 3 pSH/CON-transfected MN9D cells; Lane 4 MN9D cells; Lane 5 100 bp DNA ladder. (b) Statistical analysis of α-synuclein gene expression by real-time RT-PCR. α-Synuclein mRNA levels were decreased by approximately 90% in pSH2/α-SYN-transfected cells compared with normal MN9D cells [n = 5, F = 216.167, df(total) = 14, **p < 0.001 compared with MN9D cells, **p < 0.001 compared with MN9D/CON cells]. α-Synuclein mRNA levels were not significantly different in pSH1/α-SYN-transfected cells compared with MN9D and MN9D/CON cells. (c) Immunofluorescence staining of normal MN9D cells (a) and MN9D cells transfected with pSH1/α-SYN (b), pSH/CON (c) and pSH2/α-SYN (d) using an antibody against α-synuclein. Strong α-synuclein staining was observed in MN9D, pSH1/α-SYN-transfected and pSH/CON-transfected MN9D cells, whereas weak staining was observed in pSH2/α-SYN-transfected MN9D cells. Bar = 50 μm. (d) Western blot analysis of α-synuclein protein levels. Lane 1 MN9D cells; Lane 2 MN9D/CON cells; Lane 3 MN9D/α-SYN− cells. (e) Statistical analysis of α-synuclein protein levels in MN9D, MN9D/CON and MN9D/α-SYN− cells using the linear density ratio of α-synuclein/β-actin. α-Synuclein protein levels were significantly decreased in MN9D/α-SYN− cells (0.150 ± 0.004) compared with MN9D (0.401 ± 0.038) and MN9D/CON (0.321 ± 0.011) cells [n = 5, F = 162.034, df(total) = 14, **p < 0.001 compared with MN9D cells, **p < 0.001 compared with MN9D/CON cells] Effects of Silencing α-Synuclein Expression on Cell Viability No significant differences in cell morphology were observed between the three groups of MN9D cells (data not shown). As shown in Fig. 2, the cell viability of MN9D/α-SYN− cells was significantly decreased compared with control MN9D and MN9D/CON cells [n = 5, F = 421.457, df(total) = 14, **p < 0.001 compared with MN9D cells, **p < 0.001 compared with MN9D/CON cells]. Cell viability was decreased by 58.3 and 50% in MN9D/α-SYN− cells (0.091 ± 0.006) compared with MN9D (0.217 ± 0.012) and MN9D/CON (0.189 ± 0.005) cells, respectively (Fig. 2). These data suggest that α-synuclein may be important for the viability of MN9D cells. Fig. 2Effects of silencing α-synuclein expression on cell viability. Cell viability was measured using an MTT assay. Cell viability was decreased in MN9D/α-SYN− cells compared with MN9D and MN9D/CON cells [n = 5, F = 421.457, df(total) = 14, **p < 0.001 compared with MN9D cells, **p < 0.001 compared with MN9D/CON cells] TH Expression and Activity in MN9D Transfected Cells Previous studies have reported that TH expression may be down-regulated by over-expression of α-synuclein in MES23.5 and M17 cells [3, 42]. Here, we examined whether a similar effect occurred after the silencing of α-synuclein expression in MN9D/α-SYN− cells. TH mRNA, protein and phosphorylation levels were measured by real-time RT-PCR and Western blot analysis, respectively. Real-time RT-PCR showed that there were no significant differences in TH mRNA levels between MN9D, MN9D/CON (116.00 ± 10.23%), MN9D/α-SYN− (135.89 ± 14.58%) and MN9D/α-SYN+ (85.67 ± 5.55%) cells (Fig. 3a). These observations show that silencing α-synuclein expression did not alter TH gene expression, suggesting that α-synuclein does not regulate expression of TH. Similar TH protein levels were observed in MN9D, MN9D/CON (103.55 ± 0.84%), MN9D/α-SYN− (126.40 ± 10.81%) and MN9D/α-SYN+ (83.89 ± 4.06%) cells by Western blot analysis (Fig. 3b, c). However, TH phosphorylation levels were significantly increased in MN9D/α-SYN− (157.63 ± 8.36%) cells compared with MN9D and MN9D/CON (97.45 ± 5.06%) cells, and significantly decreased in MN9D/α-SYN+ cells (20.36 ± 2.86%) (Fig. 3b, d). To directly assess TH activity, we used NSD-1015 to inhibit AADC, thereby blocking the conversion of l-DOPA to DA. We found that l-DOPA levels were increased in MN9D/α-SYN− cells (266.33 ± 11.05 nmol/l) compared with MN9D (121.92 ± 5.61 nmol/l) and MN9D/CON (124.89 ± 8.94 nmol/l) cells (Fig. 4). In contrast, a significant decrease in l-DOPA levels was observed in MN9D/α-SYN+ cells (41.14 ± 3.89 nmol/l). Since accumulation of l-DOPA could only occur if TH actively converted tyrosine to l-DOPA, these data suggest that TH activity was increased in MN9D/α-SYN− cells and diminished in cells over-expressing α-synuclein. These data support the hypothesis that silencing α-synuclein expression does not affect TH gene expression but can enhance TH activity by increasing TH Ser40 phosphorylation. Fig. 3Effects of silencing α-synuclein expression on TH and phospho-Ser40-TH levels. (a) Statistical analysis of TH gene expression by real-time RT-PCR. Differences in TH mRNA levels between the transfected MN9D cells were not statistically significant (n = 5). (b) Western blot analysis of TH, phospho-Ser40-TH and β-actin protein levels. Lane 1 normal MN9D; Lane 2 MN9D/CON; Lane 3 MN9D/α-SYN−; Lane 4 MN9D/α-SYN+ cells. (c) Statistical analysis of TH protein levels in MN9D, MN9D/CON, MN9D/α-SYN− and MN9D/α-SYN+ cells using the linear density percent of TH/β-actin. No significant differences in TH protein levels were observed between the transfected MN9D cells (n = 5). (d) Statistical analysis of phospho-Ser40-TH protein levels in MN9D, MN9D/CON, MN9D/α-SYN− and MN9D/α-SYN+ cells using the linear density percent of phospho-Ser40-TH/TH. Phospho-Ser40-TH protein levels were significantly increased in MN9D/α-SYN− cells and significantly decreased in MN9D/α-SYN+ cells compared with MN9D and MN9D/CON cells [n = 5, F = 84.505, df(total) = 19, **p < 0.001 MN9D/α-SYN− cells compared with MN9D cells, **p < 0.001 MN9D/α-SYN− cells compared with MN9D/CON cells; **p < 0.001 MN9D/α-SYN+ cells compared with MN9D cells, **p < 0.001 MN9D/α-SYN+ cells compared with MN9D/CON cells]Fig. 4Effects of silencing α-synuclein expression on l-DOPA levels. Cell lysates were prepared from transfected MN9D cells incubated in the presence of 200 μM NSD-1015 for 30 min. l-DOPA levels were measured by HPLC. l-DOPA levels were significantly increased in MN9D/α-SYN− cells and significantly decreased in MN9D/α-SYN+ cells compared with MN9D and MN9D/CON cells [n = 6, F = 140.944, df(total) = 23, **p < 0.001 MN9D/α-SYN− cells compared with MN9D cells, **p < 0.001 MN9D/α-SYN− cells compared with MN9D/CON cells; **p < 0.001 MN9D/α-SYN+ cells compared with MN9D cells, **p < 0.001 MN9D/α-SYN+ cells compared with MN9D/CON cells] Effects of Silencing α-Synuclein Expression on DA Levels To assess differences in cytosolic DA levels between the transfected MN9D cells, we prepared cell extracts and measured DA content by HPLC with electrochemical detection. As shown in Fig. 5, DA levels were significantly increased in MN9D/α-SYN− cells compared with MN9D and MN9D/CON cells. The DA content was approximately 4.8-fold higher in MN9D/α-SYN− cells (834.72 ± 24.72 nmol/l) than in normal MN9D cells (186.04 ± 31.38 nmol/l) and 2.4-fold higher than in MN9D/CON cells (341.87 ± 46.25 nmol/l). No significant differences in DA content were observed between MN9D and MN9D/CON cells (Fig. 5). These results indicate that silencing α-synuclein expression leads to an increase in DA levels in MN9D cells. Fig. 5Effects of silencing α-synuclein expression on DA levels. Changes in DA levels were detected by HPLC. DA levels were significantly increased in MN9D/α-SYN− cells compared with MN9D and MN9D/CON cells [n = 6, F = 161.774, df(total) = 17, **p < 0.001 compared with MN9D cells, **p < 0.001 compared with MN9D/CON cells]. Differences in DA levels between the MN9D and MN9D/CON cells were not statistically significant Discussion In the present study, we silenced the expression of α-synuclein in MN9D dopaminergic cells by vector mediated RNAi using the BLOCK-iT™ inducible H1 RNAi entry vector. This vector was selected for several reasons. Firstly, the vector contains a kanamycin resistance gene for selection in E.coli and a ZeocinTM resistance marker to allow generation of stable cell lines that express the shRNA of interest. Secondly, the cloning site of the vector contains a 4-nucleotide overhang on the 5′ end of each DNA strand, allowing directional cloning of the shRNA of interest. Thirdly, the H1 promoter of the vector is recognized by RNA polymerase III resulting in high-level, constitutive expression of shRNA in most mammalian cell types. We identified an effective targeting sequence for RNAi, which localized in the C-terminal coding sequence of the mouse α-synuclein gene, and used this to generate MN9D/α-SYN− cells in which the expression of α-synuclein was stably silenced. Due to the endogenous expression of α-synuclein and the dopaminergic characteristics of MN9D cells, MN9D/α-SYN− is a good cellular model for studying the normal function of α-synuclein and examining its role in PD pathogenesis. We found that the cell viability of MN9D/α-SYN− cells was significantly decreased compared with MN9D and MN9D/CON cells, suggesting that loss of α-synuclein may induce cell injury directly or indirectly. Based on subsequent observations, we propose that a marked increase in cytosolic DA levels may account for decreased cell viability. DA can readily oxidize to generate hydrogen peroxide, superoxide and reactive DA quinones [16, 35], which are cytotoxic due to their inhibitory effects on the mitochondrial respiratory system [5, 13]. In addition, superfluous DA itself may be cytotoxic via inhibition of mitochondrial complex I activity [4]. Thus, it is also possible that increased cytosolic DA levels induced mitochondrial dysfunction resulting in decreased viability in MN9D/α-SYN− cells. α-Synuclein may be involved in the regulation of TH, the rate-limiting enzyme of DA biosynthesis. Several studies have shown that α-synuclein regulates TH gene expression. In α-synuclein over-expressing MES23.5 dopaminergic cells, TH mRNA and protein levels were significantly reduced [42]. A similar effect was observed in M17 dopaminergic cells transfected with WT α-synuclein [3]. However, in the current study, no significant differences in TH mRNA and protein levels were observed between the four groups of MN9D cells. Interestingly, a previous study using MN9D cells reported that α-synuclein over-expression did not diminish endogenous TH levels [26], which is consistent with our results. We propose that the difference in TH levels between the present α-synuclein silenced system and the previously described α-synuclein over-expression system is mainly due to the different cell types and not due to the different ways in which the cells were manipulated. Although TH protein levels remained unchanged in MN9D/α-SYN− cells, phosphorylation of TH at its Ser40 site was significantly enhanced. Furthermore, l-DOPA levels were significantly increased after NSD-1015 treatments. These results suggest that TH activity was enhanced in MN9D/α-SYN− cells, which is consistent with previous reports [24, 26]. In addition, we also found that cytosolic DA levels were significantly increased in MN9D/α-SYN− cells. Our data show that TH activity was enhanced in α-synuclein-silenced MN9D/α-SYN− cells, and that this was associated with an increase in DA biosynthesis followed by increased cytosolic DA levels. The mechanisms by which α-synuclein may regulate TH activity have not been elucidated. It is known that only the phosphorylated form of TH is active, and that the phosphorylation and dephosphorylation of TH are important in regulating DA biosynthesis [29, 37]. Furthermore, phosphorylation of TH at Ser40 results in the most prominent activation of TH [37]. Thus, it has been suggested that α-synuclein regulates TH activity by regulating the phosphorylation of TH [24]. Protein phosphatase 2A (PP2A), an important enzyme required for dephosphorylation of TH, can be activated by α-synuclein, resulting in a significant increase in TH dephosphorylation and reduction in TH activity [6]. In addition, other factors may also be involved in the regulation of TH phosphorylation by α-synuclein. For example, 14-3-3 enhances TH activity by binding directly to phosphorylated TH. α-Synuclein may directly interact with 14-3-3 [22], thereby accelerating the dissociation of 14-3-3 from phosphorylated TH. α-Synuclein may also affect TH activity by directly interacting with TH [26]. In conclusion, our data demonstrate that in PD pathology, silencing of α-synuclein expression results in enhanced TH activity and increased DA levels in neurons. We propose that in cells that unable to remove excess DA from the cytosol, DA itself and its oxidized products including reactive oxygen species and reactive DA quinones may lead to cell injury or even death. Further understanding of the normal function of α-synuclein in maintaining DA homeostasis may help to identify novel preventative or therapeutic strategies for PD.

Eur_Radiol-3-1-1914269

A new approach to the assessment of lumen visibility of coronary artery stent at various heart rates using 64-slice MDCT

Coronary artery stent lumen visibility was assessed as a function of cardiac movement and temporal resolution with an automated objective method using an anthropomorphic moving heart phantom. Nine different coronary stents filled with contrast fluid and surrounded by fat were scanned using 64-slice multi-detector computed tomography (MDCT) at 50–100 beats/min with the moving heart phantom. Image quality was assessed by measuring in-stent CT attenuation and by a dedicated tool in the longitudinal and axial plane. Images were scored by CT attenuation and lumen visibility and compared with theoretical scoring to analyse the effect of multi-segment reconstruction (MSR). An average increase in CT attenuation of 144 ± 59 HU and average diminished lumen visibility of 29 ± 12% was observed at higher heart rates in both planes. A negative correlation between image quality and heart rate was non-significant for the majority of measurements (P > 0.06). No improvement of image quality was observed in using MSR. In conclusion, in-stent CT attenuation increases and lumen visibility decreases at increasing heart rate. Results obtained with the automated tool show similar behaviour compared with attenuation measurements. Cardiac movement during data acquisition causes approximately twice as much blurring compared with the influence of temporal resolution on image quality. Introduction The introduction of multi-detector computed tomography (MDCT) has permitted the non-invasive visualisation of coronary arteries with sufficient temporal and spatial resolution. Moreover, MDCT has been used to research the assessment of coronary artery stent patency and discrimination between the presence of in-stent stenosis. Re-stenosis occurs in a substantial amount of patients which have been treated with stent implantation [1]. It has been shown that no direct visualisation of coronary in-stent re-stenosis is feasible using four-slice MDCT due to partial volume effects and beam hardening caused by metal artifacts of the stents [2–6]. Recently it has been shown that despite some limitations 16-slice MDCT is sufficiently useful for the assessment and detection of in-stent restenosis in patients with a high accuracy in comparison with conventional coronary angiography (CAG) [7]. With the emergence of 40 and 64-slice MDCT systems the assessment of lumen visibility and diagnostic accuracy of in-stent restenosis has been improved considerably with respect to the 16-slice MDCT systems [8, 9]. The artificial lumen narrowing shows a decrease of approximately 5% and the CT attenuation is reduced by approximately 35 HU [10]. Visualisation of the in-stent lumen with 16-slice MDCT allows for the assessment of coronary artery stent patency based on the measured enhancement of contrast [11]. It has been shown that the best diagnostic quality images are obtained with a sharp edge-enhancing reconstruction kernel, although the quality of the obtained images is hampered by increased noise levels compared with standard kernels [11–13]. The results originate from the comparison of the lumen visibility images pairs, which are reconstructed with different reconstruction kernels. The apparent lumen width is measured using a digital measuring tool included in the visualisation software. This method, however, is subjective to the observer and it is difficult to determine the exact boundary of the stents in the gradient part of the image. In addition, the image quality of the stents is very sensitive to the movement of the heart and image quality diminishes at heart rates higher than 75 beats per min (bpm) [14, 15]. In previous studies, a patient population was used to investigate image quality of coronary artery stents at high and low heart rates on four- and 16-slice MDCT [14, 15]. To our knowledge no systematic study has been published about the image quality of coronary artery stents on 64-slice MDCT. In this study, we therefore aimed at describing the correlation of image quality of coronary artery stents and heart rate using 64-slice MDCT in an ex-vivo setting by using an anthropomorphic moving heart phantom. The purpose of our study was to assess the lumen visibility of coronary artery stents at various heart rates with an automated objective method using a moving heart phantom on a 64-MDCT system. Theory Coronary imaging is hampered by motion artefacts originating from the relative large velocity scale of the coronary arteries within the cardiac cycle compared with the temporal resolution of the MDCT acquisition technique [16]. This movement results in motion artefacts in the reconstructed images, expressed as blurring. The amount of blurring depends on (1) the amount of movement of the imaging target MS and (2) the acquisition time (AT). The amount of movement of the coronary artery is a function of the heart rate (HR) of the patient. The temporal resolution of a MDCT system is a function of the reconstruction method used and HR [17, 18]. This means that the resulting image quality (IS) can be expressed as in which MS describes the amount of movement of the coronary artery as a function of heart rate, AT describes the acquisition time as a function of heart rate and g is a weight factor. The heart rates can be ranked from low to high. With this ranking, a relative grading can be made for the function MS. In addition, the acquisition times can be ranked from high to low. With this ranking, a relative grading can be made for the function AT. The results of this grading are shown in Electronic supplementary material (ESM) Table 1, where a high score implies a high image quality. Since there is no cardiac movement at 0 bpm, this heart rate was graded with the highest score for both factors. Materials and methods Nine commercially available stents were used and stent dimensions were measured using a digital calliper. The stent properties are summarised in ESM Table 2. The stents were inserted into plastic tubes and the tubes were filled with contrast fluid (Visipaque 320, Amersham Health, Little Chalfont, UK) diluted to an attenuation value of approximately 200 HU simulating contrast enhanced blood. The tubes were wrapped in horse fat with an attenuation value of −100 HU to simulate the in vivo situation of epicardial fat (Fig. 1left). Fig. 1Left: schematic figure showing the experimental setup of the contrast fluid filled tube surrounded by horse fat in the axial plane. The numbers depict the HU values of the different relevant structures. Right: moving anthropomorphic heart phantom with artificial coronary arteries Next, the stents were attached to a silicon, moving, anthropomorphic heart phantom (Limbs & Things, Bristol, UK) with artificial coronary arteries (Elastrat, Geneva, Switzerland) (Fig. 1right). The phantom is connected to a respirator, which was used to control the heart rate. The heart rate was set at values of 0, 50, 60, 70, 80, 90 and 100 bpm. Measurements have been performed on a 64-detector CT-scanner (Somatom Sensation 64, Siemens, Forchheim, Germany). The scan parameters were 120 kV, 120 mAs, 370 ms rotation time and 64 × 0.6 mm collimation. Image reconstruction was performed using a sharp convolution kernel (Siemens B46f) with a reconstruction slice width of 0.75 mm and a 0.5-mm increment. ECG-gating was used during scanning and the images were retrospectively reconstructed at 25% of the RR-interval corresponding to maximum expansion of the heart phantom. Afterwards the stents were visualised on an Aquarius workstation version 3.3 (Terarecon, San Mateo, USA). We used two independent methods to asses the lumen visibility of the stents in the reconstructed images. In the first method, the average HU-value in the stent lumen was manually measured with a standard region of interest (ROI) technique. A window level/width of 300/800 was used for visualisation with an average ROI of 5.0 mm2 and 3.9 mm2 in the longitudinal and axial plane, respectively (Fig. 2). The lumen measurements were used to calculate the mean CT attenuation in the stent lumen. In the second automated method, screenshots were taken from every stent in the longitudinal and the axial plane with a window level/width of 512/2,048. The screenshots were used to perform the automated lumen visibility measurements using a dedicated tool. Fig. 2Measuring the CT attenuation in the lumen. Left: measurements in the axial plane, Lekton Motion at 60 bpm. Right: measurements in the longitudinal plane, Lekton2 at 90 bpm. Calculated by the Aquarius workstation are mean HU-value, standard deviation and area of ROI The automated lumen visibility measurements were performed with an Automatic Stent Visibility Calculation (ASVC), a custom-build Matlab tool (The Mathworks, Natick, USA). The tool constructs the average attenuation profile through the stent in the longitudinal and axial plane. The calculation of the average attenuation profile in the longitudinal plane is done as follows; attenuation profiles are calculated from position A to B for every image line perpendicular to the stent central axis, where the position A and B are determined by the user (Fig. 3left). The average longitudinal profile is calculated from the individual longitudinal profiles. The calculation of the average attenuation profile in the axial plane is doen as follows: after determination of the centre of the stent by the user, 180 attenuation profiles are calculated evenly distributed around the centre (Fig.3right). The average axial attenuation profile is calculated from the individual axial profiles. Fig. 3Stent images (left: Jostent Stent Graft at 60 bpm; right: Taxus at 80 bpm) showing the positions of the individual profiles for calculation of the average stent profile in the longitudinal plane (left) and the axial plane (right) The profile depth of the lumen (D), the full width at half maximum (FWHM) and the total width (TW) are calculated from both average profiles (ESM Fig. 4). We define the percentual width (Wp) as a percentage of the FWHM and TW The percentual width is used instead of the absolute width to compensate for different magnifications of the stent images. From the profile depth (D) and Wp, the lumen visibility (LV) is defined by If the left and right edge of the attenuation profile are at different heights, D is calculated from the average height. The stent images at individual heart rates were ranked according to their CT attenuation (7=least attenuation, 1=most attenuation), FWHM (7=largest width, 1=smallest width) and D (7=largest depth, 1=smallest depth) in the axial and longitudinal plane. The difference between the CT attenuation values in the axial and longitudinal plane were compared using the Wilcoxon test at a significance level of 5%. The correlation between heart rate and CT attenuation, and the correlation between heart rate and LV, were analysed by calculating the Spearman rank correlation coefficient at the significance level of 5%. Results CT attenuation in the stent lumen The results for the HU-value measurements are summarised in ESM Tables 3 and 4 for the longitudinal and axial plane, respectively. The CT attenuation in the stent lumen was calculated for each heart rate and the results are shown for the longitudinal and axial plane in ESM Figs. 5 and 6, respectively. The error margins have been omitted to improve readability of both figures. All stents showed increased attenuation with increasing heart rate in the longitudinal plane. The CT attenuation in the longitudinal plane showed an increase from 0 to 100 bpm between 75 and 221 HU for the Taxus and Bx Sonic stent, respectively. The average increase was 139 ± 49 HU. The average Spearman coefficient for all stents was 0.66. All stents showed increased attenuation with increasing heart rates in the axial plane. The CT attenuation in the axial plane showed an increase from 0 to 100 bpm between 76 and 252 HU for the Bx Sonic, Multi-link Zeta and the Jostent, respectively. The average increase was 148 ± 69 HU from 0 to 100 bpm. The average Spearman coefficient in the axial plane was 0.58. The average increase of CT attenuation over both planes was 144 ± 59 HU. Using the ASVC, the FWHM and depth of the stent profile were determined. The results for the FWHM are summarised in ESM Tables 5 and 6; the results for the profile depth are summarised in ESM Tables 7 and 8. The standard deviation for every measurement is given in parentheses. The LV was calculated using equations 2 and 3 at each HR using the measured width FWHM and depth D. The data are plotted in ESM Figs. 7 and 8 for the longitudinal and axial plane, respectively, and have been fitted to a line. Error margins have been omitted to improve readability. All stents showed decreasing LV with increasing heart rate in the longitudinal plane. The LV decreased from 0 to 100 bpm between 7.5% and 48.6% for the Taxus and Cypher stent respectively. The average decrease in LV was 26.8 ± 13.4%. The average Spearman coefficient was 0.47. All stents showed decreasing LV with increasing heart rates in the axial plane. The LV decreased from 0 to 100 bpm between 19.8% and 48.8% for the Lekton and Multi-link Zeta stent respectively. The average decrease in LV was 30.9 ± 10.4%. The average Spearman coefficient was 0.58. The average decrease of LV over both planes was 29 ± 12%. Image scoring The measurements are shown in ESM Fig. 9 after grading and averaging as described in the methods section. From a least squares fit, we found an optimal value of g = 0.63 ± 0.06. Discussion Coronary artery stenting is a successful method to treat stenosis. However, re-stenosis in the stent may happen after the procedure. This makes check-ups a necessary procedure to perform [19, 20]. MDCT is one the candidates for visualisation of the lumen of coronary artery stents. Although the results look promising, the image quality is hampered by cardiac movement and metal artefacts [21, 22]. This may result in blurred images and an exaggerated thickness of the stent struts compromising the visibility of the lumen. The 64-MDCT scanner has increased spatial and temporal resolution compared with previous CT scanner generations [23, 24] and increased LV of coronary stents is expected with these new scanners. Although 64-MDCT imaging of coronary artery stents show an improved image quality with respect to previous CT scanners, the images still show blooming artefacts and blurring [9]. The apparent width is measured in a generally accepted method to assess the stent lumen, but this method is very subjective to the user [25]. In contrast, our method, developed in this study to analyse the stent lumen, is an automated method. It enables the possibility of a systemic evaluation of the stent lumen at various heart rates. The in vivo conditions were approached as much as possible using contrast fluid and fat. Influence of increasing heart rate To our knowledge there have been no previous studies systematically investigating the relation between heart rate and image quality of coronary stents using 64-MDCT. There are, however, some studies with 16-slice MDCT assessing coronary arteries, which concluded that for successful cardiac imaging a heart rate below 75 or 70 bpm is necessary [14, 15, 26–29]. In a study with 40-MDCT by Gaspar et al. [8], patients were given an oral beta-blocker if their heart rate was higher than 65 bpm, and in a study with 64-MDCT, patients were given beta-blockers if their heart rate was higher then 70 bpm [30]. Ferencik et al. [23, 30] concluded that a low heart rate is an important prerequisite for excellent image quality. ASVC Two different methods were used to assess the relationship between heart rate and image quality of coronary stents. The first method, the CT attenuation measurement, is a commonly used method. The second method, the ASVC technique, is a new approach. Both methods show a negative correlation between image quality and heart rate as expressed by the linear fits. The fits, however, show small significance. This small significance can be explained by the fact that the image quality does not depend entirely on heart rate, which shows a linear behaviour, but also on temporal resolution, which shows a non-linear behaviour, as can be seen from ESM Fig. 9. Despite the poor quality of the fit, the results obtained with this new ASVC method are in agreement with previous studies using generally accepted methods as stated before. Therefore, we conclude that our developed tool is a valid method for the analysis of coronary stent lumen. Image scoring The image scoring results (Fig. 9) show a varying resemblance between the theoretical scoring and the measured scoring. This resemblance depends on the proportion g and varies between 0 (IS completely depends on AT) and 1 (IS completely depends on MS). The best resemblance was found for g = 0.63. This implies that the cardiac movement causes almost twice as much blurring in the CT image compared with blurring caused by the limited temporal resolution of the acquisition method. Multi-segment reconstruction (MSR) has been developed to further increase the temporal resolution [6, 18, 32]. The data acquisition at a rotation time of 370 ms and a HR > 65 bpm has been performed during two consecutive heart cycles using a two-segment MSR. At heart rates of 70 and 90 bpm, the data acquisition is approximately evenly distributed over two consecutive heart cycles. At 80 and 100 bpm, however, the majority of data is acquired in the first heart cycle, and the remaining portion in the second heart cycle. From Fig. 9, we can deduce that for HR of 70 and 90 bpm the measured IS is smaller than the theoretical IS. For HR of 80 and 100 bpm, the measured IS is larger than the measured IS. From this we can conclude that it is beneficial to the image quality to acquire during one cycle and that the MSR technique is not beneficial to image quality. In contrast to this, Halliburton et al. [31] showed that MSR is beneficial for 16-slice MDCT. Two other studies concluded that MSR was not beneficial to the image quality for four-slice MDCT, in accordance with our results [31, 32]. This can be explained by the fact that the coronary artery has to be in exactly the same position in the two heart cycles for the MSR algorithm to work properly. However, in general the starting position of the arteries at the beginning of the heart cycle will not exactly be the same as in the previous heart cycle. This phenomenon will introduce additional motion artefacts which result in extra blurring to the image, as has been shown before by Greuter et al. [33]. Reducing blurring Cardiac movement and limited temporal resolution will introduce blurring in CT images of coronary arteries. The result for image scoring showed that the influence of cardiac movement is almost twice as large as the influence of the temporal resolution. To reduce blurring, it is therefore more efficient to reduce the heart rate than to increase the temporal resolution. Limitations These results have been obtained using a moving heart phantom. It remains uncertain how well these results are applicable to patients. However, a previous study showed that the movement of the heart phantom is a good approximation to the in-vivo situation [18]. Furthermore, in order to approach the in-vivo situation as close as possible, contrast fluid and fat were used, with CT attenuation values equal to those in-vivo. Screenshots captured with the visualisation software were used to perform LV measurements. A loss of information is expected due to image compression used in the .jpg format of the screenshots. However, a comparison between the screenshot method and direct analysis using dicom files showed no observable difference in results. Conclusion We have shown that at increasing heart rates the CT attenuation in the stent increases and the lumen visibility decreases. A new approach to asses the stent lumen has been described which shows similar results to CT attenuation measurements and previous studies. The cardiac movement during data acquisition causes approximately twice as much blurring compared with the influence of temporal resolution. We conclude that a lowering of the heart rate is more beneficial to image quality than using a multi-sector reconstruction technique. In addition, we conclude that it is beneficial to image quality to acquire data in one cardiac cycle. Electronic Supplementary Material Below is the link to the electronic supplentary material. Fig. 4 Figure showinf the profile of the Bx Sonic at 80 bpm with the different variables which are determined by the ASVC; the dotted blue line depicts the error margins of the profile. TW total width; D depth; FWHM=full width half minimum (DOC 56 kb) Fig. 5 CT attenuation measured in the stent lumen at various heart rates in the longitudinal plane (DOC 76 kb) Fig. 6 CT attenuation measured in the stent lumen at various heart rates in the axial plane (DOC 77 kb) Fig. 7 Lumen visibility plotted versus heart rate in the longitudinal plane (DOC 75 kb) Fig. 8 Lumen visibility plotted versus heart rate in the axial plane (DOC 74 kb) Fig. 9 The measured IS (green), the theoretical IS for g = 0.63 (blue), the theoretical IS for g = 0 (dotted red), the theoretical IS for g = 1 (dotted orange) (DOC 53 kb) Table 1 Acquisition times for a 64-MDCT with 370 ms rotation time, number of cycles used and theoretical scores for every HR used in this study. AT acquisition time; MS the amount of movement of the imaging target (DOC 32 kb) Table 2 Commercial name, manufacturer, material, length and diameter of the stents used in the study (DOC 33 kb) Table 3 HU-value measurements in the longitudinal plane (DOC 34 kb) Table 4 HU-value measurements in the axial plane (DOC 34 kb) Table 5 Results of the measured FWHM in the longitudinal plane (DOC 34 kb) Table 6 Results of the measured FWHM in the axial plane (DOC 34 kb) Table 7 Results of the measured profile depth in the longitudinal plane (DOC 34 kb) Table 8 Results of the measured profile depth the axial plane (DOC 34 kb)

Clin_Oral_Investig-4-1-2238780

Pathological or physiological erosion—is there a relationship to age?

This conventional literature review discusses whether pathological tooth wear is age dependant. It briefly reviews the components of tooth wear and the prevalence of tooth wear in children, adolescents and adults. The emphasis on terminology relating to tooth wear varies. In some countries, the role of erosion is considered the most important, whereas others consider the process to be a combination of erosion, attrition and abrasion often with one being more dominant. The importance of tooth wear or erosion indices in the assessment and the evidence for progression within subject and within lesions is described. The data from the few studies reporting pathological levels of wear reported in children and adults are discussed, in particular its relationship with age. There is little evidence to support the concept that pathological levels of erosion or wear are age dependant. There is, however, some evidence to suggest that normal levels of erosion or wear are age dependant. Introduction There has been considerable interest recently on the epidemiology and pathogenesis of dental erosion. Much of the epidemiology has been investigated in children and adolescents rather than adults and so the validity of any association between severity and age is based on clinical observations seen primarily in these young people [1, 3, 7, 17]. The aim of this paper is to consider and investigate the concept that pathological erosive tooth wear is age dependant. There is some debate within the dental academic community on the appropriateness of terminology. Many academics recognise the term tooth wear as encompassing erosion, attrition and abrasion. However, other researchers focus upon acid erosion often using the term to describe what others would call tooth wear and use the term erosion in a context which others might not agree. Whilst the definitions of erosion, abrasion and attrition are accepted, the relative importance of these causes is not. Therefore, in this paper both terms are used where appropriate to convey meaning interpreted by many researchers working in this field. Tooth wear and erosion The causes of tooth wear are considered to be erosion, abrasion, attrition and possibly abfraction. Traditionally, erosion is associated with loss of enamel and dentine from acids either intrinsic [4] (gastric) or extrinsic (dietary) [29]. Abrasion is more commonly associated with wear from surfaces other than teeth, whereas attrition is wear from tooth to tooth contact [26]. Abfraction is a considered by some to be a component of tooth wear, but so far apart from some laboratory studies, there is little clinical evidence to provide support for its role [9]. However, different countries interpret the aetiologies in different ways [8]. Many European countries focus attention upon erosive tooth wear, whereas in North American countries, abrasion or attrition is more commonly recognised whilst the role of erosion is less understood. In the United Kingdom, although the importance of erosion is acknowledged, the impact of abrasion and attrition is also recognised, and the term tooth wear is more commonly used. When interpreting studies from different parts of the world, some consideration of these subtle differences should be understood. The major difference seems to be the impact of attrition. For many clinical situations, the impact of wear on the teeth is a combination of attrition and erosion particularly on the incisal or occlusal surfaces of teeth (Figs. 1 and 2) [10]. On the buccal or lingual surfaces, erosion is usually more dominant, but the impact of abrasion should not be forgotten. Since it is almost impossible to tell from the appearance of a lesion what is the underlying cause, it maybe more correct to use the term tooth wear. But there are circumstances in which one cause, more commonly erosion, is the dominant feature. Although the definitions of tooth wear and erosion are quite different, they can often be used to describe the same process. Fig. 1The wear on the occlusal/incisal surfaces of the upper anterior teeth. The wear is caused by a combination of erosion and attrition. The “cupped out or ditched” areas result from the action of acids, whereas the flatter surfaces are caused by attritionFig. 2The wear on the palatal/lingual surfaces of the teeth has been caused by regurgitation erosion. It is unlikely that abrasion or attrition has contributed to the process Prevalence Tooth wear indices grade the severity of wear by recording the surface characteristics of teeth with a numerical score. These data can be used to compare wear rates between individuals and between different populations. However, there is currently no agreed consensus on a universally accepted tooth wear index. Some indices [20, 24] appear to be more widely used than others, but even these have undergone many modifications since they were first published [22]. For the most part, indices record wear on all tooth surfaces: cervical, buccal, occlusal/incisal and palatal/lingual [24]. The Smith and Knight index [24] records wear on all surfaces, but no attempt is made to relate the aetiology to the outcome of the wear on the teeth. Smith and Knight and later Robb and Smith estimated acceptable levels of wear in each age cohort [23, 25]. For the majority of the population, any wear on teeth is often limited to enamel, and dentine involvement only occurs in a relatively small proportion of the population [25]. A study by Dugmore and Rock [17] reported that 59.7% of 1,753 12-year-old children had evidence of tooth wear of which 2.7% had exposed dentine and this rose to 8.9% by the age of 14 years. Another study by Bardsley et al. [3] reported that 53% of 2,385 14-year-old adolescents had exposed dentine, but this included assessment of incisal surfaces. This latter study reported higher values in contrast to most other studies [1, 7]. From most of these studies, it is clear that in children and adolescents wear of enamel is common, almost normal, but wear of dentine exposing more than 1/3 of the tooth surface is less so. Smith and Robb observed that tooth wear in adults was an almost universal experience with up to 97% of all ages experiencing some wear on their teeth with the older aged cohorts, dentine exposure became more common [25]. In their study, Smith and Robb [25] identified that between 5–7% of 1,007 adults had wear that could justify treatment, in all age cohorts. This is the only study published to date, with a large sample, which provides data on tooth wear in all age groups in adults. Other studies have reported the levels in specific age cohorts [20]; Donachie and Walls [14] reported the degree of tooth wear in a sample of 586 adults aged 45 and over in Newcastle, UK and reported no correlation between severity of wear and age. The data available to guide our knowledge on the levels of tooth wear in adults are therefore limited. Progression of tooth wear The term pathological tooth wear has been used to describe unacceptable levels of wear [18, 23, 25]. It is usually interpreted as meaning a level of wear that could justify operative treatment. However, finding a scientific way to interpret this concept is challenging. For pathological tooth wear to have meaning, the normal levels in different age groups are required to allow comparison, but the basic information on the pathogenesis of tooth wear is unknown. For instance, it is not known if tooth wear is episodic or continual throughout life. A few authors have reported data from longitudinal studies on adolescents and adults that indicate the development of new lesions [16, 19]. Lussi et al. [20] reported the results of a prevalence study on 26–30- and 46–50-year-old Swiss adults. In a later study, the same group of researchers reported the progression of wear in the same subjects over a 6-year period [19] and observed that wear progressed on the facial, occlusal and cervical surfaces and was more common in the older aged groups. However, the authors made no attempt to define what level of tooth wear was acceptable and what was not. Other studies have reported increase in the incidence of tooth wear in adolescents [16], but longitudinal data remain sparse. There are no studies that report the progression of the same lesion within the same subject apart from one study which used a tooth wear index to record wear on study models taken from the subject's teeth [5]. They observed that only 7% of tooth surfaces showed any change over a median time of 26 months (IQR 14–50 months). The paucity of data on the severity of tooth wear in different age groups and its progression means it is difficult to be able to predict what is the pathophysiological behaviour of tooth wear. A number of authors have published methods to measure tooth wear and erosion using profilometric scanners to accurately map teeth [2, 13, 21, 28]. But these methods have reported their findings on relatively small numbers, and the methods are as yet unsuitable for larger studies. Often the time needed to scan a single tooth can take up to one hour and therefore they remain research tools for small studies. However, data from these studies suggest that wear on teeth may progress between 3.7 μm at 6 months [6] and 5.56 μm/month [21] to 18.3 μm/month [28]. There are no data to indicate if particular teeth or tooth surfaces have a greater potential to wear or whether abrasion, attrition or erosion is more important in the progression. Furthermore, correlating this rather limited data to the general population is not possible, and therefore, most researchers rely upon epidemiological studies to record the severity of wear. Pathological tooth wear The concept that an unacceptable level of wear for a particular age group was first proposed by Eccles, Smith and others [18, 23, 25]. These and other authors argued that tooth wear continued throughout life as it was part of a normal physiological process. Berry and Poole [11, 12] hypothesised that in common with other mammals, humans have compensatory mechanisms that adapt to wear of teeth. They based their argument on comparative anatomy of mammals, in particular large herbivores, and suggested that tooth wear was beneficial to the efficiency of mastication. A gradual reduction in height and shape of cusps on molar teeth, they argued, led to improved masticatory efficiency. Their hypothesis remains unique and unproven but raises a number of interesting concepts. Smith and coworkers [23, 27] introduced the term unacceptable levels of wear and later re-termed it as pathological tooth wear. The authors estimated unacceptable levels of wear based on their clinical judgement. Their so-called threshold levels were calculated for different age groups and used as an indicator for the necessity of operative intervention. However, these thresholds were judgements made by the authors, albeit based on clinical experience and previous research [25]. Smith and Robb [25] reported in their paper that the ultimate decision on the threshold values was the clinical experience of the first author. The data were analysed and re-analysed until the results matched the clinical judgement of the authors. Whilst this empirical assessment may have had some value at the time of publication, the ability to reproduce agreed thresholds in subsequent investigations would be challenging and have not been wholeheartedly supported by other researchers. Donachie and Walls [15] argued strongly that the threshold levels set for older age groups were inaccurate as they were based on a relatively small sample and so in their 1996 paper modified values were used [25]. We cannot rely upon the clinical judgement of one or two researchers to define what are pathological levels of tooth wear. Scientists need to have more defined and reproducible values. There are emerging data to suggest that tooth wear is common in all age groups [3, 17, 20, 25]. Smith and Robb [25] reported in their study of 1,007 subjects, aged 16 and over, that within each age cohort a proportion had higher grades of tooth wear [mean 5.1%] than the others. Virtually every other study on the prevalence of tooth wear also reports that a small proportion, generally between 2% and 10%, have higher levels of tooth wear than the majority. If these data are representative of the population at large, it is possible that a percentage of each age cohort has higher than normal levels of wear and this could be termed pathological. The hypothesis that pathological tooth wear is age related depends on who is assessing the impact. State health care authorities, private insurance schemes, industry, dentists and patients all have different interpretations on what is pathological and what is not. For patients, loss of enamel could be considered pathological particularly if they are focussed upon the appearance of their teeth, whereas dentists may consider intervention is needed when dentine is involved, but their capacity to treat is affected by the limitations of restorative materials. On the other hand, state health care providers may take a much longer term view, with the assumption that a tooth remains functional, if not aesthetic, when operative care is not imperative. Based on the current data, it is too challenging to suggest that tooth wear is an age-related phenomenon. There is some justification to this hypothesis based on the current data from children, adolescents and adults. Clinical experience suggests that as adults age they tend to develop more wear on the occlusal and incisal surfaces of teeth. It is likely, therefore, that some progression of wear on teeth is age related. However, this assumption needs investigating. Evidence partly from prevalence studies and partly from accurate measurement of tooth wear by profilometry tends to give support to this hypothesis. It must be remembered, however, that the prevalence studies at best report on just over 1,000 subjects. This is extremely small considering the populations involved. Tooth wear indices remain the most convenient and reproducible method to grade severity but are limited by incompatible criteria. It is imperative that a consensus is developed to build a simple and reproducible index, used by researchers so that data on the prevalence of tooth wear, particularly in adults, can be investigated. Conclusion The physiological wear of teeth is probably an age-related phenomenon. As the teeth continue to function and be challenged by erosive, attritive and abrasive factors, there will be change to the surfaces of teeth. This is most commonly seen on the incisal edges of the upper and lower incisors. As the challenges continue throughout life, it is not surprising therefore to see, as reported in Smith and Robb's study, that most people have some evidence of wear [25]. Therefore, small changes or evidence of gradual wear throughout life is probably a feature of the ageing of the dentition. When pathological levels of wear are considered, the situation is less clear. There are insufficient data from epidemiological studies on adults to be definitive. But where data are present, it suggests similar proportions of severe levels of wear are observed in each age group, and it could be argued that this was independent of age [3, 17, 25]. It seems, therefore, that although tooth wear is an age-dependant phenomenon, severe tooth wear is not.

Anal_Bioanal_Chem-4-1-2359830

Investigation into mercury bound to biothiols: structural identification using ESI–ion-trap MS and introduction of a method for their HPLC separation with simultaneous detection by ICP-MS and ESI-MS

Mercury in plants or animal tissue is supposed to occur in the form of complexes formed with biologically relevant thiols (biothiols), rather than as free cation. We describe a technique for the separation and molecular identification of mercury and methylmercury complexes derived from their reactions with cysteine (Cys) and glutathione (GS): Hg(Cys)2, Hg(GS)2, MeHgCys, MeHgGS. Complexes were characterised by electrospray mass spectrometry (MS) equipped with an ion trap and the fragmentation pattern of MeHgCys was explained by using MP2 and B3LYP calculations, showing the importance of mercury–amine interactions in the gas phase. Chromatographic baseline separation was performed within 10 min with formic acid as the mobile phase on a reversed-phase column. Detection was done by online simultaneous coupling of ES-MS and inductively coupled plasma MS. When the mercury complexes were spiked in real samples (plant extracts), no perturbation of the separation and detection conditions was observed, suggesting that this method is capable of detecting mercury biothiol complexes in plants. Introduction Mercury is one of the most important elements to consider when environmental pollution is concerned, and ever since the Minamata disaster in 1959, the organic compound methylmercury has been in focus owing to its enhanced toxicity and its biomagnification properties in the food chain [1]. Following the Minamata disaster, the differentiation of inorganic mercury in the form of Hg2+ from organic mercury, namely in the form of methylmercury, has become a major task. Mercury speciation in complex matrices is not easy to achieve [2], and more and more sophisticated analytical methods have been developed in the past few years in order to obtain reliable quantitative results. In particular, species-specific isotope dilution mass spectrometry (SSIDMS) employing stable mercury isotopes has been a major breakthrough [3, 4]. Otherwise, experiments with stable isotope tracers introduced into the environment have given invaluable information on the distribution and transformation behavior of mercury in the environment [5–7]. Mercury has also been identified as a global pollutant, and the importance of new analytical methods towards a more complete understanding of species distribution, pathways and global impact has been highlighted recently [8]. As of today, the most common application of mercury speciation in biota is focused on the determination and distinction between methylmercury and inorganic mercury in the forms of MeHg+ and Hg2+. To separate the mercury species from their matrix, usually extraction or soft digestion methods are applied, which conserve the C–Hg bond, keeping the methylmercury moiety intact. However, it is more than unlikely that mercury and methylmercury occur as free cations, be it in biota or in the environment, and rather they are likely bound to sulfur-containing biomolecules, e.g. in fish [9], or present as chloride complexes, e.g. in seawater [10]. But, with the usual practice of mercury speciation, the counterion bound to the mercury species is lost and, even worse, completely disregarded. Thus, the information on the molecular entity of the mercury species is wasted—strictly speaking, this is not really speciation, but rather a fractionation method. Now, in spite of the enormous progress made in mercury speciation, many questions relating to mercury and its behavior in biota remain unsolved mysteries: Why is methylmercury highly bioaccumulated, while Hg2+ is not? Why are different organs targeted by methylmercury as opposed to Hg2+, and how can the enormous latency period (up to several months!) for methylmercury intoxication be explained [1, 11]? Little is known today about the mechanisms that enable mercury uptake into cells, and its subsequent distribution and bioaccumulation in biota. But, mercury and methylmercury are well known to bind to biothiols abundant in biota and form stable complexes via Hg–S bonds. Especially, in a model system, mercury complexes with cysteine (Cys) as a ligand have been found to be able to cross cell membranes, and the Hg(Cys)2 complex has been defined as a likely form in which Hg2+ is present. Zalups [12] and Bridges and Zalups [13] proposed that Hg(Cys)2 mimics the cystine molecule, and thus may use the active cell transport systems usually used for cystine transport into the cell. Although the theory of molecular mimicry has recently been questioned using structural comparison obtained from X-ray absorption studies of the complexes concerned [14], it is still commonly agreed that mercury and methylmercury are most likely bound to and transported as complexes formed with biothiols abundant in biota. Other studies employing direct speciation methods in biota, like extended X-ray absorption fine structure and X-ray absorption near-edge structure, underpin the assumption that methylmercury and mercury are bound to sulfur in a complex matrix [9]. Although mercury biothiol complexes have not been identified in vivo so far, mercury biothiols certainly play a key role in cell uptake, distribution and subsequently toxicity of mercury in biota [1, 11–13]. But, even state-of-the-art analytical methods for mercury speciation fail to provide us with all the necessary information, i.e. the complete molecular structure of the mercury compounds present in vivo. Therefore, our interest lies in the development of an analytical method that allows the direct detection of mercury and methylmercury biothiol complexes in biota. In this paper, we describe an analytical method allowing the species identification of mercury and methylmercury biothiols as intact molecules. As example species, Cys and glutathione (GS) complexes with mercury and methylmercury were synthesised and analysed. Cys is a small sulfur-containing amino acid, while GS is a small peptide comprising only three amino acids: γ-glutamyl (γ-Glu), Cys and glycine (Gly). Like Cys, this compound is usually abundant in living cells in millimolar concentrations, and it is a key compound for the cell’s redox state control: GS is the smallest building block for the synthesis of phytochelatins, which are produced in plants as a response to metal stress, and are believed to play a paramount role in metal detoxification in plants [15]. Plants are well known to accumulate mercury when grown on polluted soil [16, 17], and a future object of study for mercury exposure will be rice (Oryza sativa); therefore, we tested the method and the species stability in the matrix of a real plant extract derived from rice plants. Experimental Instrumentation The instrumentation used for this work consisted of an Agilent Technologies (USA) suite comprising an 1100 series high-performance liquid chromatography (HPLC) system, an electrospray mass spectrometer (ES-MS) (XCT ion-trap mass spectrometer) and an inductively coupled plasma mass spectrometer (ICP-MS) (7500 c series). The HPLC system was equipped with an automatic degasser, a gradient pump, a thermostated autosampler tray and a thermostated column device. For the chromatographic separation of the mercury biothiols, an HPLC method was adapted from that of Raab et al. [18]. Species separation was carried out using an Agilent Zorbax Eclipse XDB C-18 (4.6 mm × 150 mm, 5–μm) reversed-phase (RP) HPLC column using a gradient elution with eluent A being 0.1% formic acid in water and eluent B being 0.25% formic acid in methanol. The gradient programme went from 100% eluent A to 50% eluent B in 20 min with a flow rate of 1 mL/min, and a volume of 50 μL was injected via the autosampler. The autosampler tray was cooled to 4 °C for all experiments. Coupling of the HPLC instrument to the ES-MS and ICP-MS system was performed either individually using PEEK capillary tubing (1.6-mm outer diameter, 0.3-mm inner diameter), or simultaneously via a micro flow splitter (Upchurch, UK). In split mode for simultaneous coupling, 80% of the HPLC eluent was directed into the ES-MS system, while 20% went into the ICP-MS system. Hg(Cys)2, HgMeCys, Hg(GS)2 and HgMeGS solutions were prepared as described above in concentrations of 10 mg/L (as Hg), each in 0.1% formic acid. From these solutions, a mixture containing all four compounds was prepared at equal concentrations, resulting in a solution with a concentration of 2.5 mg/L (as Hg) of each compound. A continuous internal standard (Rh, 20 μg/L) was mixed with the HPLC effluent prior to the ICP-MS nebuliser via a Teflon T-piece. This internal standard enables the monitoring of overall instrument sensitivity and plasma conditions, and may potentially be used to correct the mercury signal for changes in intensity due to matrix effects stemming from the introduction of methanol during gradient elution [19]. The ES-MS system was also used in direct injection mode for the species’ fragmentation experiments. Here, the sample was introduced into the ES source via a syringe pump using a 1-mL glass syringe. The ES-MS and ICP-MS instrumentation parameters can be found in Table 1. Table 1ICP-MS and ES-MS parametersInstrumentSettingsICP-MSInstrumentAgilent Technologies 7500 cTorchStandard Ar gas flows  Cooling gas16 L/min Auxiliary gas1 L/min  Nebuliser gas0.95 L/min  Optional gas: (O2)5%  Spray chamberScott, cooled (2 °C)  NebuliserPFA, microconcentric   Internal standardContinuous aspiration, 20 μg/L Rh in 1% HNO3  ConesPlatinum  Isotopes monitored200Hg, 202Hg, 103Rh, 34S, 32S16OES-MSInstrumentAgilent Technologies XCT ion-trap mass spectrometerIon sourceElectrospray ionisationCapillary voltage4,500 VNebuliser pressureHPLC 50 psi (0.345 MPa); direct injection 20 psi (0.138 MPa)Drying gasHPLC 12 L/min; direct injection 5 L/min Gas temperature350 °CScan windowm/z 100–900MS2 fragmentation window m/z 4PFA perfluoroalkoxy, HPLC high-performance liquid chromatography, MS2 Ion trap Reagents All reagents used were of analytical grade and were purchased from Sigma-Aldrich, UK, unless mentioned otherwise. The water used for all the preparations was of suprapure quality (18 MΩ, ELGA water system, UK). HPLC eluents were prepared from formic acid at 0.1% in H2O and 0.25% in methanol. An inorganic mercury (Hg2+) standard stock solution at approximately 1,000 mg/kg (as Hg) was prepared by dissolving solid HgCl2 in water. A methylmercury (MeHg+) standard stock solution at approximately 1,000 mg/kg (as Hg) was prepared from solid MeHgCl in methanol. For the preparation of mercury and methylmercury biothiols, these stock solutions were further diluted in 0.1% formic acid. The water for the preparation of standards was degassed using ultrasonication (10 min) followed by purging with N2 (10 min at 200 mL/min). Stock solutions were kept in a freezer at −20 °C; further dilutions were kept for a maximum of 1 week at 4 °C in the dark. Reduced l-cysteine and reduced GS were dissolved in water at a concentration of 1 mg/mL and were prepared fresh daily. Synthesis of HgMeCys, HgMeGS, Hg(Cys)2 and Hg(GS)2 From solutions prepared as described above, mercury and methylmercury biothiols were synthesised by stoichiometrically adding the respective amounts of dissolved biothiol (GS or Cys) to a defined volume of mercury or methylmercury solution. All steps were carried out in a glove bag under a N2 atmosphere in order to avoid oxidation of the thiols. Usually, mercury–thiol compounds were prepared fresh daily. Modelling the MeHgCys conformation: ab initio and density functional theory calculations The conformation of MeHgCys might involve bonding via either an oxygen atom in the carboxylic group or a nitrogen atom from the amino group, and either an uncharged or zwitterionic form could be possible. In an attempt to predict which, if any, of these conformations/configurations might be favoured in the gas phase, a series of ab initio and density functional theory (DFT) calculations were performed on a variety of possible structures. Geometry optimisations were performed using the electronic structure software GAMESS-US [20] at both the DFT B3LYP and the ab initio MP2 levels of theory. The 7 September 2006 release of GAMESS-US was used for the DFT calculations and the 24 March 2007 release was used for the MP2 calculations. In both sets of calculations the 6-31G(d,p) basis set [21] was employed for all atoms with the exception of mercury, which was described by the SBKJC effective core potential basis [22]. A subsequent vibrational analysis at the optimised geometries showed them to be true minima and also provided zero-point energies (ZPEs) for use in correcting the energies obtained from the minimisations. The ZPEs were scaled by 0.961 (B3LYP) and 0.9646 (MP2) as suggested by the collected data available online from the CCCBDB database at http://srdata.nist.gov/cccbdb/. The relative populations of the conformers with the lowest ZPE-corrected energies were estimated by calculating the ratios of the Boltzmann factors [23] at 350 °C, which is the gas temperature inside the ES ion source. Plant preparation Rice (Oryza sativa) was grown from seeds in vermiculite medium and fed with regular Hoagland solution [24] three times a week. After 2 months of growth, the rice plants were harvested. The vermiculite was washed off the roots, and the plants were sectioned into roots and shoots, cut into pieces of approximately 5 mm length. Extracts were prepared according to the method of Raab et al. [25]. Briefly, 1 g of plant material was ground in a mortar under addition of liquid N2, the sample was transferred into a 15-mL Greiner tube, 3 mL of 1% formic acid was added and the mixture was left standing in ice at 0 °C for 1 h. Results and discussion The objective of this work was to develop an analytical method for the separation and identification of mercury and methylmercury bound to Cys and GS. Structural MS carried out here using an ES mass spectrometer equipped with an ion trap was used as a tool for the identification and structural characterisation on a molecular level. The information gained via the fragmentation pattern obtained from the ion-trap measurements proves invaluable for the identification of unknown molecules, along with some information on the molecules’ conformation, while the simultaneous element-selective detection of mercury via ICP-MS is the key to identifying mercury-containing biomolecules in a complex matrix amidst a variety of other organic molecules. More detailed information on the conformation of the HgMeCys molecule was obtained by a modelling approach, which underpins the findings of ion-trap ES-MS. In this paper, we focus on the structural identification of the selected mercury and methylmercury biomolecules via ion-trap ES-MS after their separation using RP HPLC. Here, all mercury compounds were measured under the same detection conditions, and all ES-MS parameters were kept constant for all four species during direct injection as well as in HPLC injection mode. Simultaneous detection with ICP-MS was applied with HPLC separation for mercury-selective determination. Ion-trap ES-MS measurements of HgMeCys, HgMeGS, Hg(Cys)2 and Hg(GS)2 using direct sample injection In a first approach of identification, mercury and methylmercury complexes with Cys and GS, synthesised as described earlier in 0.1% formic acid at a concentration of 50 mg/kg (as Hg) each, were injected as single standards into the ES source of the ion-trap MS instrument. The ES-MS parameters can be found in Table 1. The species identification was performed by using information obtained from the ES-MS and ion-trap MS data: The MH+ molecule clusterThe characteristic isotope pattern dominated by the mercury isotope distributionIn-source fragmentation of the mercury biomolecule Cys and GS moietiesIon trap MS (MS2) of the MH+ molecular ion peak In Fig. 1, the ES-MS spectra measured for the molecular ions (MH+) of the four compounds are shown. The vertical black lines represent the theoretical isotope ratio pattern of the molecules according to their molecular structure. Fig. 1Electrospray (ES) mass spectrometry (MS) spectra of the MH+ peak cluster for four mercury biothiol compounds (direct injection of single standards). The theoretical isotope pattern is shown as vertical black lines. Molecular structures as ball-and-stick models are shown on the left (Hg violet, S yellow, O red, N green, C grey, H not shown). Theoretical isotope abundance is matched to the highest-abundance peak, set at 100%. The exact mass (theoretical) is given for the 100% peak. a MeHgCys. b MeHgGS. c Hg(Cys)2. d Hg(GS)2. Cys cysteine, GS glutathione The mercury isotope pattern is very prominent, it dominates the overall isotope pattern of the molecules, and can therefore be used as a characteristic fingerprint: In small mercury biomolecules, the mercury isotope pattern is greatly preserved, even when the organic molecular structure, like in HgGS2, accounts for 75% of the molecular mass. The mass spectra show that under the ionisation conditions applied, the mercury compounds are protonated and detected by the ES-MS instrument as a molecule cluster. The experimentally obtained isotope cluster for MH+ match well with the theoretical isotopic molecule patterns calculated for each compound. The major isotope in the clusters is here referred to as the 100% abundance isotope, reflecting the usual practice in isotope abundance visualisation by defining the major mass as 100% and the less abundant isotopes as fractions of this. In-source fragmentation of HgMeCys, HgMeGS, Hg(Cys)2 and Hg(GS)2 Molecule fragmentation is usually observed during the ionisation step in the ES ion source. The fragmentation pattern obtained in this process gives information about the molecular structure of a compound, using the different fragments for reconstruction of the complete molecule. However, for our experiments, we are interested in keeping the in-source fragmentation as low as possible in order to get the main peak from the molecular ion MH+. One reason for this is that we try to obtain optimum detection limits for mercury biomolecules in real samples, and in-source fragmentation leads to a diminished MH+ intensity. Moreover, the MH+ peak gives information on the molecular mass and enables identification via the characteristic isotope pattern. Finally, where more information on the molecular structure is needed, e.g. to elucidate isomeric forms, further fragmentation is performed on selected masses within the ion-trap MS instrument, using collision-induced fragmentation. All mercury compounds were synthesised in solution in stoichiometric amounts (i.e. two molecules of thiol for Hg2+, one molecule of thiol for MeHg+). In order to determine whether the mercury compounds had reacted quantitatively with the organic thiols, we monitored the protonated molecular ions (MH+) of Cys (m/z 122) and GS (m/z 308). As only the reduced thiols are able to bind to mercury, we also checked for the oxidised dimers of Cys and GS with m/z 224 and m/z 613. The ratio between the molecular ions of the mercury complexes and those of the free reduced or oxidised thiols gives an indication for the reaction yield in solution, as well as the stability of the complexes. In Fig. 2, the mass spectra (MS+) of the four mercury compounds investigated are shown, highlighting the in-source fragments observed. In-source fragmentation can be influenced by ionisation parameters, such as capillary voltage. Here, the parameters used were as detailed in Table 1 for the direct-injection mode. Fig. 2ES-MS spectra (MS+) showing the in-source fragmentation for four mercury biothiol compounds (direct injection of single standards). a MeHgCys. b MeHgGS. c Hg(Cys)2. d Hg(GS)2 From Fig. 1 it is evident that from the four mercury compounds investigated only MeHgCys (Fig. 1a) shows extensive in-source fragmentation, resulting in two clusters of approximately the same peak height: the MH+ cluster with m/z 338 as the 100% abundance isotope and a second cluster with m/z 321 as the 100% abundance isotope, showing a mass loss of 17 from the MH+ molecular ion cluster. This mass loss can be attributed to the loss of either OH or NH3. Apart from the two most prominent in-source fragments, only one fragment exhibiting a mercury-like isotope pattern can be found at m/z 234. This may be attributed to a or a MeHgOH moiety, and accounts for just 3% of the combined peak areas of the two main peaks. Similar fragmentation has been described by D’Agostini et al. [26], who suggested the loss of NH3 from the MH+ ion, and hypothesised that two isomeric species of MeHgCys are present in solution, either exhibiting a Hg–N or a Hg–O interaction. Here, we show that the Hg–N interaction is the most likely form present in the ion source, and that the in-source fragment at m/z 321 is indeed most likely formed through loss of NH3. This was achieved through the ion-trap MS2 fragmentation pattern and a modelling approach, in which the most stable conformation for MeHgCys was calculated in the gas phase. These experiments are highlighted further below. A fragment with mass 119.5 may be attributed to free Cys or cystine (doubly charged), but occurs at less than 1% abundance compared with the two main clusters. The absence of free Cys also suggests that the complex is formed quantitatively in the solution. Figure 2b shows the in-source fragmentation of MeHgGS during direct injection, clearly showing the MH+ molecular peak cluster as the dominant component. Two more fragment peaks can be identified. One fragment at m/z 395, exhibiting a cluster with the typical mercury isotope pattern, can be attributed to a loss of the γ-Glu moiety from GS. A second peak appears at m/z 308, which can be clearly attributed to protonated GS. The spectrum shows the MeHgGS molecule peak prevailing with more than 95% abundance, while the two other fragments observed correspond to approximately 1% MH+ abundance only. For Hg(Cys)2 (Fig. 2c), MH+ is the most prominent peak in the spectrum. An in-source fragment obtained from a mass loss of 17 is also observed, but accounts for less than 6% of the MH+ peak only. Another fragment with the typical mercury isotope pattern and m/z 339 the 100% abundance peak may be attributed to a moiety, similar to the fragment observed for MeHgCys: a Cys group can obviously be cleaved from the molecule, leaving a Hg–N bond behind. Two more peaks can be distinguished at m/z 241 and m/z 122, corresponding to protonated cystine (Cys–Cys) and Cys. Cystine at m/z 241 seems quite abundant, but accounts for only 10% of the MH+ cluster of Hg(Cys)2. Only traces of free Cys at m/z 122 (less than 1% of the mercury molecular cluster) were found. The spectrum of Hg(GS)2 (Fig. 2d) shows two mercury-containing fragments at m/z 544 and m/z 508 besides the most prominent peak for MH+ at m/z 815. While m/z 508 can clearly be attributed to a protonated HgGS fragment after loss of one GS group, the cluster at m/z 544 cannot be clearly identified, but might be a Hg–GS cluster with two water molecules associated. A peak at m/z 407 corresponds to the doubly charged molecule peak MH2+. Two more peaks can be distinguished: m/z 613, corresponding to protonated oxidised GS, and m/z 308, corresponding to free reduced GS. The two mercury-containing fragments correspond to 10% (m/z 544) and 15% (m/z 508), respectively, of the overall abundance, while the protonated oxidised and reduced GS only account for approximately 1% each, and MH2+ accounts for less than 2%. MH+ is found to occur at more than 70%. The ionisation efficiencies differ substantially between the four different compounds: While MeHgCys exhibits the lowest intensities, with 2 × 105 counts, the MeHgGS signal is tenfold higher. Hg(Cys)2 and Hg(GS)2, each with around 5 × 105 counts, show intensities between these two. Taking into account that MeHgCys forms an abundant in-source fragment with the same intensity as the MH+ ion, these three molecules appear to have similar ionisation behaviour, leading to comparable ratios of protonation in the ES source under the conditions applied. Conformation of HgMeCys: ion-trap MS2 fragmentation and conformation modelling Collision-induced fragmentation was performed using the ion-trap device of the ES-MS instrument. The in-source fragmentation of MeHgCys showed an important fragment with a mass loss of 17 amu with abundance as high as the MH+ molecular ion. This mass loss can be explained by either loss of OH or NH3 from MH+, and both possibilities have been postulated before [26]. Here, we use the ion-trap MS2 information on the two prevailing peaks obtained combined with a modelling approach to find the most probable conformation of MeHgCys, which may explain the fragmentation behaviour observed. As shown in Fig. 3, the MS2 spectrum of the MeHgCys molecular ion (m/z 336, 200Hg) shows a predominant fragment at m/z 318 due to loss of H2O. A second fragment at m/z 231 can be attributed to either or MeHg–O+, formed through rearrangement of the molecule under loss of the residual Cys moiety, and corresponds to the fragmentation pattern obtained in source. This finding is an indication that the Hg–S bond is not the only interaction between Cys and HgMe, but that either Hg–O or Hg–N bonding is involved, i.e. either the carboxylic or the ammonia group forms a bond to the central metal. Fig. 3Ion trap MS (MS2) spectra as obtained from collision-induced fragmentation in the ion-trap device from the two main in-source fragments obtained from the direct injection of a single standard of MeHgCys. a MS2 of MH+ at m/z 336 (Me200HgCys). b MS2 of MH+ at m/z 319 (Me200HgCys–OH or Me200HgCys–NH3) A different picture is seen when MS2 is performed on the in-source fragment ion with m/z 319 (formed through loss of OH or NH3 from 200HgMH+ at m/z 336) . Here, we can distinguish several fragments: m/z 301 (loss of H2O), m/z 288 and m/z 261, formed through the successive loss of parts of the Cys moiety, and m/z 215, corresponding to MeHg+. In contrast to the MS2 of MH+ (m/z 336), the fragment at m/z 231 is not formed. This is an indication that the group forming this fragment has been lost during in-source fragmentation. Modelling the MeHgCys conformation using ab initio and DFT calculations The conformation of MeHgCys can involve either bonding via an oxygen atom in the carboxylic group or a nitrogen atom from the ammonia group, and can carry the proton at either group. A modelling approach was used to determine the total energies for the different possible conformations of HgMeCys, including correction for the ZPE. The final conformations obtained from the geometry optimisations at the MP2 level are shown in Fig. 4 (conformers 1–6). Those obtained from the B3LYP calculations differed only by small deviations in bond lengths and angles and are therefore not shown. Two of the geometry optimisations were started in zwitterionic configurations with either a carboxylic oxygen (conformer 1) or the amino nitrogen (conformer 2) positioned close to the mercury atom. The zwitterionic form of MeHgCys was found to be unstable and optimisations led to the neutral form regardless of the initial protonation state. Of the remaining calculations, conformer 3 and 4 were started with the amino nitrogen close to the mercury, whereas conformers 5 and 6 were started with a carboxylic oxygen in this position. In all of these optimisations the initial Hg–X (X is O or N) interaction was retained throughout, suggesting that this is generally favoured in the conformational behaviour of MeHgCys. Fig. 4Modelling the MeHgCys conformation: final conformations of MeHgCys obtained from geometry optimisation at the MP2 level. Model numbers (1–6) as used in Table 2 The pattern of relative conformational energies (Table 2) is basically the same at both levels of theory, with the most energetically favoured conformations displaying the Hg–N interaction. Those conformations displaying Hg–O interaction make up the second-most thermodynamically stable grouping, whereas the extended conformer where the mercury atom is free from nonbonded interaction is considerably higher in energy than all the others. The energy differences are more pronounced at the MP2 level than at the B3LYP level and this is probably due to the fact that the perturbation theory treatment is capable of describing dispersion effects, whereas DFT is not. Dispersion effects are responsible for mediating nonbonded interactions such as hydrogen bonds and van der Waals “bonds” and therefore it would be expected that these would be important in this particular case. For this reason, the MP2 results are expected to be more reliable than those obtained with DFT since the former provide a more complete physical picture of the MeHgCys system. Table 2Conformational energies obtained for the two different models usedConformerB3LYP/6-31G(d,p) ΔE (kJ/mol)aMP2/6-31G(d,p) ΔE (kJ/mol)b17.418.0212.730.330.00.047.66.9525.821.0613.721.4Scale factors obtained from the CCCBDB database at http://srdata.nist.gov/cccbdb/aZero-point energies scaled by 0.9610bZero-point energies scaled by 0.9646 The results of the Boltzmann factor calculations based on the corrected MP2 energies indicate that at the experimental temperature approximately 98% of the MeHgCys would be present as the form displaying the Hg–N interaction, with the remaining 2% consisting almost exclusively of the Hg–O forms. Using the B3LYP energies, these calculations suggest that the slightly lower figure of 87.5% for the Hg–N forms, with one of the Hg–O conformers making up most of the remaining population. As the MP2 energies are thought to be more accurate on the basis of physical considerations, it is reasonable to assume that the figure of 98% for the Hg–N population is similarly more accurate; however, even at the B3LYP level the figures clearly point to this being the form of the molecule expected to be dominant in the gas phase at 350 °C. The use of larger and/or more flexible basis sets might lead to alterations in these results owing to improved description of the Hg–X interactions but it is felt that this would be unlikely to significantly alter the distribution of energies in this case. Assuming that the Hg–N form is dominant in the ion source, it is reasonable to assume that the in-source fragment obtained at m/z 234 (202Hg) corresponds to a (protonated) ion. This is confirmed by the occurrence of the same fragment in MS2 at m/z 231 (). The in-source fragment at m/z 319 (200Hg) does not exhibit the m/z 231 fragment during MS2, but loses the Cys completely, with MeHg+ remaining as the smallest mercury-incorporating fragment. This is suggestive of the possibility that the in-source fragment with m/z 319 has lost NH3 rather than OH – a MeHg–N fragment cannot be formed from this mother ion. However, it should be noted that besides the Hg–N interaction, a small contribution of Hg–O is possible. Ion-trap MS2 of the MH+ cluster of Hg(Cys)2, HgMeGS and Hg(GS)2 Figure 5 depicts the ion-trap fragmentation patterns obtained for the three remaining mercury-containing biomolecules. The m/z values of the MS2 spectra shown here in Fig. 5 are each defined for the 200Hg isotope. However, the chosen trap window ion width of 4 amu ensures that a major part of the ion cluster is trapped and undergoes collision-induced fragmentation. Thus, easier identification of mercury-containing fragments is possible, as the mercury pattern is greatly conserved. However, the fragment incorporating 200Hg will be trapped with the highest abundance. Fig. 5MS2 spectra as obtained from collision-induced fragmentation of the MH+ molecular cluster in the ion-trap device from three mercury biothiols. a MeHgGS. b Hg(Cys)2. c Hg(GS)2 Hg(Cys)2 The ion-trap collision-induced fragmentation of Hg(Cys)2 exhibits a most abundant fragment at m/z 434, corresponding to the loss of H2O, and a second important fragment at m/z 337, which corresponds to the loss of one Cys molecule leaving a nitrogen atom bound to mercury (CysHg–N+). This behaviour is similar to what was found for the fragmentation of MeHgCys, and seems to be typical for mercury bound to a Cys moiety. No other fragments containing mercury can be observed, and no unbound Cys molecules were detected. MeHgGS The MS2 of MeHgGS shows one high-abundance fragment at m/z 375, corresponding to the loss of a Glu moiety and OH, plus two low-abundance fragments at m/z 509 (loss of H2O) and m/z 393 (loss of Glu). GS is not lost during this fragmentation, but rather the Glu moiety is chopped off the GS. Interestingly, a MeHg+ ion fragment that it is formed during MS2 of HgMeCys cannot be detected either; hence, mercury is probably stabilised by the Gly/Cys moiety. Hg(GS)2 The MS2 of Hg(GS)2 shows two main fragments with m/z 686 (loss of Glu) and m/z 508 (loss of GS), plus four less abundant fragments: m/z 795 (loss of H2O), and m/z 666, m/z 557 and m/z 547 formed by successive loss of GS amino acids. HPLC separation of HgMeCys, HgMeGS, HgCys2 and HgGS2 with simultaneous ES-MS and ICP-MS detection For the chromatographic separation of the mercury biomolecules, HPLC and the simultaneously coupled ES-MS and ICP-MS system was used as described in “Instrumentation”. Hg(Cys)2, HgMeCys, Hg(GS)2 and HgMeGS solutions were prepared accordingly, as well as a mixture of four compounds with a concentration of 2.5 mg/L (as Hg) each. Single injections of each standard revealed the retention times for each compound, and these injections were followed by injection of the mixture of the compounds. The elution order obtained was Hg(Cys)2, HgMeCys, Hg(GS)2 and HgMeGS, with HgMeGS being eluted at approximately 9.5 min. All compounds were eluted at a gradient composition of less than 15% eluent B, showing that relatively soft elution conditions can be applied. Figure 6 shows the combined ICP-MS (202Hg, 103Rh) and ES-MS traces for the extracted ion chromatograms of m/z for MH+ for the four mercury species. Fig. 6Simultaneous ICP-MS and ES-MS spectra from high-performance liquid chromatography injection of MeHgCys, MeHgGS, Hg(Cys)2 and Hg(GS)2 The ES-MS traces of the extracted ion chromatograms show distinct peaks of the protonated molecular ion masses. For each compound, the same isotope cluster was found as was obtained in the flow-injection experiments with the single species. Moreover, the MS+ spectra (in-source fragmentation) are identical to those obtained in the direct-injection experiment, as are the MS2 spectra of the MH+ molecular ions (data not shown). All ion traces in ES-MS are on the same y-scale, and it is evident that the intensities measured in ES-MS are species-dependent, like in the direct-injection experiments. MeHgGS shows the highest intensity, approximately 10 times higher than MeHgCys. Although a methanol gradient was used in the HPLC experiment (12.5% methanol at 10 min retention time), the relative peak intensities are comparable with those obtained with the direct injections. A different picture is seen for the ICP-MS signal for mercury. In ICP-MS, all compounds should be completely destroyed by the hard ionisation conditions in the plasma. Therefore, the signal intensities for the different compounds should only be governed by the absolute mercury concentration. As all the compounds were made up in the same concentration (as Hg), the ICP-MS response should be very similar for all compounds. Figure 6 shows that for the four compounds the intensities obtained are much more similar than those obtained by ES-MS, but are not ideally equal: the peaks corresponding to the first and the last species eluted (HgCys2 and MeHgGS) are approximately twice the peaks obtained for MeHgCys and HgGS2. Several reasons can be responsible for this finding: Overall intensity changes due to the input of methanol into the plasma. As shown in Fig. 6, the signal for the internal standard is constant up to approximately 4 min of runtime; afterwards it decreases to about 50 % of the initial value at 10-min runtime, when the last mercury compound is eluted. If the mercury intensities are influenced by this process only, the signals for the first compounds eluted (both less than 4 min retention time) should be equal, whereas the signals for the GS complexes should be only about half. This is not the case; in fact the last compound eluted, MeHgGS, exhibits a signal equal to the first compound eluted. However, the behaviour of mercury with its high ionisation potential (10.4 eV) is greatly influenced by organic matter in the plasma, and the mercury signal intensity may change in a different way from the intensity of rhodium. The dependence of the mercury intensity on the methanol content of the eluent must be determined, but this was not performed during the experiments reported here.The volatilities of the compounds may be different, so different amounts of each compound may be introduced into the plasma during pneumatic nebulisation: this effect is well known for volatile species like HgMe2, and has to be investigated further for the compounds used in this experiment.MeHgCys and HgGS2 may partly decompose on the column, so not all of the compound injected may reach the plasma: in this case, we would expect broad, tailing peaks and substantial amounts of free Cys and reduced GS. However, these have not been observed.MeHgCys and HgGS2 are not present to 100% in the standard, and thus the concentrations of the mercury reaching the plasma are diminished: in this case, signals for unbound Cys and GS should show also significant intensity. In fact, some oxidised Cys and oxidised GS can be distinguished in the ES-MS, but is not clear whether the amounts may sum up to explain the intensity loss in MeHgCys and HgGS2. However, the corresponding mercury part of the molecules (Hg2+ and MeHg) should show up in the ICP-MS trace, and this is not the case. As shown further later for the spiking experiments (Fig. 7), Hg2+ and HgMe+ are eluted from the column with distinct retention times differing from the retention times of the other complexes.Fig. 7Overlaid chromatograms for injections of free and biothiol-complexed mercury species in a plant extract spiked with different Hg and HgMe biothiol complexes. Peak assignment: 1 Hg(Cys)2, 2 Hg2+, 3 MeHgCys, 4 MeHg+, 5 MeHgGS, 6 Hg(GS)2 Thus, a final explanation for the difference in intensity of the four compounds cannot be given at this point. Compound stability during spiking experiments of plant extracts The mercury biomolecules synthesised and characterised here shall be detected and identified in real-world samples. One future application is the determination of such compounds in plants exposed to mercury contamination. Mercury can readily be taken up by plants, can accumulate in the roots and can be translocated into shoots and fruits [16, 17, 27, 28]. For the determination of such complexes in plant material, the latter has to be extracted using a minimally invasive method which does not destroy or transform the compounds we are looking for. Here, we tested the stability of the four example compounds in extracts from roots and shoots of rice plants. The plant extracts, prepared as described earlier, were spiked with the four compounds and the signals obtained were compared with the corresponding signals when the compounds were prepared in 0.1% formic acid. Simultaneous ES-MS and ICP-MS detection was applied using the same setup as described earlier. Additionally, plant extracts were spiked with “free” Hg2+ and HgMe+ in order to determine their retention behaviour on the column, and to see whether any complexes would form in situ with any biothiols possibly present in the plant extracts. In Fig. 7, the 202Hg traces monitored by ICP-MS are shown for MeHgCys, Hg2+, Hg(Cys)2, MeHg+, Hg(GS)2 and MeHgGS spiked into the shoot extract of a rice plant at a concentration of 10 mg/kg (as Hg) each. It is evident that the elution order of the four mercury biothiol complexes matches very well the one obtained in a standard mixture (Fig. 6). Surprisingly, Hg2+ is eluted later than HgCys2, whereas MeHg+ is eluted with a broad peak with a retention time of approximately 5 min between the Cys and GS complexes. The elution order and the retention times are practically the same for the standard, root and shoot extracts, and this also remains over time: a second injection of all samples, done 7 h after the first run, showed no significant change in retention times. A similar picture is seen for the peak areas. Except for Hg2+ and MeHgCys, the peak areas measured after 7 h are more than 80% of the initial value (peak areas corrected for instrumental drift using the internal standard signal). Surprisingly, MeHgCys turns out to be more stable in the extracts than in the standard solution. This might be due to the presence of some unidentified chelating agents. The peak area for Hg2+ varies considerably, showing a 50% rise for the standard in 0.1% formic acid, whereas a 30% decrease is observed in the plant extract. While no explanation can be given here for the intensity rise of the Hg2+ peak in the standard, in the shoot extract the formation of other, unidentified mercury complexes may be proposed. The results for retention time and peak intensity reproducibility are summarised in Tables 3 and 4. Table 3Retention times for consecutive injection of six mercury compounds with 7 h differenceSampleHgCys2Hg2+MeHgCysMeHg+HgGS2MeHgGSStandard 1120.7152213.5302458.4529.1Standard 2120.7152218.5303454.1527.7Roots extract 1125.7NA213.5286457.8529.8Roots extract 2126NA212.8284.9452.7526.9Shoots extract 1125.7150.6213.5295.6457.7529.1Shoots extract 2125151.4214.2292.8455.5526.2Average123.9151.5214.3294.1456.0528.1σ2.50.662.17.72.31.4RSD (%)2.00.40.92.60.50.3NA not available, RSD relative standard deviationTable 4Peak area (as percentage of first measurement) recoveries for consecutive injection of six mercury compounds with 7 h differenceSampleHgCys2Hg2+MeHgCysMeHg+HgGS2MeHgGSStandard 9115910989283Roots extract93NA67898786Shoots extract967749958686 The addition of MeHg+ to the extracts did not result in any additional peaks in the ICP-MS trace, while for the addition of Hg2+ a small, very broad peak can be distinguished around the retention time of HgMeGS. An elevated mercury background is seen after the introduction of Hg2+ to the standard and the extract, pointing to some kind of exchange on the HPLC column, possibly through formation of unidentified, labile complexes. However, the mercury counts are at the normal background level again prior to injection of the next sample. Regarding the ES-MS measurements (data not shown), the same MH+ ion cluster patterns are found for the four spiked mercury complexes as in the standards. As already seen from the ICP-MS traces, addition of Hg2+ or MeHg+ did not result in the formation of any other distinguishable mercury biothiol compounds. In the unspiked extracts, only trace amounts of free Cys or GS were found, but there was some oxidised and thus inactive GS. Conclusion and outlook In this work, we could successfully show the synthesis, structural identification and HPLC separation of mercury and methylmercury complexes with Cys and GS. An ES-MS system equipped with an ion trap for MS2 measurements revealed characteristic MH+ and MS2 fragmentation patterns for these compounds, enabling the identification of these or similar complexes in biota samples. The simultaneous coupling of HPLC with ES-MS and ICP-MS with its invaluable combined information on molecular structure and quantitative mercury detection proved to be suited for this kind of compounds. The method described seems to be acceptably robust and reproducible, and also suited for real samples. This was shown through spiking experiments of plant extracts. However, the quantification approach via the ICP-MS trace needs further investigation, especially in terms of mercury intensity changes due to the gradient introduction of methanol into the plasma, and also in terms of decomposition on column as has been described for As(GS)3 [18]. The work presented here is the first step in the determination of mercury species in biota on the molecular level. Ongoing experiments are aimed at plant exposure to mercury and methylmercury. Here, we can also expect mercury complexes with larger biothiols, especially phytochelatins: phytochelatins are small peptides of the general structure (γ-Glu–Cys)n –Gly (n = 2–11), which can bind to metal ions and play an important role in metal detoxification and translocation processes in plants. Other biota samples, e.g. fish, will be targeted as well. Finally, this study presents the first identification of mercury and methylmercury biothiols in spiked plant extracts; hence, this method is a novel tool to investigate whether mercury and methylmercury indeed form complexes with biothiols such as Cys and GS, or if they rather bind to larger entities such as phytochelatins in plants or proteins in fish and other biota.

Anal_Bioanal_Chem-3-1-2117337

A low perfusion rate microreactor for continuous monitoring of enzyme characteristics: application to glucose oxidase

This report describes a versatile and robust microreactor for bioactive proteins physically immobilized on a polyether sulfone filter. The potential of the reactor is illustrated with glucose oxidase immobilized on a filter with a cut-off value of 30 kDa. A flow-injection system was used to deliver the reactants and the device was linked on-line to an electrochemical detector. The microreactor was used for on-line preparation of apoglucose oxidase in strong acid and its subsequent reactivation with flavin adenine dinucleotide. In addition we describe a miniaturized version of the microreactor used to assess several characteristics of femtomole to attomole amounts of glucose oxidase. A low negative potential over the electrodes was used when ferrocene was the mediator in combination with horseradish peroxidase, ensuring the absence of oxidation of electro-active compounds in biological fluids. A low backpressure at very low flow rates is an advantage, which increases the sensitivity. A variety of further applications of the microreactor are suggested. Introduction Conventionally, characterizing enzyme properties in terms of kinetics, conversion rate, and substrate specificity requires substantial amounts of pure enzyme. We aim to develop a versatile and robust microreactor to monitor on-line the activity of small amounts of enzymes, and that can be used under harsh conditions if required, for instance, to prepare modified forms of glucose oxidase (GOx) in which the flavin adenine dinucleotide (FAD) cofactor has been replaced by artificial flavins. GOx uses β-d-(+)-glucose with high specificity as substrate, converting it to gluconolactone and H2O2. Preparation of apoGOx with low residual activity requires partial unfolding of the protein under strongly acidic conditions followed by removal of the flavin by size-exclusion chromatography [1]. The activity of the enzyme can be restored by applying a solution of FAD or FAD analogues [1–4]. Enzyme reactors can be created by covalent immobilization [5, 6]. Another option is to co-polymerise the enzyme in a flow system, trapping the enzyme in a polymer matrix [7, 8]. With these approaches assessment of enzyme properties, inhibitor screening [7], or conversion rates of stereoselective or enantiomeric substrates [9, 10] have been reported. Although convenient, covalent attachment and co-polymerisation require substantial amounts of protein to obtain a good response [11]. Upon immobilization, enzyme characteristics are often altered and enzyme activity may be lost. Especially when rare or expensive enzymes have to be used, it would be beneficial to have a system that uses far less enzyme, does not use chemical immobilization, and is easy to handle. We propose a device where the enzyme is confined in a small space on top of one filter or between two filters and continuously perfused. The device, based on our previously described biosensor technique [12], is robust, easy to handle, and loss of activity and consumption of enzyme are kept to a minimum. With this device we were able to prepare apoGOx on-line by retaining GOx on a single polyether sulfone (PES) filter using acid treatment. Restoration of the enzyme activity was achieved in the same system using a solution of FAD. Furthermore, we were able to miniaturize the microreactor, using less enzyme, and increase the sensitivity of the system using a combination of ferrocene and horseradish peroxidase (HRP) as a mediator for electrochemical detection. Experimental Materials GOx from Aspergillus niger (EC 1.1.3.4, grade 1) and horseradish peroxidase (HRP; EC 1.11.1.7, grade 1) were obtained from Roche (Almere, The Netherlands). Ferrocene carbolic acid was from Lancaster Chemicals (Lancaster Synthetics UK, Morecambe, Lancs, UK). Other chemicals were of pro analysis quality and purchased from Merck (Amsterdam, The Netherlands). Double quartz-distilled water was used for all aqueous solutions. Before and after acid treatment, the running buffer (a) of the microreactor was 0.15 mol L−1 sodium phosphate, pH 7.0, containing 1 mmol L−1 NaCl and 0.1% Kathon GC (Rohm and Haas, Croydon, Surrey, UK). Glucose solution (50 mmol L−1) prepared in running buffer was left to stand for several hours to reach mutarotational equilibrium. For electrochemical detection of GOx in combination with HRP, the running buffer (b) also contained 0.5 mmol L−1 ferrocene carbolic acid. Design of the microreactors The overall design of the microreactors is shown in Fig. 1. They were micro machined from Delrin by the instrumental workshop of Groningen University. For preparation of apoGOx and reconstitution of GOx activity, a microreactor as described in Ref. [12] was used. In contrast to the original device, only one PES filter with a cut-off value of 30 kDa (Sartorius, Göttingen, Germany) and punched to a diameter of 13 mm, was used. Performance studies were done with our miniaturized design. The dimensions of the miniaturized microreactor were: outer length 3 cm, outer diameter 1.5 cm. A PES membrane filter was punched to a diameter of 4 mm and placed in the microreactor (effective diameter 3 mm, effective volume 1 μL) and connected between the injection valve and the detector with fused-silica tubing (FST) (150 μm OD, 50 μm ID) (Polymicro Technologies, Phoenix, AZ, USA) pinched off in Teflon tubing 1/16 inch OD and 170 μm ID (Aurora Borealis Control, Schoonebeek, The Netherlands) with use of finger-tight fittings, to avoid high dead volumes. Fig. 1Schematic design of the reaction compartment of the microreactor. (a) sketch; (b) schematic diagram. Shown here is the microreactor with one PES ultrafilter which is then connected to the flow system using FST and finger-tight fittings. The mesh screen is used to support the ultrafilter Set-up of the system The perfusates of the microreactor were introduced into a flow-injection system and monitored electrochemically (VT-03 electrochemical flow cell and Decade Digital Electrochemical Amperometric Detector; Antec Leyden, Zoeterwoude, The Netherlands), essentially as previously described [13–15]. For the preparation of apoGOx and subsequent reconstitution of the holoenzyme a voltage of +0.5 V was applied between the working and reference electrodes. GOx/HRP activity monitoring For flows >1 μL min−1, a LC10ADvp solvent-delivery pump (Shimadzu Corporation, Kyoto, Japan) was used; for flows <1 μL min−1 a Harvard 22-syringe pump (Harvard Apparatus, Holliston, MA, USA) was used. A voltage of −150 mV was applied between the working (glassy carbon) and reference (Ag/AgCl) electrodes. The injection valve (Vici Cheminert C4, Valco Instruments, Houston, TX, USA) was equipped with a 20 nL internal loop; injection cycles were designed using software incorporated in the Decade system, which also thermostatically controlled the reaction temperature at 37°C. The current was registered using a flatbed recorder type BD41 (Kipp & Zonen, Delft, The Netherlands) and/or the signal was integrated using Chromeleon Software (Dionex Corporation, Sunnyvale, CA, USA) on a PC using an RS232 interface. After the filter was installed in the system, it was left to equilibrate to a flow of 50 nL min−1. Glucose solution injection was sequenced to load/inject 2/3 min (cycle time 5 min) and repeated until a stable baseline was obtained. Preparation of apoGOx and restoration of the enzyme activity Preparation of apoGOx was essentially the same as described in Ref. [16], with a solution of 0.1 mol L−1 glycine, 0.1 mol L−1 NaCl, and 0.1 mol L−1 HCl, pH 1.5 mixed with glycerol to a final concentration of glycerol of 30% (v/v), cooled in ice. This solution was applied to the GOx containing reactor. In detail, a 10 μL solution of 100 mg mL−1 GOx (625 μmol L−1) was pipetted on to the microreactor’s PES filter, which was thus loaded with 6.25 nmol. After stabilisation of the signal, the initial enzyme activity was determined (=100%). The reactor was detached from the detector. The detached reactor was cooled in ice, and perfused with the cooled acidic glycerol solution at a rate of 25 μL min−1 for 15 min. After application of running buffer (a) until the effluent was pH 7, the reactor was taken out of the flow system, re-attached to the detector cell, and the residual activity of the apoGOx was measured in buffer system (a) with a flow of 25 μL min−1. After the reactor was released from the flow system, ca 150 μL of an FAD solution (3.4 mmol L−1) in 0.15 mol L−1 sodium phosphate, pH 7.0, at room temperature, was applied manually during ca 15 min, except for time-dependent tests. Afterwards, the reactor was re-attached to the detector cell and the restored activity of the holoenzyme was measured as described above. Determination of several characteristics of the miniaturized enzyme reactor Increasing amounts of GOx in running buffer (b) (range 625 amol to 625 pmol) were injected into the system, providing the amount of GOx retained on the membrane. After equilibration, a sequence of load/inject of the glucose solution was applied. Subsequently, a solution of HRP in running buffer (b) was injected into the system providing 0.68 pmol (30 ng) of HRP. After equilibration the sequence of load/inject of glucose was repeated. The efficiency of conversion at the electrode was calculated according to Eq. (1): where h = peak height (pA), σ = peak width at 60.7% (s), I = injection volume (μL), c = concentration (μmol L−1), n = number of electrons, F = 96485 (Faraday’s constant, C mol−1) and √(2π)hσ is peak area (C) In Eq. (1) the charge that is transferred to the electrode (area under the curve) is related to the real charge that is available in the reaction, e.g. the concentration of the substrate, the number of electrons involved, and Faraday’s constant. Results Preparation of apoGOx and restoration of the enzyme activity We followed the response to glucose, the residual enzyme activity of apoGOx after acid treatment, and restoration of the enzyme activity after application of a solution of FAD. After acid treatment, negligible activity of apoGOx was observed. Application of the FAD solution restored the enzyme activity. The time dependency of enzyme activity restoration is shown in Fig. 2, composed of individual data from five apoGOx preparations. This time dependency of the reactivation is in accordance with Eq. (2). where k = 0.035 min−1 and t is the reaction time (min). Fig. 2Restoration of apoGOx activity: circles, observed relative reactivation with FAD; solid line, fitting according to the equation f(t) = 1 − exp(−0.035t) Approximately 80% of the enzyme activity was restored within 60 min, which is above average compared to other procedures [1, 2]. Performance Several properties of the system were investigated. These included backpressure of the reactor at various protein loads, its stability and reproducibility, the efficiency at the electrode, system sensitivity, and ease of handling. The relationship of backpressure to the flow and the amount of protein retained on the filter are depicted in Figs. 3a and 3b. The level of the backpressure is acceptable with these flows and these amounts of protein. The stability and reproducibility of the system, as tested with 625 fmol GOx on a series of glucose injections, showed an intra-assay standard deviation of less than 3%. The efficiency of the reaction is inversely proportional to the amount of protein on the filter. The sensitivity of the set-up was determined by retaining various amounts of GOx, ranging from 62.5 amol to 625 pmol on the ultrafilter, and testing the response. A measurable and reproducible response was obtained, even with the lowest amounts of enzyme tested (Table 1). One load of enzyme in the microreactor can be used at least 10 times and the system can be regenerated simply by replacing the filter and injection of a fresh aliquot of protein solution. Fig. 3System backpressure. (a) Backpressure in relation to the flow at various protein loadings: diamonds, 0 μg; squares, 13 μg; triangles, 24 μg; circles, 39 μg; stars, 61 μg GOx. (b) Backpressure in relation to the amount of protein retained on the filter at various flow rates: circles 1 μL min−1; squares, 5 μL min−1; triangles, 10 μL min−1; diamonds, 25 μL min−1Table 1Measured detector signal and relative conversion of glucose in relation to the amount of enzyme retained on the filter in the miniaturized microreactor. Indicated are the amounts of GOx (in fmol) on the PES filter and the signals of the detector (in nA) indicating the amount of converted glucose, calculated as described in the method sectionProtein (GOx) on the filter (fmol)Signal (nA)Conversion (%)0.06250.0160.040.6250.0280.0066.250.0510.0016250.530.00016250002.40.0000006 Conclusions The low perfusion rate microreactor is suitable for the new application described here—preparation of minute amounts of apoGOx and reconstitution of enzyme activity with a solution of FAD. In comparison to the conventional preparation of apoGOx using size-exclusion chromatography [1, 2, 16], this device requires neither complicated handling nor expensive materials and equipment. The enzyme is simply retained on the PES ultrafilter. A scale-up of the microreactor can easily be performed, allowing the preparation of larger amounts of apoGOx to be used, for example, in studies with modified FAD [2–4] or as the basis of a biosensor with which a wide variety of analytes can be detected [1]. The combination with immunological principles allows the analysis of small molecules when no specific enzyme is available, combining molecular recognition with enzyme enhancement of the signal. The sensitivity is improved even more by using electrochemical detection. Low perfusion rate microreactors may facilitate the study of enzymatic properties and modifications, including substrate inhibition, competitive and non-competitive inhibition, and allosteric activation. Because of the mild fixation conditions, they may enable investigation of receptor activity also. An advantage of the microreactor presented here is the small amount of protein required, thus allowing the use of expensive and rare enzymes. Applications using related devices, e.g. Refs. [7, 10, 17] usually require micrograms to milligrams of protein. Glucose sensors using glucose oxidase or glucose dehydrogenase have also been developed for clinical use. One example of such an application uses a volume of 300 nL blood for glucose measurements in diabetics [18]. Such reactors still use larger volumes as compared to the 20 nL we used here. Although we used electrochemical detection, other modes of detection including capillary electrophoresis, fluorescence, or chemiluminescence, are possible. In addition, with the current interest in proteomics, applications can be found where minute amounts of protein can be digested reproducibly and analysed directly by LC–MS–MS [19–21].

Emerg_Radiol-3-1-1914302

Advanced Trauma Life Support®. ABCDE from a radiological point of view

Accidents are the primary cause of death in patients aged 45 years or younger. In many countries, Advanced Trauma Life Support® (ATLS®) is the foundation on which trauma care is based. We will summarize the principles and the radiological aspects of the ATLS®, and we will discuss discrepancies with day to day practice and the radiological literature. Because the ATLS® is neither thorough nor up-to-date concerning several parts of radiology in trauma, it should not be adopted without serious attention to defining the indications and limitations pertaining to diagnostic imaging. Introduction In many countries, trauma care is based on Advanced Trauma Life Support® (ATLS®) [1]. Although the ATLS® manual and course are neither evidence based nor up-to-date concerning several parts of radiology in trauma, surgeons use the ATLS® recommendations, if present, routinely to support indications for diagnostic imaging. In addition, surgeons refer to the ATLS® unjustly with indications for imaging that are not supported by the ATLS® at all. Radiologists must be aware of this to intervene appropriately when sub-optimal imaging indications are presented. In this respect, knowing the content and the language of the ATLS can be helpful. The objective of this review is to familiarize radiologist with the ATLS®. For this purpose, the rationale and indications of diagnostic imaging is assessed where it pertains to the ATLS® protocol. Instances of disagreement with the evidence in the literature and daily practice are highlighted [1]. Purpose of ATLS® Accidents are the primary cause of death in patients aged 45 years or younger. In The Netherlands, 22 out of 100,000 people die each year because of accidental injury. For every one patient who dies, there are three survivors with serious disabilities [1, 2]. The purpose of adequate trauma care is to decrease this morbidity and mortality, which is expected to be achieved by fast, systematic, and effective assessment and treatment of the injured patient. Contrary to the ATLS guidelines, we think that imaging should play a prominent role in this process. History of ATLS® In 1976, an airplane with an orthopedic surgeon, his wife and children crashed in a corn field in Nebraska. The wife died. The surgeon and three of his four children were seriously injured. Although they survived, he considered the standard of care in the local hospital insufficient and decided to develop a system to improve the care for trauma victims, and thus, ATLS® was born. Since the first ATLS® course in 1978, the concept has matured, has been disseminated around the world and has become the standard of emergency care in trauma patients in 46 countries [1]. The ATLS® concept is also used in the pre-hospital phase of trauma patient care and has been adopted for non-trauma medical emergencies and implemented in resuscitation protocols around the world. Originally, ATLS® was designed for emergency situations where only one doctor and one nurse are present. Nowadays, ATLS® is also accepted as the standard of care for the first (golden) hour in level-1 trauma centers. The priorities of emergency trauma care according to the ATLS® principles are independent of the number of people caring for the patient. ATLS® course The ATLS® course is organized under license of the American College of Surgeons. Before the course, the students peruse the course manual. During a 2-day course, 16 students, mostly residents in surgery and anesthesiology, are trained by eight instructors. These instructors now number more than 100 in the Dutch ATLS® section, mostly surgeons and anesthesiologists but also two radiologists. During the course, all emergency measures are taught and reviewed. By means of observing, practicing, and repeating the ATLS® concepts, the object of the course is that the students are capable to perform the necessary measures independently with the correct priorities. The course concludes with a written and practical examination, which has a pass rate of 80–90%. During the course, attention is also given to the multidisciplinary character of trauma care and the organization and logistics of trauma care in hospitals and surrounding area. Radiology has a minor part in the course There are only 50 min to teach the systematic evaluation of chest radiographs and another 50 min to teach in cervical spine radiographs, with the objective for the student to be able to identify life-threatening and potentially life-threatening injuries on chest radiographs and identify fractures on the radiographs of the spine. There is no lecture or skill set concerning computed tomography (CT). Essentials of ATLS® ATLS® is a method to establish priorities in emergency trauma care. There are three underlying premises. (1) Treat the greatest threat to life first. (2) Indicated treatment must be applied even when a definitive diagnosis is not yet established. (3) A detailed history is not necessary to begin evaluation and treatment. Therefore, the assessment of a trauma patient is divided in a primary and a secondary survey. In the primary survey, life-threatening injuries are diagnosed and treated simultaneously. All other injuries are evaluated in the secondary survey. Primary survey In the primary survey, the mnemonic ABCDE is used to remember the order of assessment with the purpose to treat first that kills first (Table 1). Airway obstruction kills quicker than difficulty of breathing caused by a pneumothorax, and a patient dies faster from bleeding from a splenic laceration then from a subdural hematoma. Table 1In the primary survey, the mnemonic ABCDE is used to remember the order of assessment with the purpose to treat first that kills firstThe ABCDEAAirway and C-spine stabilizationBBreathingCCirculationDDisabilityEEnvironment and Exposure Injuries are diagnosed and treated according to the ABCDE sequence. Only when abnormalities belonging to a letter are evaluated and treated as efficacious as possible can one continue with the next letter. In case of deterioration of a patient’s condition during assessment, one should return to ‘A.’ Imaging should not intervene with or postpone treatment. A: Airway The airway is the first priority in trauma care. All patients get 100% oxygen through a non-rebreathing mask. The airway is not compromised when the patient talks normally. A hoarse voice or audible breathing is suspicious; facial fractures and soft tissue injury of the neck can compromise the airway, while patients in a coma are not capable of keeping their airway patent. Endotracheal intubation is the most definite way to secure the airway. In ‘A,’ the cervical spine needs to be immobilized. As long as the cervical spine is not cleared by physical examination, with or without diagnostic imaging, the spine should remain stabilized. For the evaluation of ‘A,’ no diagnostic imaging is necessary. Imaging of the cervical spine is just an adjunct to the primary survey and not part of the ‘A,’ specifically because, as long as the spine is immobilized, possible spinal injury is stabilized and diagnostic imaging can be postponed. When ‘A’ is secure, one can continue with ‘B.’ B: Breathing Breathing is the second item to be evaluated in trauma care. Tension pneumothorax, massive hemothorax, flail thorax accompanied by pulmonary contusion, and an open pneumothorax compromise breathing acutely and can be diagnosed with physical examination alone and should be treated immediately. Most clinical problems in ‘B’ can be treated with relatively simple measures as endotracheal intubation, mechanical ventilation, needle thoracocentesis, or tube thoracostomy. The lack of a definitive diagnosis should never delay an indicated treatment. To evaluate the efficiency of breathing, a pulse oximeter can be applied. Injuries, like a simple pneumothorax or hemothorax, rib fractures, and pulmonary contusion, are often more difficult to appreciate with physical examination. Because these conditions have less effect on the clinical condition of the patient, they can be identified in the secondary survey. A chest radiograph is an adjunct to the primary survey and can be helpful in evaluating breathing difficulties and is necessary to evaluate the position of tubes and lines. When ‘B’ is stabilized, one can continue with ‘C.’ C: Circulation Circulation is the third priority in the primary survey. Circulatory problems in trauma patients are usually caused by hemorrhage. The first action should be to stop the bleeding. Hemorrhage can be external from extremity and facial injury or not visible from bleeding in chest, abdomen, and pelvis. Instable pelvic fractures can be temporarily stabilized with a pelvic band to decrease blood loss. Blood pressure and heart rate are measured; two intravenous lines are started, and blood is obtained for laboratory investigation. In the search for internal blood loss, imaging can be very helpful. Radiological investigations such as a chest radiograph, when not already performed, ultrasound of the abdomen (focussed abdominal sonography in trauma, FAST) and a pelvic X-ray can suggest the localization of the bleeding. A tension pneumothorax can be the cause of circulatory distress but is usually diagnosed and treated in ‘B.’ When a patient’s condition deteriorates, this diagnosis must be reconsidered. Hemodynamic instability can, infrequently, be caused by pericardial tamponade. Therefore, ultrasonography of the pericardial sac is part of a FAST examination. Other less frequently occurring causes of circulatory problems in trauma patients are myocardial contusion and loss of sympathetic tone caused by cervical and upper thoracic spinal cord injuries. When it is not possible to stabilize the patient in the trauma suite, other intervention like operation or embolization should be performed. The remainder of the primary survey will be finished thereafter. When ‘C’ is stabilized, one can continue with ‘D.’ D: Disability Disability should be assessed as the fourth priority in the primary survey, and this includes assessment of the neurological status. The Glasgow coma score (GCS) is used to evaluate the severity of head injury. This score is arrived at by scoring eye opening, best motor response, and best verbal response. Patients who open their eyes spontaneously, obey commands, and are normally oriented score a total of 15 points. The worst score is 3 points. A decreased GCS can be caused by a focal brain injury, such as an epidural hematoma, a subdural hematoma, or a cerebral contusion, and by diffuse brain injuries ranging from a mild contusion to diffuse axonal injury. To prevent secondary injury to the brain, optimal oxygenation and circulation are important. Also, impaired consciousness can be caused or aggravated by hypoxia or hypotension for which ABC stabilization is essential. If a cranial CT is indicated, it should be done in the secondary survey. E: Environment and exposure Environment and exposure represent hypothermia, burns, and possible exposure to chemical and radioactive substances and should be evaluated and treated as the fifth priority in the primary survey. At the end of the primary survey, before continuing with the secondary survey, the ABCDEs should be re-evaluated and confirmed. Secondary survey During the secondary survey, the patient is examined from head to toe, and appropriate additional radiographs of the thoracic and lumbar spine and the extremities are performed when indicated. CT scans, when indicated, are also done in the secondary survey. If, during the secondary survey, the patient’s condition deteriorates, the primary survey should be repeated beginning with ‘A.’ The rigid spine board should be removed as early as possible because it is a serious risk for decubitus ulcer formation. Removing the hard backboard should not be delayed for the lone purpose of obtaining definitive spine radiographs. Diagnostic imaging Radiographs of the chest, pelvis, C-spine, and FAST are adjuncts to the primary survey. Imaging is considered helpful but should be used judiciously and should not interrupt or delay the resuscitation process. When appropriate, radiography may be postponed until the secondary survey. CT, contrast studies, and radiographs of the thoracic spine, lumbar spine, and extremities are also adjuncts to the secondary survey. Imaging is most useful and efficient if consulting with a radiologist becomes routine [3, 4]. We extrapolate this advice to the trauma setting and endorse consultation with clinicians strongly; however, consulting a radiologist is not mentioned once in the ATLS® manual! Contrary to (ever-increasing) daily practice, CT plays a minor role in the ATLS®. With the increasing use of CT in the evaluation of trauma patients, radiation exposure should be a major issue in the field of emergency radiology. CT scanners using an automatic exposure control technique can help to reduce radiation dose [5, 6]. Blunt trauma Thorax A chest radiograph must be obtained to document the position of tubes and lines and to evaluate for pneumothorax or hemothorax and mediastinal abnormalities. When not obtained in the primary survey, it should be done in the secondary survey. From the ATLS® manual, it is not clear if a chest radiograph should be performed in every patient [1]. However, this is in accordance with the literature. At present, no clinical decision rule is available concerning the indication for chest radiography in trauma patients. A CT of the chest is considered an accurate screening method for traumatic aortic injury. If a contrast enhanced helical CT is negative for mediastinal hemorrhage and aortic injury, no additional diagnostic imaging is necessary [1]. If a CT is positive, the ATLS® manual states that the trauma surgeon is in the best position to determine which, if any other, diagnostic imaging is warranted. The possibility to construct multiplanar reconstructions (MPRs), maximum intensity projections (MIPs), volumetric, and virtual angioscopic three-dimensional views from MDCT data, making diagnostic angiography superfluous, is not stated [7]. The same post-processing tools can be used to differentiate between traumatic aortic injury and normal variants [7]. Neither consulting with a radiologist nor endovascular treatment of traumatic aortic injury are mentioned in the ATLS® manual. Although it is recognized that the severity of pulmonary contusions does not correlate very well with the chest radiograph, a CT for the evaluation of pulmonary contusion is not mentioned. The superiority of CT in the detection of pneumothoraces and evaluation of the position of chest tubes is not stated [8, 9]. Abdomen FAST is used in hemodynamic abnormal patients as a rapid, non-invasive, bedside, repeatable method to document fluid in the pericardial sac, hepato-renal fossa, spleno-renal fossa, and pelvis or pouch of Douglas. When FAST is available, it replaces diagnostic peritoneal lavage (DPL) [10]. FAST is a good performing screening tool in evaluating hypotensive trauma patients to differentiate those patients who do need urgent laparotomy from those who do not [11]. An abdominal CT is the most sensitive and specific investigation for the diagnosis of visceral and vascular injury; however, according to the ATLS®, an abdominal CT can only be performed in hemodynamically normal patients because a CT is considered time consuming. This is no longer true with the helical CT available today. The rate-limiting step has become the movement of the patient to the CT suite and on and off the CT table [12]. According to the ATLS® manual, an upper GI contrast study is the imaging method of choice in suspected diaphragm rupture. CT is not mentioned as an option. On the contrary, it is stated that CT misses diaphragmatic injuries. Although CT is not 100% sensitive, neither are GI contrast studies. A comparative study is not available. MDCT has the advantage that it is much easier and quicker to perform in trauma patients [1, 13]. Although no consensus of opinion exists, coronal, and sagittal multiplanar reconstructions (MPRs) might improve the accuracy of MDCT for the diagnosis of blunt traumatic diaphragm rupture [14, 15]. A final omission, and contrary to daily practice in many hospitals, is that interventional radiology is not mentioned as an adjunct to non-operative management in patients with abdominal visceral injury [16, 17]. Pelvis It is recommended that a pelvic radiograph should be performed when the mechanism of injury or the physical examination indicates the possibility of a pelvic fracture. Evaluation of the pelvis on an abdominal CT is not mentioned [18]. Compared to conventional radiography, CT has a higher sensitivity and specificity for the diagnosis of pelvic fractures, and MPRs can be used to delineate the full extend of the fracture [19, 20]. In a hemodynamically abnormal patient with a pelvic fracture and no indication for intra-abdominal hemorrhage on FAST or DPL, angiography with embolization is advised preceding surgical pelvic fixation. In patients with an unstable pelvic fracture, inability to void, blood at the meatus, a scrotal hematoma, perineal ecchymoses, or a high-riding prostate, there is a suspicion of a urethral tear, and in these patients, a retrograde urethrogram should be performed before inserting a urinary catheter [1]. To exclude an intraperitoneal or extraperitoneal bladder rupture in patients with hematuria, a conventional or a CT cystogram can be performed [1, 21]. Cervical spine Cervical spine radiographs are not indicated in patients who are awake, alert, sober, neurologically normal, have no neck pain or midline tenderness, can voluntary move their neck from side to side, and flex and extend without pain. In all other patients, a lateral, AP, and open-mouth odontoid view should be obtained. Although it is not mentioned in the ATLS® manual, this seems to be a combination of the Canadian C-spine rules and the Nexus criteria but the criterion ‘painful distracted injury’ has disappeared between the sixth and the seventh edition of the ATLS manual [1, 22–24]. Possibly, this was done because the definition of painful distracting injury is difficult, but if omitted, this reduces the sensitivity of the clinical decision rule [25]. On the lateral view of the cervical spine film, the base of the skull to the first thoracic vertebra must be assessed. If not all seven cervical vertebrae are visualized, a swimmers view must be obtained and is considered sufficient and safe [1]. Supine oblique views and, contrary to available literature, performing a CT scan of this area is not mentioned [26–28]. Further, of all suspicious areas and all not adequately visualized areas, an axial CT with 3-mm intervals should be obtained. In the cervical spine section, multidetector CT assessment with coronal and sagittal MPRs is not mentioned at all [1]. Performing a CT of the cervical spine without a preceding conventional radiograph as the screening method of choice is not mentioned. The recent ACR appropriateness criteria suggest otherwise [29]. To detect occult instability in patients without an altered level of consciousness, or those who complain of neck pain, flexion-extension radiographs of the C-spine may be obtained [1]. As flexion-extension radiographs are often non-diagnostic and necessitate movement of the spine that is potentially dangerous, at least, performing a CT first to exclude osseous injury or a magnetic resonance imaging (MRI) for the detection of ligamentous injury should be recommended today [27, 29, 30]. MRI is recommended in patients with neurological deficits to detect an epidural hematoma or a traumatic herniated disc. Contrary to the ACR appropriateness criteria, the ATLS states that, when a MRI is not available, CT myelography may be used [1, 29]. Angiography or CT angiography for the evaluation of injury to the carotid or vertebral artery is not mentioned [1]. Head Again, according to the protocol, a cranial CT should be considered in all head-injured patients with a focal neurologic deficit of which the cause can be localized in the brain, a Glasgow coma scale less than 15, amnesia, loss of consciousness of more than 5 min, or severe headaches. This is insufficient to detect all clinical relevant brain injury. There is no reference to evidence-based clinical decision rules such as the Canadian head CT rule, the New Orleans head CT rule or the CHIP prediction rule [1, 31–34]. Thoracic and lumbar spine The indications for diagnostic imaging are the same as for the cervical spine. AP and lateral radiographs should be performed with additional CT of suspicious areas [1]. It is not mentioned that the thoracic and lumbar spine can be reliably evaluated on a CT of thorax and abdomen. When a CT of thorax and abdomen has already, or will be, performed, conventional radiography does not have any additional value especially when MPRs of the spine are obtained [35]. Penetrating trauma Chest Pneumothorax and hemothorax can be diagnosed with a chest radiograph. Even in patients with a normal chest radiograph, a CT is advocated for the evaluation of heart, pericardium, and great vessels in patients with a suspicion of mediastinum transversing injury. For the heart and pericardial sac, a CT can be replaced by ultrasound, and for the major vessels, an angiography can be performed. For the evaluation of oesophageal injury, esophagography using a water-soluble contrast agent and complementary esophagoscopy should be performed. The trachea and bronchial tree can be evaluated by bronchoscopy. Patients with penetrating injury of the lower chest below the transnipple line anterior and the inferior tip of the scapula posterior are considered to have abdominal trauma as well until proven otherwise [1]. Abdomen A hemodynamically abnormal patient with a penetrating abdominal wound does not need diagnostic imaging but should undergo laparotomy immediately. In a hemodynamically normal patient, an upright chest radiograph can document intraperitoneal air and is useful to exclude hemothorax or pneumothorax. An abdominal radiograph (supine, upright, or lateral decubitus) may be useful in hemodynamically normal patients to detect extra-luminal air in the retroperitoneum or free air under the diaphragm. In all patients with penetrating abdominal injury, an emergency laparotomy is a reasonable option, especially in patients with gunshot wounds. In initially asymptomatic patients with a lower chest wound or injuries to the back or flank, the ATLS® considers double or triple contrast CT, DPL, and serial physical examination less invasive diagnostic options, equivalent to each other [1]. For asymptomatic patients with anterior abdominal stab wounds, DPL, laparoscopy, and serial physical examination are mentioned as diagnostic options. However, although there is a significant body of evidence that this may not be optimal, CT is not mentioned as a diagnostic option in these patients [36]. Conclusion ATLS® is a well-tried systematic approach for the assessment of trauma patients. In multidisciplinary trauma care, it is beneficial and, maybe, even mandatory for effective communication that all members of the trauma team, including the radiologist, speak the same ATLS® language. Although imaging should not intervene with or postpone treatment, a chest radiograph, pelvic radiograph, and FAST can direct treatment decisions and should be performed in the primary survey when indicated. Imaging of the cervical spine is also an adjunct to the primary survey but can be postponed as long as the spine is immobilized. All other imaging should be done in the secondary survey. Unfortunately, according to the ATLS®, CT plays a minor role in the evaluation of trauma victims. In the ATLS®, chest CT is only mentioned for the diagnosis of traumatic aortic injury but, in our experience, chest CT is valuable for the evaluation of pulmonary contusions and hemothorax and pneumothorax. Nowadays, abdominal CT is less time consuming than the ATLS® states and can be used to evaluate the extent of the abdominal injury in patients in whom no immediate laparotomy is indicated to evaluate the possibilities for non-operative management with or without endovascular embolization. The indications for head CT according to the ATLS® are insufficient to diagnose all patients with significant head injury. CT of the cervical spine can be used as a primary investigating tool and not only as an adjunct to conventional radiography. When a CT of the chest and abdomen is indicated, the thoracic and lumbar spine, as well as the pelvis, can be evaluated on the axial CT images combined with coronal and sagittal multiplanar reconstructions, and in these cases, conventional radiography of the spine and pelvis do not have any additional diagnostic value. Because the ATLS® is neither thorough nor up-to-date concerning several parts of radiology in trauma, it should not be adopted without questions to define indications for diagnostic imaging. Consultation between clinicians and radiologists can improve the efficiency and quality of diagnostic imaging in trauma patients.

Int_J_Hematol-4-1-2276242

Nasal NK/T-cell lymphoma: epidemiology and pathogenesis

Nasal NK/T-cell lymphoma (NKTCL) is an uncommon disease, but usually shows a highly aggressive clinical course. The disease is much more frequent in Asian and Latin American countries than in Western countries, and is universally associated with Epstein–Barr virus (EBV) infection. Analyses of gene mutations, especially p53 and c-KIT, revealed the different frequencies by district. Epidemiological studies revealed the changes of the disease frequency in Korea during the period from 1977–1989 to 1990–1996. Case-control study showed that the exposure to pesticides and chemical solvents could be causative of NKTCL. Further studies including HLA antigen typing of patients is necessary to further clarify the disease mechanism. Rapid destruction of the nose and face (midline) was firstly described by McBride in 1897 [1]. Macroscopically the lesions usually looked like necrotic granuloma and the patients showed aggressive and lethal course, therefore the term “lethal midline granuloma (LMG)” or “granuloma gangrenescens” were used for this condition. Later the term LMG is popularly used. It had became evident that the LMG is composed of three different types of histology, i.e. Wegener’s granulomatosis (WG), polymorphic reticulosis (PR) or midline malignant reticulosis (MMR), and malignant lymphoma [2, 3]. These diseases could be histologically differentiated from each other by taking clinical findings into account. WG is usually characterized by generalized necrotizing vasculitis involving both arteries and veins and the presence of glomerulitis [4]. Cancer and specific inflammation such as tuberculosis also cause a condition indistinguishable from LMG. PR exhibits a polymorphous pattern of proliferation consisting of large atypical cells with mono- or multinucleus, small lymphocytes, plasma cells, benign-appearing macrophages, neutrophils and much less frequently eosinophils. PR had been considered as a variant of malignant lymphomas because the disease is frequently disseminated [2]. The nature of proliferating cells in PR had been controversial. Once the disease was termed as nasal T-cell lymphoma because the proliferating cells showed a positive immunoreactivity for polyclonal antibodies against T-cells [5, 6]. However, monoclonal rerrangement of the T-cell receptor genes was seldomly found in cases with “nasal T-cell lymphoma” [7]. Later Ng et al. [8] reported that the tumor cells showed a positive immunoreactivity for natural killer (NK) cell marker CD56. Subsequently it was shown that proliferating cells in PR had large granular lymphocyte morphology [9], which is a character of NK cells or cytotoxic T-lymphocytes. There have been accumulating evidences that PR is a neoplasm of activated NK cells [10–13]: the proliferating cells usually show CD2+ , CD56+ , CD3ε+, CD7−, CD16−, cytotoxic granule-associated proteins+ and do not exhibit rearrangement of T-cell receptor or immunoglobulin genes (Fig. 1). Meanwhile rearrangement of T-cell receptor genes was recorded in the rare cases of PR [14, 15]. Although the upper respiratory tract, especially nasal region, is the common site of presentation, NK/T-cell lymphoma of nasal type may present in diverse extranodal sites such as gastrointestinal tract, skin, testis, liver, and spleen [16]. In the World Health Organization classification, NK/T-cell lymphoma encompasses the lymphomas involving nasal area and nonnasal area. Until present, there have been many review articles on the clinical, pathological, immunohistochemical, and immunogenetical aspects of NK/T-cell lymphomas involving upper respiratory tract. This review focuses on the epidemiology and molecular pathogenesis of nasal NK/T-cell lymphoma (NLTCL). Fig. 1HE. Polymorphous pattern of proliferation in the nasal cavity. Large cells show positive immunoreactivity with CD3ε, TIA-1, and CD56. ABC method, ×400 Association with Epstein–Barr virus (EBV) The association between EBV and human malignancies, including endemic Burkitt’s lymphoma, Hodgkin lymphoma and non-Hodgkin’s lymphoma (NHL) of either B- or T-immunophenotypes, has been reported. Etiological role of EBV for development of NKTCL was firstly reported in 1990 [17]. Subsequent study revealed the constant association of NKTCL with EBV in the world (Fig. 2) (Table 1) [18–23]. Sino-nasal lymphomas are immunophenotypically classified into NK/T-(CD56+), T-, and B-cell type with distinctive clinical features [24]. CD56 positivity, therefore NKTCL, was closely associated with EBV positivity among sino-nasal lymphomas [22]. EBV could be subtyped based on the difference in sequence of EBNA2 region, i.e. type A and type B [25]. Almost all of the NKTCL in Korea and Japan had type A EBV, which is identical with the previous literature reporting that most cases of EBV-associated malignancies of immunocompetent patients in Asia had type A EBV [26]. Predominance of type A EBV was also found in NKTCL in Malaysia [27], indicating the predominance of type A EBV in NKTCL in Asia. Several studies indicated the occurrence of type B EBV in lymphoma of immunocompromised patients [28]. Borisch et al. [29] reported that three of six cases of NKTCL in Switzerland had type B EBV, although no findings suggestive of immunodeficiency were found in these patients. These findings suggest a geographic difference in the distribution of EBV subtype in the NKTCL. Fig. 2In situ hybridization with EBER-1 probe reveals positive signals in the nucleus of proliferating cell Table 1EBV in nasal NK/T-cell lymphomaSourceCountryEBV positive rate (%)EBV subtype Expression of latent[18]a France7/7 (100)NA7/7 LMP+[20]China21/21 (100)NA5/21 LMP-1+[21]Japan11/12 (92)10/11 typeA8/12 LMP-1+[22]Korea15/16 (94)15/15 typeA7/15 LMP-1+[24]Indonesia18/20 (90)NANAEBV Epstein–Barr virus, LMP latent membrane protein, NA not availableaFresh tissue samples were used in the analyses As shown in Table 1, the proliferating cells in NKTCL frequently express latent membrane protein (LMP) as revealed by immunohistochemistry. Latent infection gene products of EBV, EBNA-2 and LMP-1, serve as target antigens for the elimination of EBV infected cells by host cytotoxic T-lymphocytes (CTL) [30]. Shen et al. [31] showed that NKTCL cells are able to provide target epitopes of EBV for CTL. In immunocompromised hosts, proliferating cells expressing EBNA-2 and LMP-1 can escape from immune surveillance by the host CTL, which might result in the development of malignant lymphomas [32]. However, systemic immunosuppression is not noted in patients with NKTCL, thus suggesting an unknown underlying mechanism for escape of LMP-1-expressing tumor cells from the CTL. Several mechanisms such as downregulation of the immunogenic EBV nuclear antigens and preferential selection of the deletion genotype of LMP-1 was reported by Chiang et al. [33, 34] and expression of IL-10, an immunosuppressive cytokine, was reported by Shen et al. [31]. The cells expressing viral antigens are eliminated primarily by CTL in a MHC-class-I-restricted manner [35]. Two CTL-epitopes were identified in LMP-1 that are possibly pan A*02-restricted [36]. It is possible that NKTCL patients show lower frequencies of A*02 allele compared with those in the normal population. Indeed high-resolution genetic typing revealed significantly lower frequency of HLA-A*0201 in NKTCL than in normal population [37]. These findings suggest that HLA-A*0201-restricted CTL responses may function in vivo to suppress the development of NKTCL, or in other words, role of EBV for NKTCL development. Genetical changes Lymphoma arises from clonal expansion of lymphoid cells that are transformed by the accumulation of genetic lesions affecting oncogenes and tumor suppressor genes. In general, amount of samples from NKTCL lesions available for genetical analyses is small, and samples usually contain massive necrotic areas. Therefore information for genetical changes in NKTCL has been relatively limited until present. Alterations of tumor suppressor genes and oncogenes Polymerase chain reaction (PCR)—single strand conformation polymorphism (SSCP) followed by direct sequence method was employed for analyses of gene alterations in NKTCL. The genes analyzed by this method were p53, k-ras, c-kit, and β-catenin on the NKTCL cases from Asian countries [38–42] and Mexico [43]. p53 is a well-known tumor suppressor gene that causes cells with damaged DNA to arrest at the G1 phase of cell cycle or stimulating expression of the BAX gene, the protein that promotes apoptosis [44]. In a wide variety of human cancers, p53 gene mutations have been detected mainly in exon 5 through 8 [45]. K-RAS, c-KIT, and β-catenin genes are oncogenes. Greenblatt et al. [46] identified 50 studies in which sequencing of the entire coding region of p53 had been reported. Of the 560 mutations reported in those papers, 87% were found in exons 5–8, and most of the others were in exon 4 (8%). In the studies for NKTCL in Asia, exons 4–8 or exons 5–8 were examined in one institute (Table 2). The frequency of p53 mutations was various by district: high in Japan and Indonesia and low in Mexico, Korea, and Shenyang. Shenyang situates at north China, which is adjacent to the Korean peninsula, suggesting that environmental and genetical factors might generate the differences in frequency. Transitions (G:C to A:T) were the predominant pattern of mutations except for Mexico, in which number of cases with mutations was only five [43], thus the data from Mexico seemed to be not conclusive. Predominance of transition mutations suggests that some “endogenous” mutagens act in lymphomagenesis. The transition pattern of p53 mutations is known to be more susceptible to spontaneous genetic instability than transversion. While genetic instability as revealed by widespread microsatellite instability was not found in the cases with NKTCL [47]. Table 2p53 mutations in nasal NK/T-cell lymphomaNumber of cases Exons examinedFrequency of mutations (%)Predominance of transition mutationAsia Japan [41]584–862%yes Korea [41]424–831%yes China Beijing, Chengdu [38]425–848%yes Shenyang [40]204–840%yes Indonesia [42]274–863%yesMexico [43]215–824%no Quintanilla-Martinez et al. reported the association of p53 overexpression with poor prognosis, and p53 mutations with large cell morphology and advanced stage [43, 48]. However these findings were not confirmed by other studies. [15, 38] The c-KIT proto-oncogene encodes a receptor tyrosine kinase, which is involved in normal hematopoiesis, gametogenesis, and melanogenesis via the c-kit receptor-ligand system. [49]. Because the development of acute leukemia or malignant lymphoma was reported in transgenic mice expressing KIT [8, 14, 50], NKTCL in Asian countries was examined for the c-kit gene mutations [41]. Frequency of c-kit mutations was significantly higher in China (Beijing, Chengdu) (10 of 14 cases: 71.4%) [39] than in Japan (9 of 58 cases: 15.5%) [41], Korea (5 of 42 cases:11.9%) [41], northeast China (Shenyang) (2 of 20 cases:10%) [40], and Indonesia (3 of 27 cases :11.1%) [42]. These findings suggest that location-specific differences in etiological factors cause specific mutations in c-kit gene. FAS Gene Mutations Fas (Apo-1/CD95) is a 45 kDa membrane protein belonging to the tumor necrosis factor receptor family, and mediates programmed cell death (apoptosis) through binding of FAS ligand (Fas L) [51]. Fas consists of 325 amino acids with a single transmembrane domain, including signal peptide. The 80-amino acid portion in the cytoplasm, designated as a death signaling domain, is essential for the apoptotic signal transduction. FAS gene mutations were reported in about 10% cases with sporadic non-Hodgkin’s lymphoma [52]. NKTCL frequently co-express FAS and FAS ligand (Fas L), but the tumor cells seldom undergo apoptosis. Some mechanisms for resistance to FAS/FAS L - induced apoptosis might work in the development of NKTCL, thus FAS gene mutations could be one of the mechanisms. Two reports support this notion: NKTCL cells in 9 of 15 (60%) cases [53] and 7 of 14 (50%) cases [54] showed mutations of FAS gene. Mouse T-cell lymphoma cells transfected with mutated FAS genes were resistant to apoptosis (Fig. 3, Fig. 4) [54], indicating the mutations to be loss of function mutation. These findings suggest that accumulation of lymphoid cells with FAS mutations provides a basis for the development of NKTCL. Fig. 3Summary of the FAS gene mutations found in patients with nasal NKTCL. The shaded rectangles at the COOH-terminus of the protein represents the small peptide added because of the frameshift in the gene encoding FAS.TM: transmembrane domain Fig. 4The mouse WR19L cell line expressing recombinant human FAS protein with (T1102C, A978G, 1095 ins A) or without (wild type) mutations were incubated with various concentrations of anti-FAS antibody at 37.0°C for 16 h. Clones expressing FAS receptor with any mutations (A978G, 1095 ins A, T1102C) were resistant to apoptosis induced by the anti-FAS antibody Others Various cytogenetic alterations have been reported, of which deletions of 6q are the common [55]. Epidemiological features It had been reported that NKTCL seemed to be relatively common among non-Hodgkin’s lymphoma in Hong Kong [56–58]: malignant lymphomas affecting nose and nasopharyngeal region constituted 7.2% of extranodal lymphomas [56] and 45 of 70 cases with malignant lymphomas in the nose, nasopharynx, and larynx had PR morphology [57]. This disease is occasionally encountered in the hospitals in Japan [5, 59, 60]. When one of the authors (KA) visited the USA in 1987, he noticed that the disease was quite rare in the USA, but they had consultation cases from Peru. Because Japanese and a part of Peruvian belong to Mongolian ethnic group, it was postulated that the Mongolian group might be much more frequently affected by this disease. Then they started to examine the frequency of NKTCL in Japan, Korea (Seoul), and China (Shanghai) during the period from 1987 to 1993. The results are summarized in Table 3 [61–63], in which the frequency of each disease constituting LMG is shown as the frequency per 100,000 patients who visited the Ear–Nose–Throat (ENT) clinic in 37 university hospitals in Japan, Ryukyu University Hospital in Okinawa, Japan, Yonsei University Hospital in Seoul, and Shanghai Medical University Hospital. All of the histological sections were reviewed by one of the authors (KA). Frequency of PR ranged from 8 to 40.8. That in the Institute of Laryngology and Otology, London (1966–1987) was four, showing two to ten times higher frequencies of the PR in the east Asian countries [61]. The disease is rare in the USA [64] and Europe [65]. The patients with NKTCL seem to be clustered also in Latin American countries and Indonesia [66, 67]. Information from other parts of the world such as Africa, the Middle and Near East, and Rusia is helpful to elucidate the etiology of this disease. Table 3Frequency of lethal midline granuloma in Japan, Korea (Seoul), and China (Shanghai)DiseaseNumber of patients (frequency per 100,000 ENT patients)Japan other than Okinawa (1965–1986)Okinawa (1973–1991)Seoul (1979–1989)Shanghai (1979–1990)Wegener’s granulomatosis64(4)1(3)0(0)1(4)Polymorphic reticulosis114(8)9(27.4)56(40.8)73(9.8)Malignant lymphoma82(6)11(33.5)15(10.9)54(7.2)Others42(3)1(3)6(4.4)0(0)Total302(21)22(69.9)77(56)128(17) At the late 1990s, otorhinolaryngologists in both Korea and Japan had the impression that the frequency of PR appeared to be decreasing. Then the changes in frequency of PR with time among cases from Seoul and 59 university hospitals in Japan were examined [68]: the frequency rate of PR per 100,000 outpatients of ENT clinics in Seoul decreased from 40 to 20 between the periods of 1977–1989 and 1990–1996. However, there were no significant changes in Japan during the period studies. Life-style and environmental factors Epidemiological studies have revealed that the NKTCL occurs much more frequently in Asian countries than in Western countries and it is closely associated with EBV infection. There are differences in frequencies of p53 and c-kit gene mutations among patients with NKTCL in Japan, China, and Korea. Recently the first case of familial NKTCL affecting a father and one of his six children was reported [69]. They used large amounts of pesticide in a green house. An increase in the risk of developing NHL among individuals exposed to pesticides was reported [70, 71]. In addition, a correlation of exposure to certain pesticides and organochlorines with increased titers of antibodies to EBV was reported [72]. All these findings might suggest a causative role for some genetical, environmental and life style factors in the development of NKTCL. Therefore, the epidemiological study to elucidate whether socioenvironmental ambient factors contribute to the development of NKTCL was conducted as a collaborative study of Japan, Korea and China (Table 4) [73]. The odd ratio (OR) of NKTCL was 4.15 (95% confidence interval (CI), 1.74–9.37) for farmers, 2.81 (CI 1.49–5.29) for producers of crops, and 4.01 (CI 1.99–8.09) for pesticide users. The ORs for crop producers, who minimized their exposure to pesticides by using gloves and glasses, and sprinkling downwind at the time of pesticide use, were 3.30 (CI 1.28–8.54), 1.18 (CI 0.11–12.13), and 2.20 (CI 0.88–5.53), respectively, which were lower than those for producers who did not take these precautions. Exposures to pesticides and chemical solvents could be causative factors for NKTCL. Previous studies showed the increased risk of NHL in individuals using pesticides, especially phenoxyacetic acid-type herbicides [74]. Association of pesticides with risk of developing t(14;18) positive-NHL, but not t(14;18) negative-NHL, was reported [75]. Table 4Risk of nasal NK/T-cell lymphoma in relation to cultivation of crops and pesticide useOdds ration (OR)a Number of cases N = 88Number of controls N = 30595% CILowerUpperCultivation of crops At present2.8127361.495.29 More than 5 years5.0824192.4710.43Pesticides Users4.0123231.998.09Type of pesticide Herbicide3.1713161.367.38 Insecticide3.4520211.677.13 Fungicide6.051061.9818.46Precautions Gloves used3.3010111.288.54 Gloves not used 4.7613121.9311.72 Mask used5.4414102.2013.47 Mask not used 2.829131.087.37 Glasses used1.18130.1112.13 Glasses not used4.5222202.179.42 Sprinkling downward attended2.209160.885.53 Sprinkling downward not attended8.451473.0123.70CI confidence intervalaAdjusted for age (<40, 40–59, >60), sex (male/female) and country (Japan, Korea and China) Conclusions Clinical course of patients with NKTCL is usually highly aggressive. Therefore clarification of risk factors for disease development is especially important to establish a strategy for disease prevention. Because EBV infection and pesticides could be risk factors for NKTCL, investigation on effects of pesticides for EBV activation is needed. Employment of the similar kind of the epidemiological study is desirable in other areas than East Asia. Patients with NKTCL cluster in Asia and Latin American countries, therefore some genetical factors might be involved in the disease development. Further studies including HLA antigen typing of patients is important to further clarify the mechanism for disease development.

J_Hum_Genet-3-1-1915643

The 894G>T variant in the endothelial nitric oxide synthase gene and spina bifida risk

The 894G>T single nucleotide polymorphism (SNP) in the endothelial NOS (NOS3) gene, has recently been associated with embryonic spina bifida risk. In this study, a possible association between the NOS3 894G>T SNP and spina bifida risk in both mothers and children in a Dutch population was examined using both a case-control design and a transmission disequilibrium test (TDT). Possible interactions between the NOS3 894G>T SNP and the MTHFR 677C>T SNP, elevated plasma homocysteine, and decreased plasma folate concentrations were also studied. The NOS3 894TT genotype did not increase spina bifida risk in mothers or children (OR 1.50, 95%CI 0.71–3.19 and OR 1.78, 95%CI 0.75–4.25, respectively). The TDT demonstrated no preferential transmission of the NOS3 894T allele (Χ2 = 0.06, P = 0.81). In combination with the MTHFR 677TT genotype or elevated plasma homocysteine concentrations, the NOS3 894GT/TT genotype increased maternal spina bifida risk (OR 4.52, 95%CI 1.55–13.22 and OR 3.38, 95%CI 1.46–7.84, respectively). In our study population, the NOS3 894GT/TT genotype might be a risk factor for having a spina bifida affected child in mothers who already have an impaired homocysteine metabolism. Introduction Nitric oxide synthase (NOS) catalyzes the generation of nitric oxide (NO), an important signaling molecule that mediates many of its biological effects by activating the enzyme guanylyl cyclase, thereby increasing cyclic GMP synthesis (Murad 2006). Besides its important role in cardiovascular control, NO has been suggested to play a role in development (Lee and Juchau 1994). NO has been demonstrated to be present in the neural tube of chick embryos at the time of neurulation (Traister et al. 2002) and regulates the balance between mitosis and programmed cell death (Plachta et al. 2003). Other studies show that NO can inhibit methionine synthase (MTR) enzyme activity, thereby interfering with homocysteine remethylation (Danishpajooh et al. 2001) and proper neurulation (Nachmany et al. 2006; Weil et al. 2004). The 894G>T single nucleotide polymorphism (SNP) in the endothelial-derived nitric oxide synthase (NOS3) gene, identified by Hingorani et al. (1999), was shown to be associated with elevated plasma homocysteine levels in nonsmokers with low serum folate concentrations (Brown et al. 2003). More recently, it was demonstrated that the NOS3 894GT genotype is an embryonic risk factor for spina bifida (OR 1.63, 95%CI 1.09–2.42) (Brown et al. 2004). In this study, we examined the relation between the NOS3 894G>T SNP and spina bifida risk in both mothers and children using a case-control design and a transmission disequilibrium test (TDT). An effect of the NOS3 894G>T SNP on plasma homocysteine or plasma folate concentration was examined as well as possible interactions between the NOS3 894G>T SNP and the MTHFR 677C>T SNP, elevated plasma homocysteine, and decreased plasma folate concentrations. Materials and methods Study population The study population has been described in detail before (van der Linden et al. 2006). Briefly, the study population was recruited in collaboration with the BOSK and was extended by a group from the Pediatric Neurology Department of the Radboud University Nijmegen Medical Center. The study group included 109 spina bifida patients (61 girls and 48 boys, mean age 16.4 ± 11.3), their mothers (n = 121, mean age 42.9 ± 10.9) and fathers (n = 103, mean age 44.9 ± 10.2). The control group consisted of 500 volunteers recruited from a general practice in The Hague (den Heijer et al. 1995). In the present study we only included the 292 women from this control group (mean age 50.6 ± 13.5). The pediatric control group included 234 children (119 girls and 115 boys, mean age 8.4 ± 6.4) (van Beynum et al. 1999). The children of secondary school age were healthy volunteers, and the younger children were, for ethical reasons, recruited in a hospital setting. The local medical ethics committee approved the study. For the present study, DNA of 102 spina bifida patients, 116 mothers, 101 fathers, 265 control women and 211 pediatric controls was available for genotyping. Biochemical determinations Plasma total homocysteine concentration was measured in EDTA plasma by HPLC and fluorescence detection, as previously described by te Poele-Pothoff et al. (1995). Plasma folate concentration was determined using the Dualcount Solid Phase Boil Radio assay (Diagnostic Product Corporation, Los Angeles, CA). PCR amplification and genotype determination NOS3 894G>T genotyping was performed according to Hingorani et al. (1999). Primer annealing took place at a temperature of 58°C. The PCR product of 206 bp was cut with the restriction enzyme MboI, which cuts the T allele in fragments of 119 and 87 bp. Statistical analyses Case-control study Odds ratios (ORs) and their 95% confidence intervals (95%CI) were calculated by means of logistic regression analysis. The effect of the NOS3 894G>T SNP on plasma homocysteine and plasma folate concentration was studied by means of linear regression analysis. Since the distributions of plasma homocysteine and plasma folate concentrations are positively skewed, these variables were logarithmically transformed prior to linear regression analysis. Deviation from Hardy–Weinberg equilibrium was calculated to assess the accuracy of the genotyping assay. The power of the case-control study was calculated using the Genetic Power Calculator (Purcell et al. 2003). All tests were performed using SPSS software package version 12.0.1. Statistical significance was accepted at a two-tailed P < 0.05 or 95%CI of an odds ratio that did not include 1.0. Transmission disequilibrium test The relation between the NOS3 894G>T genotype of the spina bifida patients and spina bifida risk was assessed by evaluating the transmission of alleles from heterozygous parents to their affected offspring using the TDT, a family-based association test (Spielman et al. 1993). The power of the TDT was calculated using the Genetic Power Calculator (Purcell et al. 2003). Results Case-control study The NOS3 894G>T genotype could be determined in 115 mothers of a spina bifida affected child and in 259 female controls. Genotype distribution did not differ from that expected under Hardy–Weinberg equilibrium (P = 0.83), and the power to detect a twofold increase in maternal spina bifida risk was 59%. Table 1 demonstrates that neither the NOS3 894GT genotype nor the NOS3 894TT genotype increased maternal spina bifida risk relative to the NOS3 894GG genotype. Since the effect of the NOS3 894GT genotype and the NOS3 894TT genotype on spina bifida risk was the same relative to the NOS3 894GG genotype, the NOS3 894GT and TT genotypes were combined to increase power, which resulted in a nonsignificant 36% increased risk of having a spina bifida affected child relative to the NOS3 894GG genotype (OR 1.36, 95%CI 0.87–2.11). The NOS3 894G>T SNP was not associated with plasma homocysteine or plasma folate concentrations in mothers and female controls (data not shown). Table 1The NOS3 894G>T genotype distribution and unadjusted ORs in mothers of a spina bifida affected child and female controlsNOS3 894G>TMothers (%)Female controls (%)OR (95%CI)GG49 (42.6)130 (50.2)1aGT53 (46.1)106 (40.9)1.33 (0.83–2.11)TT13 (11.3)23 (8.9)1.50 (0.71–3.19)aReference group In 95 spina bifida patients and 207 pediatric controls, NOS3 894G>T genotyping was performed successfully. The NOS3 894G>T genotype distribution did not differ from that expected under Hardy–Weinberg equilibrium (P = 0.76), and the power of the study was 51%. The NOS3 894GT genotype increased spina bifida risk in children whereas the NOS3 894TT genotype did not (Table 2). Combining the NOS3 894GT and 894TT genotypes resulted in a 66% increase in spina bifida risk relative to the NOS3 894GG genotype (OR 1.66, 95%CI 1.02–2.72). In the pediatric controls, the NOS3 894GT and 894TT genotypes were associated with increased plasma homocysteine concentrations relative to the NOS3 894GG genotype (14.7% increase, 95%CI 4.2–26.2% and 23.5% increase, 95%CI 3.9–46.8%, respectively). There was no association between the NOS3 894G>T SNP and plasma folate concentration in children (data not shown). Table 2The NOS3 894G>T genotype distribution and unadjusted ORs in spina bifida patients and pediatric controlsNOS3 894G>TSB patients (%)Pediatric controls (%)OR (95%CI)GG39 (41.1)111 (53.6)1aGT46 (48.4)80 (38.6)1.64 (0.98–2.74)TT10 (10.5)16 (7.7)1.78 (0.75–4.25)aReference group Transmission disequilibrium test In this study there were 75 complete mother–father–child triads, and the power to detect a twofold increase in spina bifida risk was 32%. In 36 out of 70 informative transmissions (51.4%), the NOS3 894T allele was transmitted to the child, which resulted in a Χ2 of 0.06 (P = 0.81). Gene-gene interaction Data on both the NOS3 894G>T genotype and the MTHFR 677C>T genotype were present for 110 mothers of a spina bifida affected child and 259 control women. Since the MTHFR 677CT genotype did not increase spina bifida risk relative to the MTHFR 677CC genotype, we decided to combine the MTHFR 677CC and CT genotypes to increase power. In combination with the MTHFR 677TT genotype, the NOS3 894GT/TT genotype increased the risk of having spina bifida affected offspring 4.5 times relative to the NOS3 894GG genotype combined with the MTHFR 677CC/CT genotype (OR 4.52, 95%CI 1.55–13.22) (Table 3). Table 3Maternal spina bifida risk for the combination of the NOS3 894G>T SNP and the MTHFR 677C>T SNPNOS3 894G>TMTHFR 677C>TMothers (%)Female controls (%)OR (95%CI)GGCC/CT42 (38.2)114 (44.0)1aGGTT6 (5.5)16 (6.2)1.02 (0.37–2.77)GT/TTCC/CT52 (47.3)123 (47.5)1.15 (0.71–1.85)GT/TTTT10 (9.1)6 (2.3)4.52 (1.55–13.22)aReference group In children, there was no interaction between the NOS3 894GT/TT genotype and the MTHFR 677TT genotype (OR 1.23, 95%CI 0.31–4.91) (data not shown). Gene-metabolite interactions Since the MTHFR 677C>T SNP is a genetic determinant of plasma folate and plasma homocysteine concentrations, possible interactions between the NOS3 894G>T SNP and these metabolites were examined in mothers. In combination with decreased plasma folate concentrations (<8.52 nmol/L), the NOS3 894GT/TT genotype did not increase maternal spina bifida risk relative to the NOS3 894GG genotype in combination with normal plasma folate concentrations (≥8.52 nmol/L) (OR 1.14, 95%CI 0.49–2.67) (data not shown). The risk of having spina bifida affected offspring increased to 3.4-fold when the NOS3 894GT/TT genotype was present in combination with elevated plasma homocysteine concentrations (≥12.56 μmol/L) (OR 3.38, 95%CI 1.46–7.84) (Table 4). Table 4Maternal spina bifida risk for the combination of the NOS3 894G>T SNP and elevated plasma homocysteine concentration, adjusted for ageNOS3 894G>TPlasma Hcy conc (μmol/L)aMothers (%)Female controls (%)OR (95%CI)GG<12.5642 (36.8)100 (38.6)1bGG≥12.567 (6.1)30 (11.6)0.66 (0.26–1.65)GT/TT<12.5647 (41.2)108 (41.7)1.17 (0.69–1.96)GT/TT≥12.5618 (15.8)21 (8.1)3.38 (1.46–7.84)aThe 80th percentile of the control plasma homocysteine distribution was used to define elevated plasma homocysteine concentrationsbReference group Discussion In this study, the NOS3 894TT genotype did not significantly increase spina bifida risk, and the TDT demonstrated no preferential transmission of the NOS3 894T allele. However, in combination with the MTHFR 677TT genotype or elevated plasma homocysteine concentrations, the NOS3 894GT/TT genotype increased maternal spina bifida risk 4.5- and 3.4-fold, respectively. Using a likelihood ratio test (LRT) for both proband and mother trios, Brown et al. (2004) demonstrated the NOS3 894GT genotype, but not the NOS3 894TT genotype, to be an embryonic risk factor for spina bifida (OR 1.63, 95%CI 1.09–2.42). Data from our case-control study might be in line with this observation of Brown et al. (2004); however, based on the effect of the heterozygous NOS3 894GT genotype on spina bifida risk, a larger effect of the NOS3 894TT genotype on spina bifida risk would be expected. The observation that the NOS3 894GT genotype increases spina bifida risk and the NOS3 894TT genotype does not could be explained as a chance finding, but could also be the result of lack of power, as also suggested by Brown et al. (2004). Despite the small numbers on which the analyses are based, the possible interactions between the NOS3 894GT/TT genotype and the MTHFR 677TT genotype or elevated plasma homocysteine concentrations are interesting and may suggest a role for NOS3 uncoupling or S-nitrosation. The NOS3 894G>T SNP (Glu298Asp) has been suggested to impair NOS3 protein function and/or activity resulting in decreased NO production (Sofowora et al. 2001; Tesauro et al. 2000), although results are contradictory (Fairchild et al. 2001). Elevated homocysteine concentrations have been demonstrated to switch the NOS3 enzyme to an uncoupled state directed to the synthesis of reactive oxygen species (ROS) rather than NO synthesis (Topal et al. 2004). The combination of the NOS3 894GT/TT genotype and elevated plasma homocysteine concentrations may lead to such a decrease in NO levels that neurulation is hampered. NO can also react with homocysteine to form S-nitrosothiols, thereby modulating homocysteine availability (Stamler et al. 1993). As previously suggested by our group (Heil et al. 2004), decreased NO levels may result in decreased S-nitrosothiol formation, resulting in less capturing of homocysteine into S-nitrosoHcy. The subsequent increase in plasma homocysteine concentrations might influence proper neural tube closure, especially when plasma homocysteine concentrations are already high. The results of our study suggest that the NOS3 894G>T SNP is a spina bifida risk factor in the mother and not in the child. Our results furthermore indicate a role for the homocysteine metabolism in the detrimental effects asserted by the NOS3 894G>T SNP. More studies on the association between the NOS3 894G>T SNP and spina bifida in larger populations are warranted.

Biochim_Biophys_Acta-2-1-2258316

The microtubule-associated protein tau is phosphorylated by Syk

Aberrant phosphorylation of tau protein on serine and threonine residues has been shown to be critical in neurodegenerative disorders called tauopathies. An increasing amount of data suggest that tyrosine phosphorylation of tau might play an equally important role in pathology, with at least three putative tyrosine kinases of tau identified to date. It was recently shown that the tyrosine kinase Syk could efficiently phosphorylate α-synuclein, the aggregated protein found in Parkinson's disease and other synucleinopathies. We report herein that Syk is also a tau kinase, phosphorylating tau in vitro and in CHO cells when both proteins are expressed exogenously. In CHO cells, we have also demonstrated by co-immunoprecipitation that Syk binds to tau. Finally, by site-directed mutagenesis substituting the tyrosine residues of tau with phenylalanine, we established that tyrosine 18 was the primary residue in tau phosphorylated by Syk. The identification of Syk as a common tyrosine kinase of both tau and α-synuclein may be of potential significance in neurodegenerative disorders and also in neuronal physiology. These results bring another clue to the intriguing overlaps between tauopathies and synucleinopathies and provide new insights into the role of Syk in neuronal physiology. Many neurodegenerative disorders are characterised by intracellular inclusions of highly insoluble proteins, and a classification based upon the main protein component of these aggregates is widely used. The term “synucleinopathies”, for instance, refers to a group of disorders including Parkinson's disease (PD) in which the synaptic protein α-synuclein (α-syn) forms neuronal or glial aggregates [1]. “Tauopathies” are another pathological entity in which the microtubule-associated protein tau self-assembles into filamentous inclusions called neurofibrillary tangles (NFT) [2]. The tauopathies include Alzheimer's disease (AD), progressive supranuclear palsy and corticobasal degeneration. Human brain tau consists of a family of six isoforms, generated by the alternative splicing of a single mRNA transcript. Tau proteins contain either three (tau 3R) or four (tau 4R) tubulin-binding domains depending on the splicing of exon 10, and either one (tau 1N) two (tau 2N) or no (tau 0N) N-terminal inserts depending on the inclusion of exon 2, exons 2 and 3, or exclusion of both, respectively. Their apparent molecular weights range from 45 kDa for tau 0N3R isoform to 65 kDa for tau 2N4R. When aggregated in NFT, tau is abnormally hyperphosphorylated by a series of serine/threonine kinases, among which glycogen synthase kinase-3β, cyclin-dependent kinase 5 and casein kinase 1 appear to be the most relevant [3,4]. It has been hypothesized that this hyperphosphorylation contributes to neurodegeneration through the destabilisation of microtubules [5]. Recent data suggest that phosphorylation of tau also occurs on tyrosine residues and this could be of potential significance in pathological conditions [6,7]. Human tau protein possesses five tyrosine residues on positions 18, 29, 197, 310 and 394 (numbered according to the longest 2N4R tau isoform). Tau extracted from NFT of AD patients was found to be phosphorylated on tyrosine 18 (Y18) by immunocytochemistry using a phosphospecific antibody [6], and on tyrosine 394 (Y394) by mass spectrometry [8]. Tyrosine 197 (Y197) has been shown to be phosphorylated in tau aggregates from transgenic mice overexpressing mutant tau [9]. Recently, the dual specificity kinase tau-tubulin kinase 1 has been shown to phosphorylate tau in vitro on Y197 [10]. While Abl was recently described as a candidate tyrosine kinase for Y394 [8], Fyn is considered to be the kinase for Y18 [6]. The tyrosine kinase Syk is known for its critical role in signalling through immune receptors in leucocytes [11]. Syk tyrosine kinase expression is however not confined to hematopoietic cells, and has been reported in a variety of tissues [12]. Syk expression has been confirmed in the central nervous system (CNS) by immunoblot analysis of mouse brain homogenates, and its localization in mouse neuronal cytoplasm affirmed by immunohistochemistry [12]. While its function in the CNS remains unclear, Syk was recently found to be a kinase for α-syn [13]. The direct interaction of Syk and α-syn was proven by confocal microscopy and a dual-hybrid system approach [13]. The intriguing overlap in the pathophysiology of synucleinopathies and tauopathies prompted us to assess whether tau protein was also a substrate for Syk tyrosine kinase [14]. We herein report that Syk can phosphorylate Tau on Y18, and that the kinase and its substrate directly interact. We first investigated whether Syk can directly phosphorylate tau in vitro. Recombinant human tau 2N4R was incubated with or without recombinant Syk or Abl [8]. Tau was phosphorylated in vitro by Syk and by its previously recognized tyrosine kinase Abl as judged on Western blots probed with P-Tyr-100 anti-phosphotyrosine antibody (Cell Signaling), whereas no phosphorylation was observed in experiments where tau was incubated without Syk (Fig. 1). In the presence of Syk, an additional band of approximately 70 kDa, migrating just above tau, was observed on the phosphotyrosine immunoblot. This band is likely to contain Syk since the kinase is known to autophosphorylate. Taken together, these results demonstrate that Syk can directly catalyze tau phosphorylation. To determine whether Syk can phosphorylate tau in cells, co-transfection experiments were performed in CHO cells using Syk expression vectors together with untagged or V5-tagged tau. Cells were lysed and tau was immunoprecipitated with either anti-V5 antibody (Invitrogen) or with anti-tau antibody (Tau-5, BD Biosciences). Western analysis was then performed on immunoprecipitated tau using the anti-phosphotyrosine (P-Tyr-100, Cell Signaling), anti-tau (Tau-5) or anti-Syk (N-19, Santa Cruz) antibodies as previously described [8]. In preliminary experiments, a V5-tagged tau 2N4R construct and wild-type Syk were transiently coexpressed in CHO cells. We have previously shown that V5-tagged tau 2N4R migrates at approximately 70 kDa on SDS-PAGE [8]. Because of the close molecular weights of Syk (72 kDa) and the V5-tau construct, the two proteins co-migrated using conventional denaturing gel electrophoresis (data not shown). To better resolve the two proteins, we used two complementary approaches. In a first set of experiments, we expressed V5-tagged tau 2N4R along with a Syk-GFP construct that migrates at 100 kDa. The Syk-GFP construct was proven to remain functionally active in previous studies [15]. In a second set of experiments, we coexpressed wild-type untagged Syk along with the shortest tau isoform (0N3R), which migrates at 45 kDa. Tau protein was phosphorylated on tyrosine when co-transfected with Syk-GFP construct or with its known tyrosine kinase Abl (Fig. 2A). Additional positive controls included tau-transfected cells treated with the tyrosine-phosphatase inhibitor pervanadate (100 μM for 30 min). When Syk-GFP was co-transfected with tau, tau immunoprecipitates contained a ∼ 100-kDa tyrosine-phosphorylated protein, likely to contain Syk-GFP since this enzyme is tyrosine phosphorylated (Fig. 2A). This was confirmed by blotting with Syk antibody after membrane stripping (Fig. 2A). This co-immunoprecipitation of Syk with tau is indicative of Syk and its substrate being in the same protein complex. In order to exclude any effects of the V5 and GFP tags fused with tau and Syk, we wished to repeat this experiment using vectors expressing native proteins. The untagged and shortest isoform of tau (0N3R) was phosphorylated on tyrosine when co-transfected with native 72 kDa Syk (Fig. 2B), as compared with the transfection of tau alone. As in Fig. 2A, an additional 72-kDa band corresponding to phospho-Syk was visible on the phosphotyrosine blot when Syk was co-transfected with tau. The co-immunoprecipitation is therefore confirmed with native proteins. Taken together, these results demonstrate that Syk tyrosine kinase can both phosphorylate and co-immunoprecipitate with tau in cells, consistent with the direct phosphorylation of the substrate demonstrated in vitro (Fig. 1). The binding of Syk to both the shortest and longest isoform of tau suggests that it is independent of tau splicing. Tau has been shown to be tyrosine phosphorylated on residue 18, 197 and 394 [6,8–10]. To map the tyrosine residue(s) phosphorylated by Syk in tau, Syk was co-transfected into CHO cells along with wild-type or mutant tau constructs in which individual tyrosines were replaced by phenylalanine (Y18F, Y197F and Y394F). These cDNA constructs have been described in detail previously [8]. The only such tyrosine mutation that resulted in a strong decrease in tau tyrosine phosphorylation was Y18F (Fig. 3). Tyrosine phosphorylation of the Y197F and Y394F constructs were not significantly different from the wild-type control (Fig. 3). This clearly demonstrates that Y18 is the major tyrosine phosphorylation site for Syk. In addition, the binding of Syk was independent of the tyrosine phosphorylation state of tau (Fig. 3), suggesting that the SH2 domains of Syk are not involved in its binding to tau. As Fyn and Syk phosphorylate the same tyrosine residue on tau, we decided to perform a time course of phosphorylation of tau by the two kinases under conditions designed to favour the measurement of stoichiometry. After 30 min, Syk had incorporated an average of 0.23 mol of phosphate/mol of tau and Fyn had incorporated an average of 0.25 mol of phosphate/mol of tau. These results show that Syk and Fyn phosphorylate tau with the same efficacy (Fig. 4). Our demonstration of binding of tau to Syk is consistent with tau being a specific substrate for Syk and for their involvement in a cell-signaling pathway in neurons. Interestingly, Syk plays an important role in signalling events of neurite induction and outgrowth in neuronal cell lines [16]. It is thus tempting to speculate that the phosphorylation of tau by Syk could be involved in neurite outgrowth. Our study also reinforces the role of Syk in neurodegenerative disorders. Remarkably, Y18 has been shown to be phosphorylated in NFT, suggesting that, like Fyn, Syk could be critical in the pathophysiology of AD [6,7,17]. Although early studies suggested a clear distinction between ‘tauopathies’ and ‘synucleinopathies’, more recent studies demonstrate that there is often an overlap in the pathological findings in these disorders [18–20]. Syk binds to and phosphorylates both tau and synuclein, suggesting that this kinase could be a link between synucleinopathies and tauopathies. In conclusion, our findings significantly contribute to the understanding of the signaling pathways involving tyrosine phosphorylation of tau in both physiological and pathological conditions. Further elucidation of the functional relationship between tau and the Syk in neurons could provide critical insights into the role of tau in cell signaling as well as the role of tau in neurodegenerative processes.

Eur_J_Health_Econ-_-_-1388087

Spanish health benefits for services of curative care

This contribution presents entitlements and benefits, decision criteria, and involved actors for services of curative care in Spain. It describes basic benefits included in the category of curative care defined by the central government and any additional benefits that some autonomous communities (ACs) have included to enlarge their own basket. It is concluded that there is no specific and explicit benefit catalogue. As no user charges exist for this category, waiting times serve as the main cost containment tool. There is a need for further legislation, as inequalities may increase across the territory as a matter of fact. Inequalities in access to health care resources between ACs are not due to differences in health baskets but mainly to the availability of technologies. The principal actors responsible for defining of benefit catalogues from the services of curative care in Spain are the Central Government and the autonomous communities (ACs). Law 16/2003 [1] states that the Spanish health benefit basket should serve as a minimum for all Spanish residents to ensure the equality of access and benefits among them irrespective of their location. Nevertheless, as ACs may vary the allocation of their funding, some may decide to offer additional or new benefits even including those that are explicitly excluded on national level by Royal Decree 63/1995 [2] and Law 16/2003. The central government’s main responsibilities are as follows: (a) general coordination and basic health legislation, (b) financing of the system and the regulation of the financial aspects of social security, (c) definition of a benefit basket guaranteed by the National Health System (NHS), (d) international health, (e) pharmaceutical policy, (f) health professionals undergraduate and postgraduate training, and (g) human resources policies regarding civil servants. A number of ministries (Economy and Finance, Public Administration, Labor and Social Affairs, Education and Culture, Environment, Justice, and Defense Ministries) share these responsibilities and therefore determine the health benefit basket, although the Ministry of Health and Consumer Affairs plays the main role in finally determining any health policies. Regional governments (ACs) have the responsibility for planning, financing, and providing health care services (including an enlargement of the basic health benefit basket), social and community care, and public health. Another important agent in coordinating and evaluating new benefits is the Institute of Health Carlos III. Local governments are responsible for home-based nursery and social services. Coordination takes place in the Inter-Territorial Council of the NHS, and advisory committee comprising representatives from the central and regional governments that is responsible for ensuring the equality of benefits among and access across regions. The present contribution focuses on services of curative care, excluding services such as rehabilitative care and goods dispensed to outpatients. The other categories have been studied in a detailed report as part of the Health Basket Project and are presented in Tables 1 and 2, including the benefit defining criteria. We first present an overview of the basic benefits that all ACs should provide and describe the additional benefits that some ACs include in their own basket. We then discuss the benefit catalogues and their actual implementation.Table 1 Benefit-defining laws/decrees and catalogues and the implicit regulation by sector. IGeneral Health ActRoyal Decree 63/95Law 16/03Ministerial Decree 30 April 1997Ministerial Decree 3 June 1998Ministerial Decree 3 March 1999Functional categories  HC.1.1+++–––  HC.1.2++–––  HC.1.3+++–––  HC.1.3.1+++–––  HC.1.3.2++–––  HC.1.3.3+++–––  HC.1.3.4+++––  HC.1.4+++–++  HC.2.1+++–––  HC.2.2+++–––  HC.2.3+++–––  HC.2.4––––––  HC.3–++–––  HC.4.1–++–––  HC.4.2–++–––  HC.4.3–++–––Legal statusLawPresidential DecreeLawMinistry DecreeMinistry DecreeMinistry DecreeDecision makerParliamentGovernmentParliamentGovernmentGovernmentGovernmentDegree of explicitnessa132333Positive/negative definition of benefitsPPPPPPOriginal purposeGeneral definition of entitlementsBasketEquality among citizens and ACsBasketBasketBasketCriteria used for defining benefits  Need++++++  Costs–+++++  Effectiveness–+++++  Cost-effectiveness––––––  Budget––––––UpdatingNoNoNoRegularlyRegularlyRegularlya 1, all necessary; 2, areas of care; 3, itemsTable 2 Benefit-defining laws/decrees and catalogues and the implicit regulation by sector. IIGeneral Health ActRoyal D 1663/1995Law 16/2003Decree 3157/1966Law 25/1990Royal Decree 83/1993Royal Decree 1663/1998Royal Decree 1348/2003Ministerial Decree 18 January 1996Functional categories  HC.5.1+––+++++–  HC.5.1.1––––+––––  HC.5.1.2––––+––––  HC.5.2–––––––––  HC.5.2.1–––––––––  HC.5.2.2–+––––––+  HC.5.2.3––––––––+  HC.5.2.4––––––––+  HC.6.1–++––––––  HC.6.2–+–––––––  HC.6.3+++––––––  HC.6.4+++––––––  HC.6.5–––––––––Legal statusLawPresidential DecreeLawLawLawPresidential DecreePresidential DecreePresidential DecreeMinisterial DecreeDecision makerParliamentGovernmentParliamentParliamentParliamentGovernmentGovernmentGovernmentGovernmentDegree of explicitnessa132123333Positive/negative definition of benefitsPPPPPNNNPOriginal purposeGeneral definition of entitlementBasketEquality among citizens and ACsGeneral definition of entitlementTarget ruleExclusionExclusionExclusionBasketCriteria used for defining benefits  Need+++++–––+  Costs–++–+++++  Effectiveness–++–+++++  Cost-effectiveness–––––––––  Budget–––––––––UpdatingNoNoNoNoNoRegularyRegularyRegularyRegularya 1, all necessary; 2, areas of care; 3, items Definition and structure of the main benefit basket for curative care With regards to benefit coverage the Spanish Constitutional Act stipulates Spanish citizenship as the only precondition for access to the range of health care benefits. Parliamentary ratification on 18 December 1997 [3] extended coverage to the entire resident population. The major differences between ACs are related to foreigners. The fundamental act on health benefit regulation is Royal Decree 63/1995 (20 January) on the organization of health services provided by the NHS (Fig. 1). It provides inclusion and exclusion criteria and defines the current level of provision as the guaranteed basic health care basket in a comprehensive form. It contains a checklist of services that constitute the entitlement, establishes certain exclusion criteria, and limits the provision of certain services due to the limited resources available to the NHS. It also stipulates conditions for the introduction of new technologies and procedures as well as new entitlements. Finally, it prescribes coordination between health and social care.Fig. 1 Royal Decree 63/1995 Services, activities, and procedures are excluded under any of the following circumstances: (a) There is insufficient scientific evidence of their clinical safety and efficacy or if they are clearly outdated. (b) They have not sufficiently proven their effective contribution to prevention, treatment, or cure illnesses, preservation or improvement of life expectancy, self-help, or elimination or reduction of pain and suffering. (c) They are constitute merely activities such as those of leisure, rest, comfort, sport, cosmetic improvement, or spa. The Spanish NHS has not yet in fact defined a basic basket of services, although Royal Decree 63/1995 established a general framework of benefits, as did the Law 16/2003 of 28 May (Fig. 2). This law on “Cohesion and Quality of the NHS” is a reform that built on the previous act with the purpose of coordinating a strongly decentralized activity. Its aim is to establish the legal framework for the coordination and cooperation of the Public Health Administrations guaranteeing equality, quality, and participation in the system. It also introduced the establishment of a general procedure for revisions on the content of the health benefit basket. Since 2002 all ACs have health service responsibilities. They manage their regional health care system and also have related legislative responsibilities. These regional health care baskets must include at least the benefits defined by the NHS. In this sense Law 16/2003 is intended to sustain the coordination and the basic guarantees between all the territories.Fig. 2 Law 16/2003 Chapter I of Law 16/2003 refers to health care benefits of the NHS and defines a catalogue that incorporates the benefits defined by Royal Decree 63/1995, public health benefits. The catalogue of benefits is defined as a set of preventive, diagnostic, therapeutic, rehabilitative services, and direct health promotion to citizens, which includes public health, primary and specialized care, social and community care, dietetic urgencies, pharmacy, orthoprosthesis, products, and sanitary transport benefits. It guarantees the right of all citizens to obtain a second medical opinion, the right to receive medical assistance in one’s own AC of residence within a maximum time along, and the right to receive the defined benefits of the NHS under the same conditions and guarantees as residents in other ACs. As a further development of the 16/2003 Law the Spanish Ministry of Health is currently drafting a Royal Decree to establish the NHS health benefit basket and the means for updating it. The purpose of the Royal Decree is to guarantee equality and appropriate care by the NHS. The ACs approve their own baskets by always including all the services of the NHS health benefit basket since these services are considered a basic need and common to all users of the NHS. They can enlarge their own baskets with additional technical services, technologies, and procedures. New benefits added to the basic basket are evaluated by the Ministry of Health through the Evaluation Agency of Health Technologies (“Agencia de Evaluación de Tecnologías Médicas”) and the Carlos III Health Institute in collaboration with other evaluation agencies proposed by the ACs and the Inter-Territorial Council of the NHS. Contents of the benefit baskets for curative care Inpatient curative care The above regulation affecting inpatient curative care defines the benefits covered by the NHS which ought to be provided by all ACs (free of any user payment) such as: specialized health care in hospitals, which includes medical attention, surgery, obstetric, and pediatric services for acute conditions, worsening of chronic conditions or delivery of recommended diagnostic treatments or procedures; organs, tissues and cells of human origin transplants according to the special legislation as long as there is the evidence for therapeutic effectiveness; palliative care to terminally ill patients; and mental health care and psychiatric assistance, which includes clinical diagnosis and follow-up and the prescribed group of family psychotherapy if it implies hospitalization when required. Hospitalizations of short or medium periods of time take place in the psychiatric departments of general hospitals. Inpatient mental long-term care services take place at psychiatric hospitals, which are part of the long-term care network (local authorities). The main benefit exclusions are mental inpatient care (included only upon referral) and transplants of hair, nails, and placenta. Additional benefits on the part of ACs include sex/gender change surgery intervention, such as that now offered in Andalusia. Andalusia also offers epidural anesthetics during birth. The other main benefit extension is related to euthanasia. Catalonia, Galicia, and Extremadura have regulated and included the vital testament as a benefit. Day cases of curative care The entitlements concerning day cases of curative care define the service groups included in the Spanish benefits basket as: specialized ambulatory health care consultations that could include minor surgical procedures; specialized ambulatory health care delivered in “day hospitals” for patients in need of continuous specialized care, physician’s or nursing services, including major surgery as long as no hospitalization is required; and hemotherapy. The following services are explicitly excluded: plastic surgery when it is not related to accident, disease, or congenital malformation; sex change surgery, except for those cases in which it is necessary to repair pathological intersexual conditions. Services of outpatient care Outpatient care is generally provided in primary care centers or specialist care centers (outpatient clinics). There is a wide disparity in the number and use of the latter among ACs. These categories of care are regulated both at national and AC levels. The outpatient benefit basket includes the following service groups (all free of user charges): health assistance for health consultation services and centers including basic medical and diagnostic services (curative) primary care; health services provided at home; indication or prescription and the compliance, in relevant cases, with examinations and basic diagnostic measures as indicated by the primary care physician; the administration of parenteral treatments, cures and minor surgery; initial mental assistance is provided in mental health centers, where patients are being evaluated, assisted, or referred to other assistance services. Benefits provided include: pharmacological and psychotherapy treatments, crisis interventions, etc. Mental emergencies can also be treated at the hospital emergency service; emergency primary health care which will be provided to persons of any age and delivered continuously 24 h a day, through medical and nursing services, on an outpatient basis or at the domicile of the patient if the situation requires to do so; primary dental health care, including dental health and hygiene information and education, preventive and assistance measures; application of topical fluoride, obstructions, sealing of fissures or other services for the infant population according to the annual financial budget and special programs for dental health, treatment of acute orthodontic problems including dental extractions, and preventive exploration of the oral cavity in pregnant women; renal lithotripsy, interventionist radiology, and radiotherapy. Certain services such as complex diet therapy are not free of charge but are also not explicitly excluded from the Spanish health care benefit basket. Other services such as spa treatments, rest cures, psychoanalysis, and hypnosis are explicitly excluded. In this category of care many ACs offer additional services as part of their benefit catalogues. At primary care level the Basque Country, Extremadura, Castilla La Mancha, and Catalonia have passed several decrees; at specialized outpatient care level Navarre offers obstetrics, gynecological and family planning such as menopause care; Valencia provides reactive strips for determining glucose in blood and urine to diabetics; and at special treatment level the Balearic Islands offer neurological reflexology in cooperation with the Kovacs Foundation. It must also be noted that Catalonia has started a pilot experience in offering acupuncture for arthritis treatment. Nine ACs also provide free dental care for some groups: Navarre, Basque Country, Andalusia, Extremadura, Castilla La Mancha, Cantabria, Castilla León, Murcia, and Galicia. Catalonia offers periodic rinsing of a fluorine solutions program for primary school students and those taking the first two courses of secondary school. As all these catalogues are quite similar, we present below the dental care benefit catalogue of Castilla La Mancha (Fig. 3). The Infant-Young Dental Care Plan defines the group of measures and activities, both preventive and welfare, as well as any procedures to provide dental care. Benefits are classified into: oral check-up, basic dental treatments, emergency dental treatments, special dental treatments, and orthodontics.Fig. 3 Dental care catalogues: the example of Castilla La Mancha Services of curative home care As part of the entitlements of services of curative home care, oxygen therapy service at home is provided at authorized and specialized centers capable of carrying out gasometry and spirometry. The Ministerial Decree of 3 March 1999 [4] elaborates these services, considering the following benefits: (a) oxygen therapy at home, (b) mechanical ventilation at home, (c) ventilation treatment of the sleep apnea symptoms, and (d) aerosol therapy. Enteral nutrition is also provided at home. This service is made available to patients who suffer from the following symptoms: swallowing, transit, digestion or absorption of foods in its natural form problems or when special requirements of energy and/or nutrients exist that cannot be covered with foods of daily consumption. Royal Decree 63/1995 has been developed with regard to benefits with diet products in two ways: regulating complex diet therapy and regulating enteral nutrition at home. Complex diet therapy is dealt with in the Ministerial Decree of 30 April 1997 [5] which specifies the relationship of congenital metabolic upheavals of carbon hydrates and amino acids included in this benefit and the type of diet in each case. It also prescribes these treatments to be performed by a special physician in hospital units, specifically authorized by the appropriate Health Administration. Enteral nutrition at home is regulated by the Ministerial Decree of 2 June 1998 [6], which defines the requirements and the patient’s clinical situation for this. There are also regulations at the national and interterritorial council levels as well as in six ACs that have developed their own dietetic regulations. The Ministerial Decree of 3 March 1999 was passed upon the proposal of a panel of experts (Fig. 4). This elaborates Royal Decree 63/1995 regarding the techniques of oxygen therapy at home financed by the NHS and clarifies and standardizes the criteria, clinical situations, and other matters for the direction, prescription, and provision of these benefits. It defines the oxygen therapy techniques that can be carried out at home in terms of their financing by the NHS and establishes the basic requirements that justify their prescription. The Ministerial Decree of 3 March 1999 thus provides a common basis upon which the health service providers must develop their own procedure for providing and supervising these benefits. The Decree actually does not provide a benefit catalogue as such because the individual physician determines the specific treatment in each case. In addition there are also specific regional oxygen therapy regulations in four ACs (Andalusia, Canary Islands, Navarre and Valencia).Fig. 4 Oxygen therapy at home Services of MUFACE catalogue All civil servants and beneficiaries are entitled to receive benefits as determined by the MUFACE catalogue. This benefit basket is virtually equivalent to that of the NHS but include some additional benefits. The principal difference is related to copayments for pharmaceuticals. In addition to particular features for medical assistance abroad, orthoprosthetic, dental care, ocular, pharmaceutical, and sociosanitary benefits, services such as vaccinations are included free of charge. Specialized health care includes all the medical and surgical specialties, both in outpatient and inpatient sectors. The specialties are structured in four levels according to their complexity and geographic scope, sanitary transport, oxygen therapy, and aerosol therapy at home. Discussion The way in which the Spanish benefit catalogue is defined is characteristic of tax-financed NHS health care systems. A system such as that in Spain which guarantees universal coverage is not completely developed until it is specifies a minimum basket of health care services. However, as no specific and explicit benefit catalogue is being developed, there remains a need for further legislation. The Ministry of Health and Consumption is moving towards an explicit process of decision making in health benefit planning instead of defining a detailed benefit catalogue. As is the case in many countries, the possibility of using regional plans as the basis for resource allocation and capacity planning has not yet been considered. An additional problem in Spain is the fact that the majority of health care plans offer unrealistically long lists of objectives which are often not sufficiently based on available epidemiological and cost-effectiveness evidence, and their efficacy is seldom evaluated. They cannot be considered as benefit catalogues since they are not sufficiently strong legal instruments, and therefore users are not able to claim the objectives contained in the health plans as benefit rights. Then purpose of the Spanish health benefits basket is twofold, as it was initially established to serve more as a budgetary than as a cost containment measure when created by the Social Security act [7]. However, on the other hand, as Royal Decree 63/1995 and the 2003 Cohesion Law recognize, they are merely an ordering of benefits as they try only to describe what is being offered in practice. Two major aspects of the Spanish health care system are: (a) A more explicit definition of the benefit basket is needed as the present vague definition leaves the final decisions to practitioners, making expenditure very volatile, and having as the sole restriction that of waiting times. (b) Greater transparency is needed in the process of approval/rejection of new benefits, towards agents in the health sector, and towards citizenship along with the need to include economic considerations in the decision process. In Spain it still remains the case: “all for everyone and almost everything for free.” This obviously leads to long waiting times and thus a dual system (public-private) for those able to skip the long queues and high public pressure over resources devoted to the NHS. Furthermore, since there are no user payments, waiting lists serve as the mechanisms restricting demand for health services. For the first time, the Catalan Health Service is now studying the possibility of introducing a copayment system in primary care (fixed payment per visit). There are still some remaining problems in the Spanish benefit basket. In terms of equality, the fact that MUFACE-type schemes have not been fully integrated to the NHS provides additional benefits for these groups. There is a lack of real control over the managers of the health care services. Autonomy of decisions by practitioners may mean an obstacle to the early evaluation of new technologies since under uso tutelado (monitored use) it is physicians who start using them. In the present financing system there is no relationship between this and the benefits provided, nor a control mechanism over what is provided in reality. Moreover, the information systems are clearly the main problem in the decentralization process. To overcome this problem the Ministry of Health has recently introduced an institution for monitoring the health system. Even the appearance of new benefit differences across ACs will not affect equality such as it may affect clinical practices and access to equipment. Therefore the main purposes of the Spanish health policy are to promote common clinical guidelines and evaluation processes with a single registry on new infrastructure and the creation and regulation of a common procedure for new benefit approvals. In the future the central government is not expected to increase benefits but rather the quality of the existing ones, through new technologies and clinical procedures. Improvements are expected regarding the provision of services (e.g., waiting times), better user information, and increased range of choices. Probably the most important task is the development of a long-term care or dependency system and a real network of mental health. Future reforms taken by the different ACs will likely address the benefits basket. Dental care and natural and alternative medicines are probably the first services that will be addressed. The definition of a basic long-term social and community care benefit basket similar to that in health care will also be a priority for the central government. Nevertheless, as we can see in Table 3, new benefits have very little impact on the health expenditure of ACs.Table 3 Spending in the provision of health care services not included in the benefit catalogue of the National Health System (Royal Decree 63/1995): proportion of total health care services spending19992000200120022003Total0.150.130.130.120.11Andalusia0.400.320.320.300.29Aragon–––––Asturias–––0.00030.0007Balearic Islands––––0.02Canary Island–––––Cantabria–––––Castilla y León–––––Castilla–La Mancha–––––Catalonia0.400.380.360.320.27Valencia–––––Extremadura––––0.04Galicia–––––Madrid–––––Murcia––––0.05Navarre0.470.440.470.460.49Basque Country0.200.180.190.180.19La Rioja–––––Ceuta–––––Melilla––––– Finally, in Spain, the growth of public health expenditure is due to both the increase in input prices and the received average benefits per capita. The growth of public health care expenditure is largely attributable to the benefits provided. The yearly change in health care benefits was responsible for 1.86% of the 6.73% annual average increase in health expenditure between 1991 and 2003.

Diabetologia-4-1-2270364

Epigenetic regulation of PPARGC1A in human type 2 diabetic islets and effect on insulin secretion

Aims/hypothesis Insulin secretion in pancreatic islets is dependent upon mitochondrial function and production of ATP. The transcriptional coactivator peroxisome proliferator activated receptor gamma coactivator-1 alpha (protein PGC-1α; gene PPARGC1A) is a master regulator of mitochondrial genes and its expression is decreased and related to impaired oxidative phosphorylation in muscle from patients with type 2 diabetes. Whether it plays a similar role in human pancreatic islets is not known. We therefore investigated if PPARGC1A expression is altered in islets from patients with type 2 diabetes and whether this expression is influenced by genetic (PPARGC1A Gly482Ser polymorphism) and epigenetic (DNA methylation) factors. We also tested if experimental downregulation of PPARGC1A expression in human islets influenced insulin secretion. Introduction Type 2 diabetes is characterised by chronic hyperglycaemia as a result of impaired pancreatic beta cell function and insulin resistance in peripheral tissues, i.e. skeletal muscle, adipose tissue and liver. Although each of these pathogenic defects could be accounted for by specific mechanisms, impaired ATP production as a consequence of reduced oxidative phosphorylation might provide an intriguing common pathogenic pathway for all these defects. The transcriptional coactivator peroxisome proliferator activated receptor gamma coactivator-1 alpha (protein PGC-1α; gene PPARGC1A) is an important factor regulating the expression of genes for oxidative phosphorylation and ATP production in target tissues through coactivation of nuclear receptors [1]. We have previously shown that the expression of PPARGC1A and a set of genes involved in oxidative phosphorylation is reduced in skeletal muscle from patients with type 2 diabetes [2]. Furthermore, a common polymorphism, Gly482Ser, in the PPARGC1A gene has been associated with increased risk of type 2 diabetes and an age-related reduction in muscle PPARGC1A expression [3–5]. In addition, genetic variation in the PPARGC1A gene was associated with indices of beta cell function [6]. Despite the central role of ATP for insulin secretion, the function of PGC-1α in human pancreatic islets and beta cells is less well established [7]. Obesity, reduced physical activity and ageing are well known risk factors for type 2 diabetes. However, all individuals exposed to an affluent environment do not develop the disease. One likely reason is that genetic variation modifies individual susceptibility to the environment. However, the environment could also modify genetic risk factors by influencing expression of a gene by DNA methylation or histone modifications. Cytosine residues occurring in CG dinucleotides are targets for DNA methylation and gene expression is usually reduced when DNA methylation takes place at a promoter. Whether DNA methylation influences gene expression in target tissues for type 2 diabetes and thereby the pathogenesis of the disease remains to be demonstrated. The present study investigated: (1) whether expression of PPARGC1A is altered in human islets from patients with type 2 diabetes; (2) if this expression is influenced by genetic (the PPARGC1A Gly482Ser polymorphism) and epigenetic (DNA methylation) factors; and (3) if expression of PPARGC1A influences insulin secretion. Methods Multi-organ donors The characteristics of the 48 non-diabetic and 12 type 2 diabetic multi-organ donors, whose pancreases were processed for islet preparation, are presented in Table 1. Pancreases were obtained and processed with the approval of the regional Ethics Committee. Table 1Clinical characteristics of type 2 diabetic and non-diabetic donors Non-diabetic donorsType 2 diabetic donorsp valuen (male/female)48 (30/18)12 (6/6) 9 (7/2)a10 (5/5)a Age (years)53.2 ± 2.466.7 ± 2.4<0.0554.2 ± 3.5a65.1 ± 2.6aBMI (kg/m2)24.8 ± 0.627.1 ± 1.0<0.0525.9 ± 1.3a26.9 ± 1.1aGly/Gly (%)b53.327.3 Gly/Ser+Ser/Ser (%)b46.772.3 Data are expressed as mean ± SEMaDonors used for DNA methylation analysisbPPARGC1A Gly482Ser polymorphism Human pancreatic islets and experimental plan Isolated pancreatic islets were prepared by collagenase digestion and density gradient purification [8, 9]. After isolation, islets were cultured free floating in M199 culture medium (Sigma-Aldrich, St Louis, MO, USA) at 5.5 mmol/l glucose concentration and studied within 3 days from isolation. Cell viability, measured by Trypan Blue exclusion, was higher than 90% in control and diabetic islets after 3 days in culture. Insulin secretion study Insulin secretion studies were performed as previously described [8, 9]. Following a 45 min pre-incubation period at 3.3 mmol/l glucose, groups of 30 islets of comparable size were kept at 37°C for 45 min in KRB, 0.5% (wt/wt) albumin, pH 7.4, containing 3.3 mmol/l glucose. At the end of this period, the medium was completely removed and replaced with KRB containing either 3.3, 16.7 or 3.3 mmol/l glucose plus 20 mmol/l arginine, or 3.3 mmol/l glucose plus 100 μmol/l glibenclamide. After an additional 45 min incubation period, the medium was removed. Media (500 μl aliquots from the 10 ml incubation volume) were stored at −20°C until insulin concentrations were measured by immunoradiometric assay (Pantec Forniture Biomediche, Turin, Italy). Animals Streptozotocin (STZ)–nicotinamide (NA)-treated male Wistar rats (2–3 months old) were administered 210 mg/kg NA i.p. (Sigma, St Louis, MO, USA) dissolved in saline, 15 min before an i.v. injection of 60 mg/kg STZ (Sigma) that had been dissolved in citrate buffer (pH 4.5) immediately before use. Control rats were injected with vehicle alone. STZ–NA-treated animals had stable hyperglycaemia (8.9–10.0 mmol/l) and they were used for the experiments 5 weeks after diabetes was induced. Pancreatic islets were isolated by the collagenase method using the procedure of pancreatic duct cannulation and density gradient purification as described elsewhere [10, 11]. GK rats were obtained from the Stockholm colony and bred as described [12]. Inbred, normoglycaemic F344 rats were purchased from Charles River Laboratories (Wilmington, MA, USA) and maintained by sister–brother mating. Transfer of GK alleles onto the genome of F344 rats by repeated backcrossing (ten generations) established the homozygous congenic strains F344.GK-Niddm1f (NIDDM1F) and F344.GK-Niddm1i (NIDDM1I). NIDDM1F carries 0.5% of the GK genotype (8 cM), based on genetic distance, on a homozygous F344 genetic background. NIDDM1F rats display hyperglycaemia accompanied by fasting hyperinsulinaemia and increased epididymal fat, implicating insulin resistance. NIDDM1I carries 0.8% of the GK genotype (14 cM), and display hyperglycaemia and insulin secretion defects [13–15]. Backcrossing was designed to introduce mitochondrial DNA and chromosomes X plus Y from F344. The congenic strains were kept constant by sister–brother mating for several generations. To avoid effects of the oestrous cycle and other sex-specific influences, only male rats were included in this study. Rats were maintained at constant temperature and humidity in a 12 h light–dark cycle with free access to standard laboratory chow pellets and water. All experiments were approved by the local Ethics Committees. Isolated pancreatic islets were prepared from rats at 8 weeks of age after a 6 h fast (08:00–14:00 hours), by injection of a collagenase solution via the bile–pancreatic duct [16]. Analysis of PPARGC1A mRNA expression in pancreatic islets Total RNA was extracted from human islets, after 3 days in culture, using the RNeasy Protect Mini Kit (Qiagen, Valencia, CA, USA). It was quantified by absorbance at A260/A280 nm (ratio > 1.65) in a Perkin-Elmer spectrophotometer (Waltham, MA, USA) and its integrity was assessed after electrophoresis in 1.0% (wt/wt) agarose gels by ethidium bromide staining. Human and rat PPARGC1A mRNA expression were quantified by RT-PCR [8]. Gene-specific probes and primer pairs for PPARGC1A (Assays-on-demands, human, Hs00173304_m1 and rat, Rn00580241_A1; Applied Biosystems, Foster City, CA, USA) were used. Each sample was run in duplicate and the transcript quantity was normalised to the mRNA level of cyclophilin A (human, 4326316E and rat Rn00574762_A1; Applied Biosystems). For each probe/primer set, a standard curve was generated, which was confirmed to increase linearly with increasing amounts of cDNA. Downregulation of PGC-1α in human islets using silencing RNA (siRNA) In order to test whether inhibition of PPARGC1A expression could directly modulate insulin release, isolated human islets were transfected with PPARGC1A siRNA by using Arrest-In Transfection (Open Biosystem; Celbio, Pero, Italy). This is a polymeric formulation developed and optimised for highly efficient delivery of siRNA into the nucleus of suspension cells in the presence of serum-containing medium. Pre-designed Silencer siRNAs and Silencer non-targeting siRNAs (negative control) for PPARGC1A (Ambion, Austin, TX, USA) were used. Transfection was performed according to the manufacturer’s instructions. Briefly, islets obtained from five pancreases were washed 12 times in KRB and exposed for 10 min to free Ca2+- and Mg2+-KRB to allow cell disaggregation. At the end of the incubation period, 400 islets per study point were re-suspended in 800 μl M199 medium (Sigma-Aldrich) added with adult bovine serum. siRNA (80 nmol/l) was diluted in 100 μl M199, while 20 μg Arrest-In solution (Celbio) was dissolved in 100 μl M199 and incubated for 10 min after rapid mixing to allow formation of transfection complexes. Finally, 200 μl of this solution were added into wells containing islets and incubation was allowed for 48 h in a CO2 incubator at 37°C. At the end of the incubation period well volume was doubled with M199 culture medium and samples were kept in the incubator for another 48 h, when islets function, viability, transfection efficiency (60% when measured by Polyfectamine tied to a fluorescent probe) and gene expression were evaluated. Genotyping Genomic DNA was extracted from pancreatic islets using a Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA). The Gly482Ser (GGT→AGT) polymorphism of PPARGC1A was genotyped using an allelic discrimination assay performed with an ABI 7900 system (Applied Biosystems), using PCR primers: 5′-CACTTCGGTCATCCCAGTCAA-3′ (forward) and 5′-TTATCACTTTCATCTTCGCTGTCATC-3′ (reverse), and TaqMan MGB probes: Fam-5′-AGACAAGACCGGTGAA-3′ and Vic-5′-CAGACAAGACCAGTGAA-3′ [5, 17]. DNA methylation A sequence starting 5,000 bp upstream from the PPARGC1A translation start was used in MethPrimer (http://www.urogene.org/methprimer/index.html) to search for regions with CpG sites and PCR designs. A PPARGC1A sequence 986–746 bases upstream from the translation start including four possible DNA-methylation sites and a putative hepatic nuclear factor 1 (HNF-1) binding site, was selected and analysed for DNA methylation (Fig. 1c). Genomic DNA, isolated from pancreatic islets of nine non-diabetic and ten type 2 diabetic multi-organ donors, was treated with bisulphite using the EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA). Bisulphite-modified DNA was amplified by nested PCR with primers designed using MethPrimer. Primer pair 1: 5′-TAGGGTATTAGGGTTGGAATTTAATG-3′ (forward) and 5′-CCCATAACAATAAAAAATACCAACTC-3′ (reverse), and primer pair 2 (used for nested PCR): 5′-TATTTTAAGGTAGTTAGGGAGGAAA-3′ (forward) and 5′-ATAACAATAAAAAATACCAACTCCC-3′ (reverse). The PCR products were then cloned into a vector (TOPO TA Cloning Kit for Sequencing, Invitrogen, Carlsbad, CA, USA) and ten colonies from each donor were purified with a Miniprep kit (Qiagen). These individual clones were sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The number of methylated sites was determined and divided by the total number of methylation sites and then multiplied by 100 to show the percentage of methylation for each donor. Fig. 1PPARGC1A mRNA expression in human pancreatic islets is influenced by type 2 diabetes, a PPARGC1A Gly482Ser polymorphism and DNA methylation. The influence of a type 2 diabetes and b the PPARGC1A Gly482Ser polymorphism on PPARGC1A mRNA expression in human pancreatic islets. c The PPARGC1A promoter sequence investigated, showing the four DNA methylation target sites; −772, −903, −936 and −961 and a putative binding-site for HNF-1. d The influence of type 2 diabetes on DNA methylation of the PPARGC1A promoter. The influence of e type 2 diabetes (T2D) and f the PPARGC1A Gly482Ser polymorphism on absolute insulin release (pmol islet−1 min−1) in response to 16.7 mmol/l glucose. g Correlations between PPARGC1A mRNA expression and absolute insulin release (pmol islet−1 min−1) in response to 16.7 mmol/l glucose in human pancreatic islets (r = 0.38, p < 0.05). Results are expressed as mean ± SEM. *p < 0.05 Statistical methods Differences in PPARGC1A mRNA expression, percentage of DNA methylation and insulin secretion between the different groups studied were analysed using Student’s t test or the non-parametric Mann–Whitney test, where appropriate. Correlations were calculated using Pearson correlation coefficients for normally distributed values and Spearman correlation coefficients when normality was rejected. Log values were used in the multivariate regression analysis. Differences in expression between GK, F344, NIDDM1F and NIDDM1I rats were analysed using one-way ANOVA, followed by a Kruskal–Wallis Z test. All p values were two-tailed and p values less than 0.05 were considered significant. Statistical calculations were performed by NCSS software (NCSS Statistical Software, Kaysville, UT, USA). Results The mRNA expression of PPARGC1A was markedly reduced in pancreatic islets from type 2 diabetic compared with non-diabetic donors (1.06 ± 0.63 vs 15.0 ± 3.0; p < 0.005; Fig. 1a). Intriguingly, non-diabetic carriers of a PPARGC1A 482Ser allele, Gly/Ser or Ser/Ser genotype carriers had markedly lower PPARGC1A mRNA expression compared with carriers of the Gly/Gly genotype [Gly/Ser + Ser/Ser 3.43 ± 0.94 (n = 17) vs Gly/Gly 26.53 ± 4.42 (n = 17); p < 0.00005] (Fig. 1b). However, there was no difference in PPARGC1A mRNA expression between non-diabetic carriers of the Gly/Ser (3.9 ± 4.0; n = 11) or Ser/Ser (2.7 ± 5.5; n = 6) genotypes. This demonstrates that the effect of the genotype on gene expression is seen already in the pre-diabetic state. The diabetic donors were significantly older and had higher BMI than control donors (Table 1). We therefore tested whether these factors might also relate to PPARGC1A expression in human islets using a multivariate regression analysis including age, BMI and disease status as covariates. Only disease status was significantly associated with PPARGC1A expression in this analysis (r = 1.04; p = 0.0065). We next evaluated whether epigenetic phenomena such as DNA methylation would influence PPARGC1A expression in islets. Cytosine residues occurring in CG dinucleotides are targets for DNA methylation, and gene expression is usually reduced when DNA is methylated at the promoter. We used bisulphite sequencing to assess DNA methylation of four methylation target sites, −772, −903, −936 and −961 in the PPARGC1A promoter in pancreatic islets from the type 2 diabetic and non-diabetic human multi-organ donors (Fig. 1c). Interestingly, there was an approximately twofold increase in DNA methylation of the PPARGC1A promoter of diabetic compared with non-diabetic human islets (10.5 ± 2.7 vs 4.7 ± 0.9%; p < 0.04; Fig. 1d). Also in this subset of donors, the PPARGC1A mRNA expression was significantly reduced in diabetic compared with non-diabetic islets (p = 0.002). There was a trend towards an inverse correlation between the level of DNA methylation and PPARGC1A mRNA expression (r = −0.48; p = 0.08, after adjustment for disease status). Insulin release (pmol islet−1 min−1) in response to 16.7 mmol/l glucose was reduced in type 2 diabetic compared with non-diabetic islets (0.32 ± 0.04 vs 0.55 ± 0.05; p < 0.01) as well as in non-diabetic islets carrying the PPARGC1A 482Ser allele (Gly/Ser + Ser/Ser) compared with carriers of the Gly/Gly genotype (Gly/Ser + Ser/Ser 0.43 ± 0.06 vs Gly/Gly 0.64 ± 0.07; p < 0.05; Fig. 1e–f). In addition, the mRNA expression of PPARGC1A correlated positively with glucose-mediated insulin release (r = 0.38; p < 0.05; Fig. 1g). Moreover, the PPARGC1A Gly482Ser polymorphism did not significantly affect basal insulin secretion at 3.3 mmol/l glucose (Gly/Ser + Ser/Ser, 0.23 ± 0.01 vs Gly/Gly, 0.29 ± 0.02; p = 0.13), or insulin response to arginine (Gly/Ser + Ser/Ser 0.48 ± 0.05 vs Gly/Gly 0.53 ± 0.08; p = 0.77) or glibenclamide (Gly/Ser + Ser/Ser 0.59 ± 0.1 vs Gly/Gly 0.56 ± 0.09; p = 0.98). Having seen the relationship between PPARGC1A expression and insulin secretion, we set out to determine whether modulation of PPARGC1A expression would influence insulin secretion. This was achieved by studying insulin secretion after experimental downregulation of PGC-1α in human islets transfected with PPARGC1A siRNA (n = 3–5 experiments). As shown in Fig. 2, transfection with siRNA was associated with a 45% reduction in the mRNA expression of PPARGC1A (p = 0.01), 32% reduction in insulin mRNA (p = 0.05) and 41% reduction of the insulin stimulation index (i.e. incremental folds above baseline insulin release; p = 0.01). Notably, no significant change was seen in glucagon mRNA expression, suggesting that the effect was primarily due to a downregulation of PGC-1α in beta cells. As a negative control, transfection with scrambled siRNA was not associated with any changes in PPARGC1A (1.26 ± 0.25), insulin (1.29 ± 0.16) or glucagon (0.99 ± 0.15) mRNA expression (p > 0.05). Fig. 2Transfection of human pancreatic islets with PPARGC1A siRNA is associated with reduced mRNA levels of aPPARGC1A (n = 3) and b insulin (n = 5) and c concomitant reduction of the insulin stimulation index (ISI), i.e. incremental fold change above basal insulin release. d Conversely, inhibition of PPARGC1A expression in human pancreatic islets has no effect on glucagon mRNA expression (n = 5). Results are expressed as mean ± SEM. *p < 0.05 The finding of reduced PPARGC1A expression in human diabetic islets was somewhat surprising given an earlier report of increased PGC-1α levels in islets from diabetic mice (ob/ob) and rat (ZDF and pancreatectomised rats) models [7]. To further explore species-specific differences we also measured PPARGC1A mRNA expression in pancreatic islets from two diabetic rat models. PPARGC1A expression was reduced in islets from STZ–NA diabetic male Wistar rats compared with control animals (3.08 ± 1.07 vs 10.26 ± 1.12; p < 0.009; Fig. 3a). Moreover, PPARGC1A expression was reduced in islets from the congenic NIDDM1I strain, which has insulin secretion defects, compared with the insulin-resistant NIDDM1F strain (7.18 ± 1.79 vs 11.30 ± 1.79; p < 0.02) of the GK rat, an animal model of polygenic type 2 diabetes (Fig. 3b). However, no difference in PPARGC1A expression was found when comparing islets from F344 or GK rat strains (Fig. 3b). Fig. 3PPARGC1A mRNA expression in rodent pancreatic islets. a Male Wistar rats (2–3 months old) were treated with STZ–NA (n = 5) or vehicles (control; n = 5) [10, 11] and PPARGC1A mRNA expression together with the internal standard cyclophilin A was analysed in pancreatic islets isolated from STZ–NA-treated animals showing a stable hyperglycaemia (8.9–10.0 mmol glucose/l) and controls. bPPARGC1A mRNA expression together with the internal standard cyclophilin A was analysed in pancreatic islets prepared from rats at 8 weeks of age after a 6 h fast. GK rats (n = 6) were obtained from the Stockholm colony and bred as described [12]. Inbred, normoglycaemic F344 (n = 6) were purchased from Charles River Laboratories. Transfer of GK alleles onto the genome of F344 rats by repeated backcrossing (ten generations) established the homozygous congenic strains NIDDM1F and NIDDM1I. NIDDM1F rats (n = 6) carry 0.5% of the GK genotype, based on genetic distance, on a homozygous F344 genetic background (8 cM) and display hyperglycaemia accompanied by fasting hyperinsulinaemia, implicating insulin resistance. NIDDM1I (n = 6) carries 0.8% of the GK genotype (14 cM) and display insulin secretion defects [13–15]. Results are expressed as mean ± SEM. *p < 0.05 Discussion The key results from the present study were that PPARGC1A mRNA expression and insulin secretion were reduced in pancreatic islets of patients with type 2 diabetes and in non-diabetic carriers of a PPARGC1A 482Ser allele, a genotype previously associated with the disease. Moreover, we demonstrate that epigenetic mechanisms are likely to be operative in the pathogenesis of type 2 diabetes since DNA methylation of the PPARGC1A promoter was increased in human diabetic islets. Experimental downregulation of PPARGC1A expression in human islets by siRNA resulted in decreased insulin secretion thereby demonstrating a causal link between PGC-1α levels and insulin secretion. Based on these observations we propose a model where combinations of genetic and epigenetic factors can influence the level of PGC-1α in human islets and subsequently glucose-stimulated insulin secretion. The transcriptional coactivator PGC-1α is an important factor regulating the expression of genes for oxidative phosphorylation in a number of tissues including skeletal muscle, liver and adipose tissue. We and others have previously demonstrated reduced expression of PPARGC1A and a set of genes involved in oxidative phosphorylation in skeletal muscle from patients with type 2 diabetes [2, 18]. However, the level and role of PGC-1α in pancreatic islets of patients with type 2 diabetes remained to be determined. The present results indicate that the situation in human islets mirrors the situation in skeletal muscle, since the expression level of PPARGC1A was reduced in human type 2 diabetic islets, thus providing evidence for a common defect that may simultaneously contribute to peripheral insulin resistance and impairment of beta cell function. In contrast to the present results in humans, elevated PGC-1α levels have been reported in islets from diabetic rodent models, ob/ob mice, ZDF rats and pancreatectomised rats [7]. In these rodent models, the increase in PGC-1α was associated with suppressed beta cell metabolism and insulin release. This could reflect true species differences, but also differences in experimental conditions. To address this issue, we determined: (1) the effect of inhibiting PGC-1α production by transfecting human pancreatic islets with PPARGC1A siRNA; and (2) PPARGC1A expression in pancreatic islets of different rodent models with experimental diabetes, i.e. STZ-diabetic Wistar rats and congenic strains from the type 2 diabetes-like GK rat. Silencing PGC-1α production was associated with concomitant reduction of insulin mRNA expression and insulin release in response to glucose. This effect appears to be highly specific for insulin regulation as it had no effect whatsoever on glucagon mRNA expression. The congenic strains have been bred from the GK rat based upon low insulin secretion or insulin resistance. In line with the results observed in human islets, PPARGC1A expression was reduced in islets from diabetic animals and in rat strains with suppressed insulin secretion. Collectively, these data support the notion that PPARGC1A expression is reduced in animal models of diabetes and human diabetes, and associated with impaired insulin secretion. The risk of developing type 2 diabetes increases with high-fat/high-energy diets, reduced physical activity and age. However, not all individuals respond to the environment in the same way. This variation in response to environmental factors has partially been ascribed to genetic factors. The common Gly482Ser variant in the PPARGC1A gene is a plausible candidate for such a genetic factor [3, 4]. We have previously shown that this polymorphism is associated with an age-related decline in PPARGC1A gene expression in human skeletal muscle [5]. The present study shows that the same PPARGC1A risk allele, 482Ser, is associated with reduced PPARGC1A expression and impaired insulin secretion in human islets. Thus, our results provide the ground for a common genetic predisposition towards type 2 diabetes contributing to both impaired insulin action and secretion. Reduced PPARGC1A expression may not simply reflect an effect of the polymorphism per se but could also occur through epigenetic phenomena such as increased DNA methylation of the PPARGC1A promoter. Cytosine residues occurring in CG dinucleotides are targets for DNA methylation, and gene expression is usually reduced when DNA methylation takes place at a promoter. Epigenetic changes are well known to influence gene expression of suppressor genes and oncogenes in cancer cells and contribute to tumour growth [19]. Increased DNA methylation of the promoter of the PPARGC1A gene in type 2 diabetic human islets may contribute to beta cell dysfunction by similar mechanisms. To our knowledge, this is the first study to demonstrate that DNA methylation may play a role in regulating gene expression in human diabetic islets. Our data suggest that PGC-1α can modulate glucose-mediated insulin secretion in human islets, most likely via an effect on ATP production as indicated by normal insulin release in response to arginine and glibenclamide, which stimulate insulin downstream of ATP. In support of such a role, PPARGC1A mRNA expression correlated with glucose-stimulated insulin release. Moreover, inhibition of PPARGC1A expression by siRNA transfection in human islets was associated with a decline in insulin mRNA and insulin release. Similarly, in human type 2 diabetic islets, reduced PPARGC1A mRNA levels were associated with impaired glucose-mediated insulin secretion. The PPARGC1A Gly482Ser polymorphism, which previously has been associated with type 2 diabetes, was also associated with reduced PPARGC1A expression and impaired insulin secretion in the human islets. One caveat of this study could be that we did not measure the protein level of PGC-1α in human islets. However, we have previously demonstrated a significant positive correlation between the PPARGC1A mRNA and PGC-1α protein levels in human skeletal muscle [5] and we therefore assume that this might also be the case in human islets. The reduction in PPARGC1A expression in non-diabetic carriers of a 482Ser allele was strong and factors other than haploinsufficiency may therefore explain this reduction. Moreover, whether the genotype influences PPARGC1A expression in diabetic islets would be of interest; however, the present study lacks statistical power to perform this analysis. These relationships should be addressed in future ad hoc studies. In conclusion, we have shown that PPARGC1A mRNA expression is reduced in islets from patients with type 2 diabetes and it is influenced by both genetic (Gly482Ser) and epigenetic (DNA methylation) factors. This reduction in PPARGC1A expression correlated with impaired glucose-stimulated insulin secretion, which is known to require ATP. Thereby, the present study provides two novel insights into the molecular mechanisms of islet dysfunction in type 2 diabetes; first that a master transcription regulator of oxidative phosphorylation, PGC-1α, may be involved and second, that epigenetic factors like DNA methylation should be considered when searching for the genetic causes of type 2 diabetes.

Soc_Psychiatry_Psychiatr_Epidemiol-2-2-1764204

Regional differences in psychiatric disorders in Chile

Background Psychiatric epidemiological surveys in developing countries are rare and are frequently conducted in regions that are not necessarily representative of the entire country. In addition, in large countries with dispersed populations national rates may have low value for estimating the need for mental health services and programs. Cross-national psychiatric epidemiological prevalence studies using similar diagnostic instruments have resulted in disparate rates for specific disorders [1]. The reasons for these differences in rates have been attributed to methodological issues between studies; socio-demographic factors such as socio-economic status differences between countries; and cultural differences. Cross-national comparative studies [2, 3] attempt to correct for socio-demographic variability; yet, differences persist. In some regions of the world, such as prevalence rates in Chinese based studies [4–6], the rates are markedly different than the rest of the world. This either suggests true differences in the rates of pathology or lack of cultural appropriateness of the diagnostic tools and the Western diagnostic systems utilized [7]. Understanding these cross-national differences may provide clues into the etiology of psychopathology. Regional differences also exist within countries as evidenced by studies where the methodology is similar across geographic areas [8–11]. Regional differences in the presence of serious mental illness have been attributed to migration of the mentally ill [12, 13]; birth in urban areas [14]; and genetic pooling [15]. The most studied regional differences are those between urban and rural populations, where urban environmental adversity is argued to contribute to pathology [16, 17]. An understanding of regional differences in countries where the population is spread across large distances is relevant for health care planning. Potentially risk factors may differ across a country resulting in the need to address mental health needs on a regional basis rather than centrally. Furthermore, an understanding of geographic variability permits allocation of resources to be distributed in a proportional basis by need. This issue is of particular importance to developing countries such as in Latin America, where frequently services are only provided centrally, or disproportionately to the wealthier regions of a country. In addition, epidemiological studies based on single regions of the country [18–20] are extrapolated to larger population bases for which they may or may not be representative. Population studies about psychiatric disorders in Latin America, as well as other developing regions of the world, are rare. They are important, however, for understanding variations in patterns of disorders, underlying determinants, and service needs. Chile, given its rather extraordinary geography provides an important test of variations in disorder rates across a spatially dispersed population, and offers perhaps the best case example of a country where national rates would seemingly have low value for estimating the need for mental health services and programs. The Chile Psychiatric Prevalence Study (CPPS) was developed to address issues regarding the prevalence and risk factors for mental illness based on a nationally representative sample, and service utilization. Chile has a population of approximately 16 millions people. The country is composed of 51 provinces grouped in 13 regions covering an area spanning 2 million km2 (including Antarctica and Insular Territories) over a length of 8,000 km. The large distances between major population centers resulted in the CPPS being conducted in four regions of the country, Bio Bio, the south-central region containing the second largest city, Metropolitana, the north-central region which includes the capital Santiago, Tarapaca, the north, and Araucania, the south of the country, in order to obtain a representative sampling of the population of the nation. This report focuses on whether regional differences in the prevalence of psychiatric disorders and service utilization, if present, are due to factors other than socio-demographic differences between population centers. Methods Sample selection The CPPS was based on a household stratified sample of people age 15 and older. A more detailed description of the methods used in the CPPS is available in earlier publications [21]. The sample frame was developed to be representative of the nation’s population. Four regions and their most representative province and comunas were selected. These were subsequently subdivided into districts, and then randomly selected blocks. The number of households available on each block was enumerated. The 1992 census of each region was used to determine the number of households required on each block. A list of the inhabitants, age 15 and older, in each household was generated. Using pre-assigned Kish tables (Kish 1965) one person per household was selected from the list to be interviewed. The survey was conducted by the University of Concepcion, Department of Psychiatry and Mental Health, between July 1992 and June 1999, with each site being completed in the following order based on funding: Bio Bio, Metropolitana, Tarapaca, and Araucania. A total of 2,987 individuals participated in the survey. Response rate did differ by site (χ2 = 11.08, df = 3, P < 0.02) with Metropolitana having the highest non-response rate 12.6% and Tarapaca the lowest 7.5%. A weight was used to account for the probability of the comuna, district, block, household, and respondent being selected. The data was adjusted to the 1992 census of each region based on age, gender, and marital status using a second weight. Diagnostic assessment Composite International Diagnostic Interview (CIDI) versions 1.0 and 1.1 [22] were used to generate the diagnoses using well-trained lay interviewers. DSM-III-R [23] diagnostic criteria were employed. A section on health service utilization in the 6-months prior to the interview was also included. The Spanish translation was conducted using the protocol outlined by the World Health Organization (WHO) [24]. A validation study of the Chilean CIDI was found to have kappas that ranged from 0.52 for somatiform disorders up to 0.94 for affective disorders [25] using a sample of patients and volunteers for each CIDI section. After double entry of data and verification for logical inconsistencies diagnoses were generated using the CIDI computer programs for 1.0 and 1.1 [26]. The DSM-III-R diagnoses included in this report are all affective disorders; all anxiety disorders defined as panic disorder, agoraphobia, and generalized anxiety disorder; substance use disorder which does not include nicotine dependence, and any diagnosis. Lifetime and 12-month prevalence rates were examined. Interviewers and training Social science university students in their senior year underwent training following the WHO protocol at the University of Concepcion, a WHO CIDI training and reference center. The 64 interviewers received over 80 h of instruction and practice sessions. Each interviewer had to conduct practice interviews with volunteer adult subjects with and without psychiatric disorders selected from local clinics, as well as a pilot interview on an individual in a non-selected household in the community. Approximately 80% of the interviews were audiotaped following the subject’s consent, and 20% randomly reviewed for quality control. Analysis procedures The SUDAAN statistical package [27], Taylor series linearization method, was used to estimate the standard errors due to the sample design and the need for weighting. The analysis was conducted using procedures without replacement for non-respondents. The comuna and district selected were used as the defined strata. Chi-square analyses were used to examine the association of disorders and service utilization between regions. Logistic regression was used to adjust for socio-demographic differences across regions accounting for differential rates or service utilization. Additional analyses were conducted to examine urban–rural differences. The logistic regression analyses included gender, marital status, age group, education, and income as potential confounders. All results are presented as weighted data. Results The distribution of income and marital status were found to differ across the four regions of the country (see Table 1). The population of Bio Bio had significantly lower incomes than the other regions (χ2 = 67.96, df = 9, P < 0.0001). In addition, Bio Bio had the lowest rate of individuals who were separated or had annulled marriages (χ2 = 25.31, df = 12, P < 0.05). In two of the regions the rural population was under-represented relative to the census. In Bio Bio 22% of population was rural the sample only included 2.7%, and in Araucania 38% of the population was rural and the sample only included 7.7%. Table 1Socio-demographic characteristics by regionDisordersBio Bio (N = 800)Metropolitana (N = 1363)Tarapaca (N = 306)Araucania (N = 509)χ2dfP%SE%SE%SE%SEGender  Male48.21.046.41.448.91.047.41.53.2330.38  Female51.81.053.61.451.11.052.61.5Age  15–2426.71.624.92.127.32.327.07.47.3150.94  25–3425.41.626.11.427.10.623.24.6  35–4418.21.918.51.121.12.017.52.6  45–5412.11.412.81.211.80.412.31.2  55–649.01.49.20.86.50.49.81.3  65+8.72.38.51.06.21.910.41.7Education  No education2.71.01.10.20.30.21.31.011.1390.31  Basic19.73.817.92.37.82.015.27.6  Medium52.33.347.91.652.71.538.27.3  High25.27.133.22.339.13.245.215.4Marital status  Married54.72.852.82.353.73.252.35.825.31120.05  Widowed5.21.14.70.73.31.35.80.2  Separated/anulled1.70.74.10.53.31.42.00.6  Never Married34.41.932.62.433.91.136.18.0  Common Law4.00.85.81.05.90.63.82.0Income  U$100–U$40070.57.154.53.912.24.847.715.467.9690.0001  U$401–U$80019.33.321.91.829.62.520.64.5  U$801–U$15006.91.911.91.335.63.312.54.0  U$1501+3.32.511.72.822.73.619.27.2Urban/rural  Urban97.33.199.20.998.91.492.35.03.130.39  Rural2.73.10.80.91.11.47.75.0 Prior to adjusting for socio-demographic differences between the regions, a number of differences in prevalence rates were noted (see Tables 2, 3). Lifetime rates for major depressive disorder were markedly elevated in Tarapaca, 17.2%, and lowest in Bio Bio, 11.6% (χ2 = 9.76, df = 3, P < 0.04). Drug abuse, but not dependence, also had the highest prevalence rate in Tarapaca, 2.4% (χ2 = 8.59, df = 3, P < 0.05). Interestingly, in Araucania the rate of non-affective psychosis was the lowest (χ2 = 11.45, df = 3, P < 0.02). When females were examined the differential rates for major depressive disorder (χ2 = 11.76, df = 3, P < 0.02) and substance use disorders were noted (χ2 = 10.88, df = 3, P < 0.03). Among males the only lifetime difference in prevalence rates was for elevated alcohol abuse in Tarapaca (χ2 = 9.17, df = 3, P < 0.04). For 12-month prevalence the increased risk for major depression in Tarapaca persisted (χ2 = 8.78, df = 3, P < 0.05) for both genders combined and for females (χ2 = 10.08, df = 3, P < 0.03). The differences noted in the prevalence of substance use disorders were no longer evident at 12-months. Table 2Lifetime prevalence rates of DSM-III-R disorders by regionBio BioMetropolitanaTarapacaAraucaniaDisorders%SE%SE%SE%SEχ2PAffective disorders    Major depressive episode7.11.311.60.817.22.49.80.89.760.04    Manic episode2.20.71.40.41.80.31.51.31.110.78    Dysthmia7.51.17.31.212.21.76.03.33.650.32    Any affective disorder13.62.215.41.223.21.914.03.23.880.30Anxiety disorders    Panic disorder1.20.61.30.34.30.81.10.51.160.77    Agoraphobia without panic14.22.79.81.39.71.85.30.85.460.16    Generalized anxiety disorder1.80.73.70.52.00.43.00.65.130.19    Any anxiety disorder19.23.714.81.617.91.78.60.96.790.11Substance use disorders    Alcohol abuse2.80.82.00.516.92.67.21.48.200.06    Alcohol dependence7.01.76.40.86.30.55.00.83.630.32    Drug abuse0.60.31.50.42.40.50.10.18.590.05    Drug dependence2.20.73.31.02.20.31.20.17.720.07    Nicotine dependence2.90.62.10.66.50.25.41.07.860.07    Any alcohol or drug use disorder11.01.811.21.024.32.912.31.81.940.59    Any substance use disorder13.02.012.91.129.62.914.61.72.430.50Other disorders    Non-affective psychosis2.10.62.30.50.80.20.10.011.450.02    Somatoform disorder2.71.14.40.83.10.63.50.71.750.63    Cognitive disorder4.51.83.70.80.70.81.00.45.700.15Any CPPS disorder32.24.130.81.644.41.928.93.12.510.49FemaleAffective disorders    Major depressive episode7.11.315.21.420.60.811.21.111.760.02    Manic episode2.61.01.80.53.00.60.50.53.620.32    Dysthmia10.72.011.61.917.21.38.75.12.430.50    Any affective disorder16.42.621.01.728.30.916.04.93.600.32Anxiety disorders    Panic disorder1.70.92.10.56.71.22.10.91.150.77    Agoraphobia without panic20.33.213.62.012.20.27.02.44.880.20    Generalized anxiety disorder2.51.06.41.03.80.84.40.45.980.13    Any anxiety disorder26.43.920.92.524.82.112.32.05.190.18Substance use disorders    Alcohol abuse0.50.41.10.62.60.41.00.42.060.57    Alcohol dependence1.70.72.00.74.20.20.40.27.530.07    Drug abuse0.00.00.80.32.30.50.00.07.380.08    Drug dependence2.40.75.01.62.70.40.20.26.860.10    Nicotine dependence2.50.72.30.810.00.62.60.42.450.49    Any alcohol or drug use disorder3.60.97.11.56.90.61.60.611.800.02    Any substance use disorder5.91.48.91.416.50.34.10.710.880.02Other disorders    Non-affective psychosis2.40.71.90.61.60.30.10.17.810.07    Somatoform disorder2.40.75.31.13.60.74.41.74.100.27Any CPPS disorder35.63.834.72.537.81.123.44.03.020.40MaleAffective disorders    Major depressive episode7.11.57.41.313.64.68.31.56.840.10    Manic episode1.81.01.00.40.50.62.62.21.180.76    Dysthmia4.01.02.40.67.13.33.12.04.100.27    Any affective disorder10.52.29.01.418.04.311.82.914.570.01Anxiety disorders    Panic disorder0.70.50.40.21.80.30.00.04.390.24    Agoraphobia without panic7.72.95.31.97.23.53.51.03.940.28    Generalized anxiety disorder1.00.70.70.40.00.01.31.01.830.61    Any anxiety disorder11.54.47.91.710.63.44.50.57.590.07Substance use disorders    Alcohol abuse5.31.53.00.831.95.614.12.89.170.04    Alcohol dependence12.53.711.61.98.41.210.21.81.110.77    Drug abuse1.10.72.40.82.60.60.20.26.930.09    Drug dependence1.91.11.50.61.60.42.30.51.400.71    Nicotine dependence3.20.71.90.92.90.58.62.26.050.13    Any alcohol or drug use disorder18.94.115.91.942.66.324.33.44.830.20    Any substance use disorder20.54.317.62.143.35.826.22.95.550.16Other disorders    Non-affective psychosis1.81.12.71.10.00.00.00.05.780.14    Somatoform disorder3.01.73.21.32.60.52.60.90.230.97Any CPPS disorder28.55.326.42.351.24.035.05.04.460.23Non-affective psychosis includes schizophrenia, schizophreniform disorder, schizoaffective disorder, delusional disorder, and atypical psychosisAny CPSS disorder does not include nicotine dependence or cognitive disorder; χ2 df = 3Table 312-Month prevalence rates of DSM-III-R disorders by regionBio BioMetropolitanaTarapacaAraucaniaDisorders%SE%SE%SE%SEχ2PTotalAffective disorders    Major depressive episode4.10.97.80.810.31.95.11.18.780.05    Manic episode1.90.71.10.31.80.31.00.92.050.57    Dysthmia3.00.74.11.27.92.23.12.26.100.13    Any affective disorder7.81.610.81.315.01.67.52.95.190.18Anxiety disorders    Panic disorder0.50.30.40.24.10.90.60.61.320.73    Agoraphobia without panic7.01.86.11.57.52.02.20.94.440.24    Generalized anxiety disorder1.20.52.30.61.10.31.70.52.410.50    Any anxiety disorder11.11.79.21.611.91.63.81.35.690.15Substance use disorders    Alcohol abuse2.10.61.80.55.81.83.30.84.050.28    Alcohol dependence5.21.54.80.73.70.42.80.54.260.26    Drug abuse0.20.20.40.21.50.40.00.03.990.28    Drug dependence1.50.81.90.51.80.30.40.42.910.42    Nicotine dependence2.10.51.90.65.60.34.41.04.180.26    Any alcohol or drug use disorder8.41.67.90.910.72.06.21.22.840.43    Any substance use disorder10.01.69.41.015.72.18.21.62.380.51Other disorders    Non-affective psychosis1.10.41.40.30.80.20.00.08.450.06    Somatoform disorder1.80.83.90.63.10.63.00.62.950.42Any CPPS disorder23.93.523.01.525.31.714.73.92.840.43FemaleAffective disorders    Major depressive episode4.80.910.51.314.80.85.32.410.080.03    Manic episode2.61.01.50.53.00.60.20.26.250.12    Dysthmia4.31.46.92.38.81.73.93.26.920.09    Any affective disorder9.61.815.22.220.00.98.25.35.720.15Anxiety disorders    Panic disorder0.50.30.60.36.61.31.01.21.370.71    Agoraphobia without panic11.93.08.42.410.40.33.41.05.780.14    Generalized anxiety disorder1.40.83.91.02.20.62.70.73.240.37    Any anxiety disorder17.32.313.02.517.51.26.31.75.900.14Substance use disorders    Alcohol abuse0.50.41.00.50.90.20.60.50.930.82    Alcohol dependence0.90.51.30.74.20.20.00.05.160.18    Drug abuse0.00.00.20.20.50.10.00.02.480.49    Drug dependence1.50.72.90.82.70.50.20.28.500.05    Nicotine dependence2.00.72.10.88.50.42.00.81.710.64    Any alcohol or drug use disorder2.91.04.50.85.10.40.80.77.980.06    Any substance use disorder4.91.46.00.613.30.92.80.714.740.01Other disorders    Non-affective psychosis1.50.71.20.51.60.30.00.07.050.09    Somatoform disorder1.90.75.01.13.60.74.21.84.500.23Any CPPS disorder23.42.724.72.928.40.912.35.12.830.42MaleAffective disorders    Major depressive episode3.41.14.71.15.53.65.00.41.300.73    Manic episode1.10.90.70.30.50.61.81.60.680.88    Dysthmia1.60.80.80.37.03.32.12.04.780.21    Any affective disorder5.91.75.71.19.82.86.71.34.460.23Anxiety disorders    Panic disorder0.60.40.20.21.60.40.00.03.070.39    Agoraphobia without panic1.70.93.41.74.53.80.90.91.940.59    Generalized anxiety disorder1.00.60.40.20.00.00.50.41.970.58    Any anxiety disorder4.41.84.71.66.13.51.10.93.260.37Substance use disorders    Alcohol abuse3.71.22.70.810.93.76.31.26.200.12    Alcohol dependence9.72.88.81.83.11.05.90.93.880.29    Drug abuse0.40.40.70.52.60.60.00.03.470.34    Drug dependence1.51.20.80.50.90.20.60.80.330.95    Nicotine dependence2.20.71.80.82.50.57.02.03.080.39    Any alcohol or drug use disorder14.43.211.82.016.64.412.11.61.250.74    Any substance use disorder15.43.213.32.118.24.014.12.81.070.78Other disorders    Non-affective psychosis0.80.41.60.70.00.00.00.05.750.14    Somatoform disorder1.61.22.61.02.60.51.80.82.050.57Any CPPS disorder21.85.320.52.522.03.017.33.41.120.77Non-affective psychosis includes schizophrenia, schizophreniform disorder, schizoaffective disorder, delusional disorder, and atypical psychosisAny CPSS disorder does not include nicotine dependence or cognitive disorder; χ2 df = 3Table 4Mental health service utilization in the past 6-months by region among those with DSM-III-R 12-month prevalent disorderBio BioMetropolitanaTarapacaAraucaniaDisorders%SE%SE%SE%SEχ2PAny MH service17.61.121.12.525.90.312.01.910.090.03Non-specialized MH service15.21.017.11.824.30.410.71.612.630.02Specialized MH service5.20.96.71.35.61.13.41.14.260.26Substance service0.10.10.30.20.40.10.10.12.870.43χ2 df = 3, MH = Mental HealthNon-Specialized MH Services = primary care physicians; Specialized Mental Health Services = inpatient or outpatient services provided by a psychiatrist or a psychologist or formal substance abuse services; Substance abuse services included inpatient and outpatient detoxification or Alcoholics Anonymous Using logistic regression controlling for socio-demographic variables the regional differences for major depression were maintained for both lifetime and 12-month prevalence, as well as among females in both prevalence periods. Males with lifetime prevalent affective disorders were also at increased risk in Tarapaca. In addition regional differences in lifetime prevalence for alcohol abuse, drug abuse, and nicotine dependence were found. Among females, regional differences in lifetime prevalence were noted for drug abuse, drug dependence, any alcohol or drug use disorder, and cognitive disorders, and among men alcohol abuse with increased risk among those residing in Tarapaca. The statistical differences in regional lifetime prevalence of non-affective psychosis for both genders combined, and females in both prevalence periods, persisted with the rates for Araucania remaining low. As the rates for not only major depression, but also alcohol and drug use disorders were elevated in Tarapaca, additional analyses were conducted controlling for comorbidity in the logistic regressions, the regional differences noted were not altered. Differences in service utilization across the four regions were also found. Araucania had the lowest use of mental health services utilization (χ2 = 0.03, df = 3, P < 0.03), in particular in the non-specialized health care sector (χ2 = 12.63, df = 3, P < 0.02). The rates of service utilization by region are presented in Table 4. The lower use of services persisted after controlling for socio-demographic variables in a logistic regression. When urban versus rural was examined across all sites no statistical differences in the rates of disorders were noted. In addition, there were no socio-demographic differences. The sample size of the rural population was small, 203. Discussion Regional differences that persisted after adjusting for potential confounders persisted in the CPPS. Major depression and substance use disorders were highly prevalent in Tarapaca. The high rates of substance use disorders, especially drugs, and were not surprising as the region bordering Bolivia and Peru is heavily involved in the drug trade. The increased rates of major depression, however, could not be accounted for by substance use disorder comorbidity. The differences in rates for non-affective psychosis, although may simply be due to a type 1 error, are nonetheless surprising as the Araucania region’s population and our sample has a sizable proportion of Mapuche indigenous people. The Mapuche in earlier psychiatric literature were thought to be at increased risk for psychosis [28]. The small sample size of the rural population precluded finding statistically significant differences. The utilization of health service was lowest in Araucania and Bio Bio. This may be consistent with the inequities in availability and access that do exist in health and mental health resources across different regions of Chile. The southern half of the country is the poorest and has the least resources; therefore, the lower rates may be due to a lack of access rather than demand. For example across the regions the number of available mental health beds 2001 in the public health service per 100,000 were Araucania 2.2; Bio Bio 4.8; Metropolitana 34.2; and Tarapaca 47.2. The number of primary care physicians per 100,000 populations also was lowest in Araucania, 57.0, compared to Bio Bio with 169.7, Tarapaca with 61.6 and Metropolitana with 185.8. It could be argued that these regional differences are simply artifact due to sampling differences. Clearly the population investigated in Tarapaca is small for a prevalence study and may have resulted in rates that may prove unstable. Another potential limitation is that the four regions were investigated sequentially, with the potential for socio-cultural influences to impact on the rates during the intervals between data collection. The high proportion of low-income individuals in the Bio Bio sample in comparison to the other sites and in particular Aracucania, the poorest region of the country, reflects the improved economic conditions in Chile during the course of data collection and supports a cohort effect. A cohort effect, however, is highly unlikely to explain the rates of psychopathology given that data collection was obtained from Araucania last, yet it has the highest rate of major depressive disorder. Conclusion Regional differences across countries may exist that have both implications for prevalence rates and service utilization. Planning mental health services for population centers that span wide geographical areas based on studies conducted in a single region may be misleading, and may result in areas with high need being underserved. Psychiatric epidemiological studies that are nationally representative of developing nations are needed that have a sufficient sampling frame to examine populations believed to be at high risk and regions where increased inequities may exist. Even the most recent epidemiological studies representing Latin America [6, 29] have ignored large segments of the population, such as those countries and regions of countries with large indigenous populations or segments of the population that are very poor. Fewer studies in the region have examined service needs and none have addressed regional differences in services. Data that is more representative of the Latin American population is needed in order to improve mental health services planning and addresses the large under-estimated treatment gap.

Support_Care_Cancer-3-1-2092409

Temperament as a predictor of internalising and externalising problems in adolescent children of parents diagnosed with cancer

Objective This study examined the relationship between temperament and internalising and externalising problems among children of parents diagnosed with cancer, beyond the effects of socio-demographics, illness-related variables and life events. Introduction All people are confronted with intense experiences during the course of their lifetime. Even children are not spared from them. Adolescent children, especially, are vulnerable to stressful events [1]. One stressful incident with which families with adolescent children are increasingly confronted is the diagnosis of cancer in one of the parents. Studies have shown that adolescent children in such situations, the daughters particularly, experience more emotional problems than their age peers do [2, 3]. Although the functioning of children of cancer patients has received increasing attention, research into risk and resilience factors is still in its infancy. Recent studies found evidence for the impact of parental characteristics [4], parent–child communication [5] and family functioning [6] on child functioning. Increased attention is given to the different effect of parental cancer for boys and girls and children of different ages, but research into other child characteristics is lacking. Temperament is one of the factors that may have an important influence on how children cope with stressful events, and therefore why some children are more vulnerable to the development of problems than others [7]. The primary hypothesis of most models of temperament is that specific dimensions are related to prevalence of specific problems. Research into the role of temperament can provide insight into which children are at greater risk in such situations and which children are better equipped to handle the situation. The developmental model of temperament as proposed by Rothbart and Derryberry is one of the few models that offers a measurement tool that is specifically designed for use with adolescents, and it was therefore used in the present study. Rothbart proposes that temperament is relatively stable, but that the expression of temperament can change as a result of maturation and (social) environmental influences, including life events [8, 9]. The model distinguishes between two concepts: reactivity and self-regulation. Reactivity is comprised of the physical and emotional differences that exist among individuals in reaction to stress. Children who have a high degree of reactivity are more easily upset and need more time to recover than other children do. The term “self-regulating system” refers to such processes as attention, activation and inhibition. These processes have an important influence on the regulation of emotions and behaviour. These children may be able to direct attention away from the stressor, such as parental cancer. To our knowledge, no studies have reported on the effect of different temperament dimensions on the functioning of children of parents who have been diagnosed with cancer. The aim of the current study was to investigate the effects of temperament on prevalence of problems in these children, beyond the possible effects of socio-demographics (age, gender, educational level), illness-related variables (recurrent disease and time since diagnosis) and the number of negative life events that children experienced during the year before assessment. Materials and methods Procedure Between January 2001 and February 2003, written information on this study was offered to all cancer patients who were consecutively hospitalised or who visited the outpatient’s clinic at the University Medical Centre Groningen by their physicians or oncology nurses. In addition, information was sent to patients and their family members who had contacted the researchers in response to media attention because they wished to participate in the study. Families were eligible if patients had been diagnosed between 1 to 5 years before study entry and if they had children between 4 to 18 years of age. Participants had to be fluent in Dutch. Patients discussed study participation with their partners (if present) and children. Informed consent was obtained according to the regulations of the Medical Ethical Committee of the University Medical Centre Groningen. After informed consent was received, questionnaires and prepaid return envelopes were provided. Participants were guaranteed that answers were treated absolutely confident and will be described completely anonymous. Participants The current study is part of a larger study in which 476 families with children aged 4 to 18 years were approached, and information was mailed to 110 families who had contacted us for information about participation. Of these, 205 families from the first group and 89 families from the second group consented to participate (response: 43 and 81%, respectively). Ill parents who did not participate did not significantly differ from those who participated with respect to gender, tumour type and time since diagnosis. The current study focused on the responses of children of 11 years of age and older, as they completed the questionnaires themselves. The sample for the present study consisted of 340 adolescent children (149 sons and 191 daughters), between the ages of 11 and 18 years (mean age=14.9 years, SD=2.3) and their 212 ill parents (80% mothers, mean age=45.4 years, SD=4.7). Twenty-one percent of the children were receiving education in primary school, 9% at the lower vocational level, 17% in lower general secondary education, 12% in intermediate vocational education, 38% in high school and 3% in higher vocational education or university. Nine percent of the children were from single-parent families. Parents (43 fathers; 169 mothers, mean age=45.4 years, SD=4.7) had been diagnosed with various types of cancer: breast (55%), haematological (9%), skin (9%), gynaecological (9%), urological (5%), bone tumours (4%), gastrointestinal (5%) or other cancers, such as cancer of the central nervous system or head and neck cancer (6%). The mean time since diagnosis was 2.6 years (SD=1.2). Twenty-nine percent of the parents had suffered relapses. Children and parents approached in the hospital did not significantly differ in age or gender from children and parents who had volunteered for participation. Educational level of parents of the last-named group was significantly higher (t=5.8, p≤0.001) than that of the first-named group, but this was not found for children’s educational level. Furthermore, children and parents in both groups reported similar levels of internalising and externalising problems, and children did not differ significantly in temperament. Measures Temperament Temperament was measured using the adolescent version of the Revised Early Adolescent Temperament Questionnaire (EATQ-R) of Rothbart and Derryberry [10–12]. The EATQ-R consists of 53 items and includes ten subscales that are designed to measure temperamental attention control, activation control, inhibitory control, high intensity pleasure, shyness, fear, frustration, affiliation, perceptual sensitivity and pleasure sensitivity. Answers were rated on a 5-point Likert-type scale (1 = “almost always untrue” to 5 = “almost always true”). Higher values represent a higher availability of the temperamental dimension concerned. The psychometric quality of the EATQ-R was reported to be sufficient among American adolescents with Cronbach’s alpha’s ranging from α=0.55 to α=0.78 [13]. In the present study, alpha scores ranged from α=0.36 to α=0.74, and mean inter-item correlations from r=0.11 to r=0.37. Five of the ten dimensions had mean inter-item correlations of <0.20. Therefore, factor analyses were carried out to study the extent to which the temperamental dimensions identified by Rothbart and colleagues emerged from the data of the present Dutch study (see preliminary analyses). Internalising and externalising problems The Youth Self-Report (YSR) and Child Behaviour Checklist (CBCL) were used to assess, respectively, adolescent children’s self-reported and parent’s reported behavioural and emotional problems in children [14–16]. The YSR/CBCL consists of 102/120 problem items with three response options (0 = not true, 1 = somewhat or sometimes true and 2 = very true or often true). Higher scores indicated more problems. The Internalising (TIS) and Externalising Behaviour Problem Scales (TES) were used in the present study. The internalising scale (32 items) consists of the syndrome subscales withdrawal, somatic complaints and anxiety/depression. The externalising scale (30 items) consists of the syndrome subscales delinquent behaviour and aggressive behaviour. The YSR is one of the most commonly used questionnaires in adolescent research. Cronbach’s alpha scores in the present study were high (α>0.80). The manual provides norm data based on a randomly selected Dutch sample of 560 adolescent boys and 564 adolescent girls. Life events The Questionnaire of Recently Experienced Events was used to ask children and parents about the number of life events experienced during the past year. This questionnaire is based on the Recent Life Change Questionnaire (RLCQ) developed by Rahe [17]. Questions measuring negative events (14 items; e.g., divorce, illness by other family members than the parent with cancer) were used. Analyses Factor analyses of the EATQ-R were executed using Simultaneous Confirmatory Analysis (SCA) and Exploratory Principal Component Analysis (PCA) to investigate factor validity. Chi-square and t tests were performed to compare children and parents who had been recruited in the hospital and those who had volunteered for participation on demographic characteristics and the problems reported. One-sample t tests were performed to compare prevalence of internalising and externalising problems reported by children and parents diagnosed with cancer with those of the norm group. Univariate statistics (t tests and pearson correlation analyses) were performed to investigate effects of study variables (age, gender, educational level, time since diagnosis, recurrence, number of negative life events, and temperament) and problems in children. Hierarchical regression analyses were conducted to examine the contribution of temperament dimensions to prediction of internalising and externalising problems in children. Socio-demographics and illness-related variables (first step), and number of life events (second step) significantly related to temperament were entered into regression analyses to ensure that any effect found for temperament (third step) on children’s problems would not be attributed to these variables. To examine whether multicollinearity exists between the independent variables, Pearson correlation analyses and Variance Inflation Factors (VIF) were performed. If the mean VIF is considerably larger than one and the largest VIF is greater than 10, multi-collinearity exists [18]. There is evidence that sons and daughters may respond differently to the cancer in the parent [19] and that they differ in temperament [13, 20]. Two-way-interaction terms (using standardised scores) were computed to examine whether the pattern of the relationship between temperament and prevalence of problems differ between sons and daughters. Only when the 2-way interaction accounted for a unique significant effect was it included in the model. Owing to the large number of comparisons in relation to the sample size significance was set at p≤0.01. Results Preliminary analyses Simultaneous Confirmatory Analysis (SCA) was conducted to examine differences in the percentage of variance explained by the original structure and by the exploratory structure over the same number of factors. The difference in the variance explained by the original structure (41.0%) and by the exploratory structure (46.1%) was considered too large (>2% rule of thumb, [21]) to continue with the original structure. An exploratory Principal Component Analysis (PCA) was conducted using orthogonal rotation followed by varimax procedure. The number of constituting factors was determined based on the scree test. Items that loaded consistently low (<0.30) or on varying components were excluded. In addition, all items—particularly those with loadings between 0.30 and 0.40—were critically examined for the degree to which they formed a good reflection of the dimensions on which they loaded (face validity). Dimensions were assessed as consistent by an alpha above 0.60 and an inter-item correlation above 0.20. On the basis of these criteria, 17 items were excluded, including the entire inhibition-control (five items) and the affiliativeness scale (five items). Thirty-six items remained, distinguishing seven temperamental dimensions. The first dimension was called effortful control and consists of all five items from activation control and four of the six items from attention control. One of the attention control items was excluded and the other was added to the original four perceptual sensitivity items (“I am good at keeping track of several things that are happening around me”) that represent the second dimension. The third dimension is pleasure sensitivity; just one item did not meet the criteria and was excluded. The fear/worry dimension forms the fourth dimensions. Three of the six items loaded on this scale. One of the other items was excluded, one was added to pleasure intensity (“I get frightened when I ride with a person who likes to speed”), and one was added to the original four items of the shyness scale (“Some of the kids at school make me nervous...”). Shyness was the fifth dimension. The sixth dimensions form the pleasure intensity. Three of the six items loaded low or were not consistent, and were therefore excluded. The seventh dimension forms frustration. One of the seven items did not meet the criteria and was excluded. The seven temperamental dimensions can be defined as follows. Effortful control measures the capacity to start and persist in an action and to focus attention. Pleasure sensitivity is the pleasure related to stimuli involving low intensity, complexity, novelty and incongruity. Perceptual sensitivity pointed to detection or perceptual awareness of slight, low-intensity stimulation in the environment. Pleasure intensity represents the pleasure derived from activities involving high intensity. Shyness is the behavioural inhibition to novelty and challenge, especially social. Frustration measures the negative effect related to interruption of ongoing tasks or goal blocking. Fear/worry represents the worry for occurrence of unpleasant situations. The seven factors that were formed of remaining items explained 47.2% of the variation. Alpha coefficients from the other dimensions ranged from α=0.61 to α=0.79, and inter-item correlations ranged from r=0.21 to r=0.48 (Table 1). Table 1Cronbach’s alpha EATQ-R dimensions of children in the current study and a control studyDimensionsCurrent studyControlCronbach’s alphaMean inter-item correlationsCronbach’s alphaMean inter-item correlationsNumber of itemsEffortful control0.750.260.740.259Pleasure sensitivity0.790.360.830.564Shyness0.690.300.630.255Frustration0.610.210.690.276Perceptual sensitivity0.620.250.700.315Pleasure intensity0.710.370.740.424Fear/worry0.610.340.610.353 Cross validity The same research group conducted a prospective study among children of parents recently diagnosed (1–16 weeks ago) with cancer using a similar procedure in approaching families as was used in the current study. The adapted structure was tested among 144 adolescent children (54% daughters; mean age=14.2 years, SD=2.3 years) that participated in the prospective study (comparison group). Results of PCA among children in the prospective study were similar to those found in the current study. The internal consistency and mean inter-item correlations of the two groups were comparable (Table 1). Results of the Principal Components Factor Analysis are available on request. Internalising and externalising problems in children T tests revealed no significant differences in prevalence of internalising and externalising problems between sons and norm group boys as reported by themselves and their ill parents. Daughters and their ill parents reported significantly more internalising problems than found in norm group girls (Table 2). Table 2Descriptive statistics of the YSR and one sample t tests for comparison of study and norm group SonsNorm group boystDaughtersNorm group girlstMeanSDMeanSDMeanSDMeanSDInternalising problems YSR9.67.38.65.81.613.99.910.87.14.3*Externalising problems YSR11.46.211.56.7−0.210.86.210.06.11.7Internalising problems CBCL6.16.25.75.70.88.47.26.56.03.5*Externalising problems CBCL6.66.17.17.1−0.95.96.05.55.80.9*p<0.001 Children’s problems and socio-demographics, illness-related variables and life events Socio-demographics Daughters experienced significantly more internalising problems than sons as was reported by ill parents and self-reports (t=2.9, p=.004; t=4.8, p≤0.001). Neither children’s age and educational level, nor parent’s age, gender or educational level were significantly related to the prevalence of problems reported in children. Illness-related variables Parents and children reported more internalising problems in case of recurrent disease than in case of primary disease. Time since diagnosis was not significantly associated with reports of either internalising or externalising problems (even after controlling for recurrent disease). Life events The number of negative life events children or parents experienced during the preceding year was significantly positively correlated with internalising (r=0.36, p≤0.001, r=0.25, p≤0.001, respectively) and externalising problems (r=0.25, p≤0.001) reported by or in children. Children’s temperament and socio-demographics, illness-related variables and life events Socio-demographics Sons had significantly higher mean scores on pleasure intensity (t=4.7, p≤0.001) and significantly lower scores on shyness (t=−2.8, p=0.005) than daughters did. Age of children was significantly negatively related to effortful control (r=−0.18, p≤0.001). No relationship was found between child’s educational level and temperament. Illness-related variables No significant relationships were found between length of time since diagnosis and children’s temperament. Children of parents who had recurrent disease differed significantly in fear/worry from children of parents with primary disease (t=3.1, p=0.002). Life events Number of negative life events experienced was significantly positively correlated with pleasure sensitivity (r=0.15, p=0.007), perceptual sensitivity (r=0.15, p=0.005) and fear/worry (r=0.30, p≤0.001) and negatively with effortful control (r=−0.15, p=0.008). Relationships between temperament and problems in children All temperament dimensions were significantly related to children’s self-reported internalising problems. Shyness, pleasure intensity and fear/worry were significantly related to internalising problems as reported by ill parents. Effortful control, frustration and fear/worry were significantly associated with the prevalence of externalising problems in children as reported by children and parents (Table 3). Table 3Descriptive statistics of the temperament dimensions and correlations of these variables with internalising and externalising problemsTemperamentMeanSDChildrenIll parentsInternalisingExternalisingInternalisingExternalisingrrrrEffortful control27.86.2−0.25**−0.44**−0.05−0.24**Pleasure sensitivity11.24.022**0.030.13−0.04Shyness12.03.80.30**0.000.19**−0.13Frustration18.43.80.29**0.36**0.100.14*Perceptual sensitivity16.43.50.15*0.07−0.01−0.09Pleasure intensity14.73.7−0.18**−0.02−0.19**0.10Fear/worry7.42.70.46**0.29**0.23**0.16**p<0.01**p<0.001 Predictors of internalising and externalising problems Internalising problems Child’s gender and recurrent disease explained a significant percentage of the variance in the prevalence of internalising problems in child’s (R2Ch=0.10) and parent’s reports (R2Ch=0.08). Both variables appeared to have a significant negative independent effect. Life events accounted for a significant increment in explained variance in child’s (R2Ch=0.10) and parent’s reports (R2Ch=0.07). The temperament dimensions predicted a significant percentage of additional variance in children’s (R2Ch=0.27) and parent’s reports (R2Ch=0.04). Shyness, frustration, perceptual sensitivity and fear/worry appeared to have a significant positive independent effect in children’s reports. None of the variables appeared to have a significant independent effect in parent’s reports. Interaction terms between temperament dimensions and child’s gender failed to contribute significantly to the prediction of internalising problems in children and parent’s reports. The final models accounted for 48 and 19% of the variance in child’s and parents’ models, respectively (Table 4). VIFs ranged between 1.0–1.3, suggesting that there was no problem of collinearity. Table 4Regression analyses examining temperament as a predictor of internalising problems reported by children an ill parents ChildrenIll parentsInternalising problemsInternalising problemsBetaR2R2ChFChBetaR2R2ChFChStep 10.1017.1**0.0813.5** Child’s gender−0.25**−0.18** Recurrent disease0.20**0.22**Step 20.200.1040.6**0.150.0724.4** Negative life events0.33**0.26**Step 30.480.2722.8**0.190.045.3** Effortful control−0.10– Pleasure sensitivity0.09– Shyness0.31**0.10 Frustration0.15**– Perceptual sensitivity0.12*– Pleasure intensity0.02−0.09 Fear/worry0.25**0.13The dashes indicate that variables were not entered into the model because no significant univariate relationship was found.*p<0.01**p<0.001 Externalising problems Life events accounted for a significant percentage of the explained variance in children’s reports (R2=0.07). The temperament dimensions accounted for a significant increment in children’s (R2Ch=0.22) and parent’s reports (R2Ch=0.07). Effortful control had a significant unique negative effect for both children and parent, and frustration also for children’s reported externalising problems. The interaction terms did not contribute significantly to the problems reported. The variables entered into children’s and parents’ models explained 29 and 7%, respectively, of the variance (Table 5). VIFs in both models were acceptable, ranging between 1.1–1.2. Table 5Regression analyses examining temperament as a predictor of externalising problems as reported by children and ill parents ChildrenIll parentsExternalising problemsExternalising problemsBetaR2R2ChFChBetaR2R2ChFChStep 10722.6** Negative life events0.26**–Step 2290.2233.7**0.077.9** Effortful control−0.33**−0.21** Frustration0.23**0.06 Fear/worry0.100.10The dash indicates that the variable was not entered into the model because no significant univariate relationship was found.*p<0.01**p<0.001 Discussion and conclusion The present study is the first to examine the contribution of temperament to prevalence of problems among adolescent children of parents diagnosed with cancer. The findings suggest that temperament predicted internalising and externalising problems, beyond the effects of socio-demographics (child’ gender), illness-related variables (recurrent disease) and number of negative life-events. The most powerful temperament dimensions for internalising problems were shyness and fear/worry (reactive factor). To a lesser extent, frustration and perceptual sensitivity (children’s reports only) heighten the risk for internalising problems. It was argued that shy and anxious children generally have a tendency to withdraw and are hesitant to seek support from their surroundings [22], whereas seeking support might be important, especially in situations in which a parent had cancer. High levels of frustration are related to reduced ability to regulate attention and emotions, whereby children are less able to relax and direct themselves toward matters other than the stressor [23]. Withdrawn behaviour, anxiety and depression might be consequences of these tendencies. Internalising problems reported by the child also increase when they were highly sensitive to things and people around them (perceptual sensitivity). Although, Rothbart and Bates (1998) hypothesised that children with this kind of sensitivity are more vulnerable for internalising and externalising problems, research on these dimensions is lacking [9]. The relationships between shyness, anxiety and frustration and internalising problems found in children reports have also been found in studies among the general population [9, 24–26]. Those who have a temperament characterised by a low level of effortful control (regulative factor) and a high level of frustration are more at risk to develop externalising problems. The relationship between effortful control and externalising problems has been found in other studies as well [9, 11, 26–29]. Children who have more control show more initiative in undertaking activities, have more ability to shift their attention and to focus and are less easily distracted by circumstances [27]. The control that children have over their behaviour in this regard decreases the chance of externalising problems. As mentioned above, frustration decreases emotional regulation and can lead to externalising problems, in addition to manifesting itself in internalising problems [25]. No effect was found for pleasure intensity, which was in contrast to the results of a recent study among children in the general population. That study found that children who scored high on pleasure intensity experienced more externalising problems [26]. Whether the relationship between temperament and problems is specific for situations in which children are exposed to a parent with cancer, is not clear. It is argued that reactive and regulative temperament factors really are important when the child experienced stressful events [30]. A stressful event, such as illness in the parent brings forth negative emotions, specifically in children high in emotionality. Children with low levels of effortful control may have difficulties to deal with these emotions, and may develop as a consequence emotional or behavioural problems [30]. Although the sons and daughters differed in the problems experienced and in their temperament, the relationship between temperament and problems were similar for both genders, as was found in a previous study [26]. Similar patterns were found for parent’s and self reported problems and temperament in the current study, according to a previous study [26]. Our results showed, however, a stronger relationship between temperament and the problems reported by children than parents’ reports of problems, whilst Oldehinkel and colleagues found the opposite. This inconsistency may be attributed to the informant; Oldehinkel and colleagues used parents’ reports to examine temperament, whereas the current study used self-reports from children. A number of studies found that parents and children perceived the level of problems of children under these circumstances different [19, 31, 48]. The differences in perception may have caused also the differences between parents and children found in the present study. Another interesting result from this study is that, four of the seven temperament dimensions were related to the number of negative life events experienced. This is consistent with Rothbert’s theory, which suggests that, despite its biological base, temperament is influenced by experiences. The influence of stressful environmental factors on the development of temperament is an interesting phenomenon. Because the current study uses a cross-sectional design, no causal statements can be derived from the results. The current study is one of few to use self-reports of temperament from a large number of children to examine the relationship between temperament and internalising and externalising problems. It is generally assumed that self-description is an important source of information in the field of personality research. Nonetheless, it was faced with some difficulties. First, Rothbart and colleagues paid a lot of attention to the development of the theory around temperament. The empirical implementation of the EATQ-R, however, was limited. The original structure of the EATQ-R seemed not applicable for the Dutch children in the current study. A recent study among Dutch adolescents reported also some problems with the self-reported version of the EATQ-R [26] and used, therefore, the parent version. Furthermore, the comparison of results from the current study with those of studies that used other theories of temperament was difficult, due to differences in conceptualizing and in labelling with regard to corresponding dimensions. More research using reliable, validated instruments to investigate the temperament of children is necessary. Second, some of the items used to measure temperament resemble items on the problem scale and can cause item-overlap. Previous studies show that the association between constructs remains essentially the same when correcting for possible overlapping items [26, 32, 33] indicating that they are separate concepts. To examine whether patterns were similar among different informants, parent’s reports were used also. Fourth, the current study is cross-sectional. Longitudinal designs may provide more insight into the causality of relationships. Fifth, most of the relations between temperament and problems found in the current study were similar to those found in other studies. The use of a control group may have provided more detailed information about whether the relationships between temperament and problems differed for children of parents with cancer and those of children in the general population. Finally, the current study did not pay attention to the interaction between children’s temperament and other potential predictors, such as parenting, the parent–child relationship and coping. Thomas and Chess introduced the “goodness-of-fit” concept, which means that problems in children arise only when temperament and the expectations of the surroundings are not well adjusted to each other [34]. The fact that temperament of children can have an impact on the prevention of problems by children is important information. Health care providers can use this knowledge to assist parents to take the individual characteristics of the child into account and by means of this to understand their children’s behaviour better. Additionally, parents might be supported to improve the ‘fit’ between the temperament of the child and the consequences of having a parent with cancer.

Skeletal_Radiol-3-1-2141652

Ultrasound of the small joints of the hands and feet: current status

The aim of this article was to review the current status of ultrasound imaging of patients with rheumatological disorders of the hands and feet. Ultrasound machines with high-resolution surface probes are readily available in most radiology departments and can be used to address important clinical questions posed by the rheumatologist and sports and rehabilitation physician. There is increasing evidence that ultrasound detects synovitis that is silent to clinical examination. Detection and classification of synovitis and the early detection of bone erosions are important in clinical decision making. Ultrasound has many advantages over other imaging techniques with which it is compared, particularly magnetic resonance. The ability to carry out a rapid assessment of many widely spaced joints, coupled with clinical correlation, the ability to move and stress musculoskeletal structures and the use of ultrasound to guide therapy accurately are principal amongst these. The use of colour flow Doppler studies provides a measure of neovascularisation within the synovial lining of joints and tendons, and within tendons themselves, that is not available with other imaging techniques. Disadvantages compared to MRI include small field of view, poor image presentation, and difficulty in demonstrating cartilage and deep joints in their entirety. Contrast-enhanced magnetic resonance provides a better measure of capillary permeability and extracellular fluid than does ultrasound. The ability to image simultaneously multiple small joints in the hands and feet and their enhancement characteristics cannot be matched with ultrasound, though future developments in 3-D ultrasound may narrow this gap. Magnetic resonance provides a more uniform and reproducible image for long-term follow-up studies. Introduction The small joints of the hands and feet play a central role in the diagnosis and classification of arthropathy. Ultrasound can be used to assess involvement in areas that are clinically occult as well as determine the precise structures involved. Whilst a systematic approach should include a full examination of extra-articular structures, including skin, subcutaneous tissue, the tendon and tendon sheath, the enthesis and associated bursae, this article focuses on intra-articular components of disease, specifically the early detection and classification of effusion, synovitis and erosions. In the patient with arthritis, serial examination can assess current activity and disease distribution, as well as monitoring progression or therapeutic response. Technique The hands are best examined with the patient and examiner seated. The examination table is adjusted to a comfortable height for both, taking account of coexistent shoulder and elbow disease. The examiner sits at 90° to the patient, giving easy access to both the patient and the ultrasound controls. Good contact and near field resolution require liberal quantities of contact jelly, preferably warmed to body temperature (Fig. 1). Many gel stand-off pads are rather awkward and can limit access to the lateral recesses of small joints; soft pads work best. Some operators like to immerse the patient’s hand in warm water; if this method is used, it is preferable to leave the water to stand for some while so that all air bubbles are dissipated. Fig. 1Position for examination of the hands. The probe is in position for a sagittal examination of the metacarpo-phalangeal joint (MCPJ) on the extensor (a) and flexor (b) sides. The probe is held between thumb and forefinger, with the ulnar border of the hand used as support (arrow). Note the liberal quantity of gel used to provide some stand-off (arrowhead). c Position for examination of radial aspect of second MCPJ. d Probe position to examine the collateral ligaments and lateral compartments of the interphalangeal joints High-frequency linear-array probes are mandatory, probes with operating frequencies of 10 MHz or more providing the best images. The probe should be held lightly between thumb and forefinger (Fig. 1), and the little finger allowed to rest on the table or patient to reduce contact pressure. Excessive probe pressure can obliterate small quantities of fluid, reduce the sensitivity for detection of blood flow and may obscure synovitis. Sagittal images are the mainstay for diagnosis, with axial [metacarpo-phalangeal joint (MCPJ)] and coronal [proximal interphalangeal joint (PIPJ)] images used in support. The superficial structures, including skin, subcutaneous tissue tendon and tendon sheath, should be assessed prior to the joints themselves. Within the joint, the capsule, extra-synovial connective tissue structures, synovium and visible cartilage are examined in turn. The joint should then be moved gently, as it is only during movement that some of the interfaces between the normal structures become sharply defined. Movement may also facilitate detection of low-volume synovial thickening, which bunches up in the proximal extensor recess on flexion. A comprehensive examination of the small joints of the hands and feet can be time-consuming; however, efficiency can be improved without compromising diagnostic impact, by omitting joints that are commonly negative or misleading. The first ray of both hands and feet often contain effusions, synovial thickening and osteophyte formation in the asymptomatic population. Clinically occult synovial thickening is found more commonly in the PIPJs than in the distal interphalangeal joints (DIPJs) of patients with rheumatoid arthritis (RA) [1]. Unless specifically symptomatic, the DIP joints are not routinely examined in patients with RA, but are included in patients with osteoarthritis or seronegative arthritis or where the diagnosis is unknown, in which cases the tendon insertions should also be included to look for enthesopathy. For a routine rheumatological examination of the hand, the author examines the second to fifth rays. The first ray is included only in specific clinical situations (Fig. 2). The extensor side of the metacarpo-phalangeal joints are examined first, followed by the extensor, ulnar and, in particular, the radial aspects of the IPJs, as indicated (Fig. 1). The dorsal aspects of the wrist and extensor tendons are studied before turning to the palmar side. The flexor aspect of the metacarpophalangeal and proximal interphalangeal joints are examined, with particular attention paid to those that have been normal on the extensor side. Fig. 2Iatrogenic synovtis (arrowheads) of the carpo-metacarpal joint due to silastic implant (arrow) Other authors debate whether both the flexor and extensor sides need to be examined. Scheel and co-authors found that, in most cases, synovitis was detected in the palmar and proximal sites of the MCP and PIP joints, with only 14% of affected joints not showing synovitis in these locations [1] Because of this preponderance, they propose that the examination be streamlined to include only the palmar side. Not all authors agree, and other published work suggests that a significant proportion of synovitis would be overlooked if the examination were to be limited to either one or the other. Ostergaard and Szkudlarek found that only a third of patients had synovitis on both palmar and dorsal aspect of the PIPJ. In the majority, synovitis was limited to one or other compartment, with 43% limited to the palmar [2] and 27% to the extensor sides. The prevalence is reversed in the metacarpo-phalangeal joints; 80% of synovial thickening will be detected on the extensor aspect. The distribution of synovitis within MCPJs has also received attention [1–3]. Tan et al. used contrast-enhanced MRI and divided the volume of synovitis surrounding each MCP joint into eight sections. They detected a predilection for radial-sided synovitis in the second and third MCPJs, with equal distribution in both sides in the fourth and fifth MCPJs. The distribution on the dorsal versus palmar sides was not published. Hau and colleagues also found a predilection for radial-sided synovitis in the PIPJs [4]. Scheel et al. tested various combinations of joints to determine the most efficient method of providing an overall synovitis score. Synovitis and effusion were not differentiated. Of the various joint combinations tried, counting the medial four MCPJs only was least sensitive, counting the flexor side of the MCP and PIPJs of fingers 2-4 was most sensitive. Whilst this may work well for an overall synovitis score, omitting the fifth ray may reduce the sensitivity for detecting erosions, as the fifth MCPJ and fifth MTPJ are often involved early. Normal anatomy Metacarpo-phalangeal joints A sagittal image of the palmar aspect of the metacarpo-phalangeal joint is shown in Fig. 3. Note the presence of a layer of contact gel between the probe and the underlying skin (Fig. 3b). This provides good resolution of skin and subcutaneous tissues, as well as reducing the amount of probe pressure, which can interfere with the assessment of subtle joint effusion and blood flow. Abnormalities in the superficial layers include increased thickness in psoriatic arthritis and calcification. Fig. 3a Long axis (sagittal) view of the flexor aspect of the metacarpo-phalangeal joint. The flexor tendons (asterisks) are lying on the anterior aspect of the joint capsule (arrowheads) held in place by the cribriform (c) pulley (open arrowhead). The volar plate (arrow) and articular cartilage of the metacarpal head (open arrow) are visible. Extensor aspect in full extension (b) and flexion (c). Flexing the MCPJ compresses the connective tissue in the proximal recess (between arrowheads) and may make subtle synovial thickening more conspicuous. MC metacarpal, PP proximal phalanx Deep to this is the flexor tendon in its tendon sheath reinforced by the cruciform pulley. The superficial and deep flexor tendons should be independently identified as they pass over the metacarpo-phalangeal joints into the flexor tendon sheath of the fingers. Differences in tendon excursion on finger movement allows the two tendons to be separated easily. The tendon should pass in close proximity to the proximal phalanges where they are held in position by the annular (A2) pulley. The pulleys can be seen as thin poorly reflective linear structures (Fig. 3). Small fluid collections are often seen in relation to them, which are not usually clinically significant. The joint capsule on the flexor side is reinforced by several connective tissue structures, which can be identified on ultrasound. The collateral ligament runs obliquely from posterolateral to anterolateral and is best appreciated on coronal images (Fig. 1). The accessory collateral ligament has its origin on the head of the proximal phalanx, between the collateral ligament and volar plate, with an insertion on the volar plate itself (Fig. 4). The volar plate is a centrally positioned fibrocartilagenous structure that has a broad-based attachment to the base of the proximal phalanx (Fig. 3). It inserts by two slips onto the neck of the adjacent metacarpal. These are called the check-rein ligaments. Within the joint, the proximal recess on the flexor aspect of the metacarpo-phalangeal joints is identified. Overlying this is the capsule of the MCP joint, which inserts on the adjacent metacarpal neck some distance from the joint surface. Hyporeflective articular cartilage is identified deep to the volar plate on the metacarpal head. Fig. 4Coronal image of the second metacarpo-phalangeal joint, showing the radial collateral ligament (arrow). MC metacarpal, PP proximal phalanx The proximal recess is the area between the volar aspect of the metacarpal neck and the joint capsule, which abuts the deep surface of the overlying tendon. It is filled with intra-capsular but extra-synovial fat, which normally keeps the two layers of synovium closely approximated to one another. This layer of fat is very much more prominent in the proximal recess on the extensor aspect of the joint, which can extend up to 2 cm. from the joint level. This is to allow for finger flexion [5]. The distal recess is much smaller, as the extensor tendon conforms to the shape of the proximal phalanx. Absolute measurements of normal joints have not been universally agreed, largely because different workers have used different joints, different parts of joints and different anatomical structures within the joint to define normal dimensions. The variation in the normal was demonstrated by Scheel and co-authors, who looked at the consensus between ultrasonologists involved in training programmes for the European League against Rheumatism (EULAR) [6]. Many of the differences in interpretation were the consequence of failure to appreciate normal findings. Schmidt et al. proposed a standard measurement, using the distance between the sub-chondral cortex on the head of the metacarpal and the volar plate in the second MCPJ. A mean of 1 mm was determined in a group of 102 asymptomatic volunteers [7], but variation was found to be quite wide. The extensor compartment was not examined, though this area is commonly screened for synovitis [8]. Furthermore, this measurement does not include the proximal recess, where early and prominent synovial thickening may occur. There are also differences between the proximal and distal recesses and some differences between radial and ulna sides, which cannot be encompassed by this single midline measurement. For these reasons, others use the point of maximal joint distension and compare it with the normal measurement for that location. This, however, varies with the area of the joint being examined, but most authors agree that an increase in joint dimension of more than 1 mm above normal is sufficient to suggest abnormality. On the extensor aspect of the MCPJs, the proximal recess can be measured from bone to the deep surface of the extensor tendon, a distance of approximately 2.5 mm. The distal recess is much smaller, measuring less than 1.5 mm. In the transverse plane, the synovial space is limited on its lateral and medial sides, proximally by the extensor hood and distally by the extensor slips. There should be no posterior bulging of the hood or slips; this finding would suggest focal synovial disease. On the flexor side, the proximal recess is more distended and easier to visualise than the distal recess. The proximal recess measures approximately 3 mm from bone to the deep surface of the tendon. This space includes fat, capsule and two synovial layers, so the space may be increased by hypertrophy of any of these. On high-resolution equipment, the joint cavity itself can be identified between surrounding fat and connective tissue. Occasionally, a small quantity of fluid can be identified, separating the two synovial layers. With increasing practice, ultrasonologists quickly gain an appreciation of the range of normal for the small joints of the hands and feet. The bony contours of the metacarpal head and proximal phalanx should be smooth, though it is not uncommon to identify a normal indentation on the dorsal aspect of the metacarpal head. This depression is smooth, well demarcated and has reflective bone at its base. It occurs at the site of the fused growth plate and is not associated with overlying synovial thickening. These features allow differentiation from a metacarpal head erosion. Interphalangeal joints The flexor side anatomy of the proximal interphalangeal joints is similar to that of the metacarpophalangeal joints. The volar capsule is reinforced by similar connective tissue structures. A centrally positioned volar plate has a broad-based attachment to the base of the middle phalanx. It also inserts by two slips onto the neck of the adjacent proximal phalanx. On either side of this lie two collateral ligaments. The accessory collateral ligament has its origin on the head of the proximal phalanx between the collateral ligament and volar plate. The capsule and the flexor tendons lie superficial to these. The medial and lateral extensor tendon slips of the superficial flexor tendon can be followed as they insert just distal to the PIPJ (Fig. 5). The deep flexor tendon can then be followed to its insertion on the base of the distal phalanx. In most cases, it is possible to identify a small quantity of fluid surrounding the tendon, seen as a poorly reflective halo. The thickness of this halo varies from person to person, and comparison with other tendons is helpful, assuming they are uninvolved. Another useful tip is that normal fluid surrounding the tendon is continuous on a longitudinal image, and there is no increase in vascularity, other than at the mesotenon. Fig. 5Long-axis parasagittal view of the proximal interphalangeal joint showing the insertion slips of the superficial flexor tendon (arrow), just distal to the proximal interphalangeal joint (asterisk). PP proximal phalanx, MP middle phalanx Loose adipose tissue, which surrounds the synovial membrane, lies between flexor tendon and bone. Care must be taken not to misdiagnose this fat as synovial thickening, particularly when its reflectivity is artefactually reduced. On high-resolution equipment, the proximal recess on the flexor side can be differentiated from surrounding fat and connective tissue. The sagittal images are augmented by coronal views, which provide the best depiction of the medial and lateral joint recesses and collateral ligaments. The coronal images are obtained by asking the patient to hyperextend the MCP of the finger being examined (Fig. 1d). Particular attention should be paid to the radial aspect of the joint, where synovial hypertrophy and erosions predominate. For the PIPJ, measurements can be obtained from the flexor, extensor, radial and ulnar sides. On the extensor side, the distance between the proximal phalanx, at the junction with the head, and the deep surface of the extensor tendon is 1 mm in the central sagittal plane. On the flexor side the same measurement is 2 mm, with most of the space taken up by intra-capsular but extra-synovial fat. A small quantity of fluid is commonly encountered in this recess (Fig. 6), and slight movement of the joint can make this more conspicuous. Under normal circumstances, the quantity of fluid should not be thicker than the joint capsule and should not extend out of the recess in which it is contained. Fig. 6Long-axis view of the second proximal interphalangeal joint flexor aspect. A small quantity of fluid can be present in the normal joint (arrow). The distal end of the A2 pulley is just visible (open arrow). The fluid is transonic, and the Doppler field shows no flow. PP proximal phalanx, MP middle phalanx, R2P annotation on image for right second PIPJ Measuring the joint space on the radial and ulnar side is difficult. This is due to the variable appearances of the ulnar and radial collateral ligaments due to anisotropy. For this reason it is often best to measure from the bone surface to the outer aspect of the collateral ligament. Under normal circumstances this distance is less than 2.5 mm. The wrist joint is often included in rheumatological examination of the hand, although a full description is outside the scope of this review. The joint is best appreciated from its dorsal aspect, where a small amount of fluid is frequently identified in the radiocarpal joint. Because of the undulating surface of the first carpal row there is normally a variation in the bone-to-capsule distance. To provide some standardisation, measurements can be obtained using the deep surface of the extensor tendons or bony structures as landmarks. The radius, lunate and capitate axis are particularly easy to identify (Fig. 7). Normal ranges have not been established by large population studies; however, the author uses a guide measurement of 7 mm. It is emphasised that for larger joints, such as the wrist joint, measurements should only be used as a loose guideline, as normal variation is wide and synovial proliferation may be focal. Once again, comparison with the contralateral side may be of value in unilateral disease. Fig. 7Sagittal image of the normal wrist joint. Standard sections obtained along the third extensor tendon (asterisks) in the axis of the lunate (arrow) and capitate (arrowheads) The small joints of the forefoot Technique The metatarso-phalangeal joints and proximal interphalangeal joints of the toes are best examined from the extensor aspect. The big toe is usually not included in the general rheumatological screen, as effusion and bone irregularity are frequently encountered in the asymptomatic population. Normal anatomy On the dorsal aspect of the metatarso-phalangeal joint, extensor digitorum longus and brevis lie superficial to the joint. The extensor digitorum brevis tendon divides into four slips, one to each toe, which run alongside the extensor digitorum longus tendon before inserting into it. The longus tendon itself inserts into the dorsal aspects of the middle and distal phalanges, via the dorsal digital expansions. The dorsal digital expansions have three slips, one central and two collateral. The central slip inserts into the base of the middle phalanx. The collateral slips pass on either side of the inserting central slip and receive reinforcement from the interosseus muscles, the extensor digitorum brevis and the lumbricals. Deep to the extensor tendons, the metatarso-phalangeal joints are reinforced by collateral ligaments that run from dorsal to plantar, crossing the radial and ulnar aspects of the joint. These are difficult to identify individually.Flexor digitorum longus and brevis tendons lie on the plantar aspect of the forefoot. The flexor digitorum brevis divides into four tendons, one to each of the lateral four toes, and each of these in turn splits into two at the level of the base of the proximal phalanx. The two divisions reunite before dividing again to attach on either side of the middle phalanx. The first division of this tendon is to allow the deeper flexor digitorum longus to pass distally. The tendon sheaths are fibrous tunnels, reinforced by annular and cribriform condensations mimicking the pulleys of the fingers. Running between the metatarso-phalangeal joints are fibrous condensations termed transverse metatarsal bands. Taken as a unit, these are often referred to as the deep transverse metatarsal ligament. Lying directly on the plantar aspect of the joint capsule are the plantar plates (Fig. 8), which are similar to the volar plates of the upper limb. On their deep surface, these blend with the joint capsule. The plantar plates are firmly attached distally to the base of the proximal phalanges. Proximally, there is a rather loose attachment just proximal to the articular cartilage of the head of the metatarsal. Fig. 8Long-axis (sagittal) view of the plantar aspect of the metatarso-phalangeal joint. The extensor tendon (asterisk) runs over the plantar plate (arrow). MT metatarsal head, PP proximal phalanx Ultrasound pathology Intra-articular pathology Effusion and synovitis There is an increasing trend for early and more aggressive treatment of synovitis with disease-modifying anti-rheumatic drugs (DMARDs), which may demonstrate benefit when prescribed even within weeks to months of the onset of disease [9]. The emphasis in early disease detection has now moved from the detection of erosion to the earlier detection of synovitis, and erosions are now generally regarded as being a stage too late. The earliest detectable abnormality within the small joints of the hands and feet is effusion. Definitions Simple effusion should be completely transonic, compressible, and with no increase in Doppler signal (Fig. 9). Synovial hypertrophy is defined as non-displaceable, intra-articular, poorly compressible tissue, which may exhibit Doppler signal (Fig. 10). Dynamic examination using the probe to compress the joint will cause fluid to be displaced away from the probe, whereas thickened synovium will be much less compressible. The most important pitfall is that normal anatomical structures may have low reflectivity and mimic synovitis if careful attention is not paid to technique, particularly with lower resolution equipment. In particularly, anisotropy of adjacent capsule ligaments and tendons should be avoided. Fig. 9Small effusion on the flexor aspect of the proximal interphalangeal joint. Fluid thickness is measured at 1.6 mm and extends out of the proximal recess (arrowheads). Compare with Fig. 6. PP proximal phalanx, MP middle phalanxFig. 10Moderate synovitis and bloodflow. Compare synovial thickening (arrowheads) with effusion (asterisk). Synovial thickening is hyper-reflective, non-compressible and demonstrates increased vascularity when compared with free fluid Classifying abnormal Synovial hypertrophy has been measured in a number of ways. Szuldarek categorised changes in synovial thickness by comparing it to bony structures. Grade 1 is minimal synovial thickening (considered normal), grade 2, synovial thickening bulging over the line linking the tops of the bones forming the joint without extension along the bone diaphyses, grade 3, with extension to one of the metadiaphyses and grade 4, extension to both metadiaphyses.The author classifies joint disease using semi-quantitative measurements of synovial thickness, vascularity and association with erosions. Synovial thickness is recorded on a 3-point scale (1–3 = mild, moderate and severe), with moderate synovial thickening between 2 mm and 4 mm above normal. If desired, mild grades of synovitis can be further classified into focal and diffuse, with focal involvement limited to one recess. Moderate and severe synovial thickening is less often focal. This sub-classification may be of value in monitoring more subtle changes, compared with the more two-dimensional classification. A 3-point scale is also used to record blood flow: mild is defined as a few scattered vessels only (Fig. 11), moderate as less than 50% vascularity in the synovium (Fig. 12) and severe as more than 50% (Fig. 13). Finally, 0 and 1 are used to denote the presence or absence of erosions, though where necessary for record or research purposes, a more detailed description of the proportion of bone involvement by erosions can be used. The score is recorded on the ultrasound image, together with annotation of the joint being measured. In summary, the second right MCPJ, with 3 mm of synovial thickening, diffusely involving the joint, with marked increased in blood flow and without erosions, would be classified as R3M 230. Images are printed as hard copy or stored on the department’s picture-archiving communication system (PACS) for future comparison and to monitor the patient’s progress. Fig. 11Sagittal image of the extensor aspect of an inflamed metacarpo-phalangeal joint. Mild hypervascularity with a few scattered vessels visible in the moderately thickened synovium. MC metacarpal head, PP proximal phalanxFig. 12Sagittal image of the extensor aspect of an inflamed metacarpophalangeal joint (asterisk). Detectable blood flow on power Doppler is seen in less than 50% of the severely thickened synovium. MC metacarpal head, PP proximal phalanxFig. 13Sagittal image of the extensor aspect of an inflamed metacarpo-phalangeal joint (asterisk). Detectable blood flow on power Doppler is seen in more than 50% of the moderately thickened synovium. Note the apparent defect in the metacarpal head (arrow). This is a normal finding and should not be missed or diagnosed as an erosion. Note the well-demarcated floor and lack of through sound transmissionSeveral studies have shown good inter- and intra-observer reliability of ultrasound classification. The classification by Szkudlarek et al. has been most tested and shown to have good inter-observer agreement. An inter-observer correlation coefficient ( ICC) of 0.61 was determined in a review of 150 small joints of the hand in 30 patients [10]. Naredo and co-workers coordinated a study of 22 ultrasonologists and 28 patients. For the detection of effusion and synovitis in the small joints of the hands and wrists, a mean kappa value of 0.61 was calculated [6]. In a study of 204 hands in normal subjects, Schmidt et al. found a reliability of 0.96 in repeat examinations of the same test subject [7]. Ultrasonography (US) classifications have also been shown to correlate reasonably well with findings on MRI in patients with synovitis, but there have been no studies of the variation that may be found in individuals with normal joints. Angiogenesis High-frequency US can differentiate solid from fluid elements within an enlarged joint space but is less effective at distinguishing inflamed synovium from inactive pannus, fibrous tissue and joint debris. One method of making this differentiation is to use Doppler colour flow, though there has been some variation in the reported findings in individuals with normal joints. Klauser et al. [11] found that healthy joints showed no intra-articular vascularisation, either before or after the administration of ultrasound contrast medium, while Terslev and colleagues [12] found vessels in the synovium of the MCPJs of 11 healthy subjects, in 18% before and 50% after administration of contrast agent. Different ultrasound equipment was used, with small differences in probe and Doppler frequencies. Though there is no method of determining for certain whether these differences account for the variation in findings, it is well recognised that sensitivities of ultrasound equipment from different manufacturers vary considerably, and the identification of the occasional blood vessel should probably not be regarded as abnormal when sensitive equipment is being used. Response to inflammatory changes within the synovium results in the secretion of a large number of inflammatory mediators. Vascular endothelium growth factor (VEGF) is one of the pro-hormones secreted and results in proliferation of the vascular endothelium to form new vessels, a process termed angioneogenesis. Histological studies confirm that power Doppler changes correlate quite well with pathological changes in the synovium [13, 14], although great care should be taken to avoid interface artefact, which can be present at the bone synovium interface if gain settings are too high. Increased Doppler signal correlates especially with polymorphonuclear leucocyte infiltration and surface fibrin deposition [15], though, as yet, no direct correlation between serum VEGF measurement and neovascularisation has been identified. Colour flow assessment should, therefore, accompany all ultrasound examinations of the swollen joint. The pulse repetition frequency should be kept low, and the region of interest should also be kept as small as possible, to maximise the detection of abnormal blood flow within the synovium. False positive readings can occur, due to patient or operator movement or at highly reflective interfaces, such as between bone and soft tissue (Fig. 14). Fig. 14Static image from video-loop. Apparent Doppler signal from the bone surface of the proximal interphalangeal joint (arrow) in the patient with mild synovial thickening (asterisk). When colour flow settings are set to high sensitivity, flash artefacts can be identified where there are strong reflecting interfaces such as the bone surface. PP proximal phalanx, MP middle phalanx There are two ways of measuring synovial blood flow, power Doppler ultrasonography (PDUS) and colour flow Doppler ultrasonography (CFDUS). Power Doppler is more sensitive but also more prone to artefact. It is sensitive to intravenously administered contrast agents which can be used to augment very slow rates of flow. The software that measures power Doppler is proprietary; therefore, results from one manufacturer may not be comparable with another. There is also a concern that serial studies on the same equipment may be incomparable following software or platform upgrades. Unlike CFDUS, PDUS does not carry specific directional information and will therefore register any flow within the region of interest. Although more recent advances in PDUS mean that some directional information can be imparted in the colour read out, quantification is still based on measuring the amount of colour pixels within the image, rather than an absolute measure of blood flow within the synovial tissue itself. These semi-quantitative measures can be scored either visually or by software analysis. Visual scales are most practicable in the clinical setting. As outlined above, a simple grading system is mild (few scattered vessels), moderate (<50%) or severe, >50% of the synovial exhibiting increased flow. Various other semi-quantitative scales have been proposed, including that of Klauser et al., who counted the number of visible vessels. Grade 1 is 1–5 visible signals, grade 2 6–10 and grade 3 more than 11 vessels in the field of view [11]. Computerised methods involve capturing the ultrasound colour image, importing it into a proprietary photo-package and using the software to differentiate and count the number of colour pixels against the greyscale background [16]. The software method requires standardisation of magnification and field of view and is, therefore, troublesome to apply retrospectively. CFDUS differs from PDUS in that it is dependent on the direction of flow of blood (Fig. 15). The signal acquired carries specific information about the vessel from which it has been obtained in the form of a pressure trace. The precise measurement that is most commonly calculated is the resistive index (RI) (Fig. 16). RI is the difference between systolic and diastolic pressure divided by systolic pressure. Most manufacturers include software within the ultrasound system to calculate the resistive index. A good spectral trace from a vessel within the synovial hypertrophy is acquired. The operator selects one or two pulse cycles from within the trace for analysis. RI is a good marker of high flow resistance: under normal circumstances it is 1 and is reduced by angiogenesis induced by synovitis. In contrast to PDUS, resistive index is a physiological parameter and is, therefore, independent of the equipment used and manufacturer’s software. Despite some of the limitations of power Doppler, particularly with regard to serial studies, it is a sensitive measure, which is easy to obtain and broadly reproducible between different operators on the same machine. It appears to correlate reasonably well with contrast-enhanced MRI and clinical findings and allows a rapid assessment, within days, of improvement following treatment. Fig. 15Colour flow Doppler image from a patient with synovitis of the third metatarso-phalangeal joint. Flow away from the ultrasound probe is depicted in blue and flow towards the probe is red. MT metatarsalFig. 16Screen capture during colour Doppler examination of the finger , showing automated calculation of resistive index CFDUS is less sensitive to motion artefact and is a more objective measurement than is PDUS. Problems include difficulties in acquiring a good trace when angiogenesis is sparse and maintaining the fix as measurements are made, particularly if the equipment freezes the image during measurement. The use of beam steering can sometimes help with vessel fixation. CFDUS is insensitive to contrast agents, which diminishes its usefulness in patients with subtle or early synovitis. Changes in RI can also be induced by increasing probe pressure and by changes in temperature. Serial studies should be carried out at controlled temperature. In most cases this can be achieved simply by ensuring the patient has not just come from an abnormally hot or cold environment to room temperature, though some argue that the use of a water bath is the only means of ensuring reliable temperature control. Probe pressure can be controlled in the usual manner by ensuring that there is always a layer of contact gel between probe and skin. Terslev et al. demonstrated that Doppler ultrasound using changes in resistive index may be used as a quantitative measurement of blood flow and to detect changes following intra-articular steroid injection [17]. The same group also showed that estimates of synovial activity by Doppler ultrasound was comparable with post-contrast MRI, though there was no association between MRI, ultrasound and estimates of inflammation and pain visual analogue scale (VAS) scores. Changes in RI following treatment with anti-tumour necrosis factor (anti-TNF) have also been demonstrated [18]. Varsamidis and co-authors measured resistive index in the wrists of patients with rheumatoid arthritis. The RI improved as the patients went into clinical remission [19]. The authors also suggest that ultrasound may be able to predict relapse, as patients who had a flare up within 6 months had significantly lower RI following treatment than did patients who remained in stable remission. Ultrasound contrast agents Colour Doppler signal from small vessels within the synovium can be augmented by the intravenous administration of contrast agents. When mixed and injected, these agents release bubbles of gas that are sufficiently small to enter the microcirculation. The earliest compounds were mixtures of galactose and palmitic acid microparticles. When hydrated, galactose releases micro-bubbles of air, which adhere to the irregular surface of the palmitic acid microparticles. The air bubbles are approximately 8 μm in diameter and, consequently, are able to enter small capillaries. They remain as a stable level in the circulation for up to 5 min after injection and increase the Doppler signal by approximately 20 dB [20]. Second generation US contrast agents use gases other than air, for example sulphur hexafluoride, to provide ultrasonic contrast. Because ultrasound involves assessment of a relatively small field of view, the short duration of optimal activity of micro-bubbles following a bolus injection means that there will be different concentrations in the synovium of a joint viewed at the beginning of an ultrasound examination compared with one examined at the end. This compares poorly with the single-time snapshot that can be achieved with intravenous administration of MRI contrast agent, where uptake in all of the joints and tendons within the larger field of view can be assessed simultaneously. There are other disadvantages of bolus administration of ultrasound contrast agents, which include a blooming artefact that can occur due to the sudden arrival of a large quantity of micro-bubbles into the ultrasound field and uneven destruction of micro-bubbles by the US wave acting on the large bolus itself. Administering the contrast agents by infusion rather than bolus injection can overcome some of these issues and yield more stable enhancement for up to 20 min. Three-dimensional ultrasound, with maximum intensity projection algorithms, may also help in overcoming some of these disadvantages and provide a better single-time snapshot image of synovial enhancement. Contrast-enhanced power Doppler ultrasound (CEPDUS) has been used in a number of clinical studies to demonstrate improved detection, over unenhanced ultrasound, of vascularised synovial tissue. Klauser et al. have done much of this work and have classified neovascularisation in patients with rheumatoid arthritis before and after the administration of contrast agent [11]. In the unenhanced study, 65% of subjects were classified as having normal synovial tissue, grade 0, and 5% as severe, grade 4. Following administration of contrast medium by the infusion technique, only 21% were deemed to have normal synovial tissue, whilst 22% increased to grade 4, with increases in the intermediate groups. Other studies have also demonstrated synovial enhancement and increased detection of synovitis [21] [16]. CEPDUS shows improved correlation over unenhanced power Doppler compared with arthroscopy [22]. Despite this, the therapeutic impact and outcome value of contrast agents requires further study. Bone erosions The identification of bone erosion is a significant step in the natural history of erosive arthritis. The outcome measurement in rheumatoid arthritis and connective tissue (OMERACT) group has defined bone erosion as an intra-articular discontinuity of the bone surface that is visible in two perpendicular planes. Acute erosions generally have an irregular margin and a poorly defined base, which allows through transmission of sound (Figs. 17 and 18) and are associated with active synovitis. Bone defects that do not have synovitis adjacent to them should be regarded with suspicion, though some may turn out to be true chronic erosions [23]. A common pitfall is the normal depression that is present on the dorsal aspect of the head of the metacarpal. This is smooth, with a clearly defined floor that does not allow through sound transmission and is unassociated with overlying synovitis (Fig. 13). Fig. 17Sagittal image of a large erosion in the head of a metacarpal. Note the poorly defined floor of the erosion (open arrow) and through sound transmission (arrowhead). Compare with Fig. 13. Note the adjacent synovial thickening (arrow) and the extensor tendon (asterisk). MT metatarsal head, PP proximal phalanxFig. 18Moderate synovial thickening (arrowheads) with underlying metacarpal head (MC) erosion (arrow) Erosions in RA are most commonly detected on the radial aspects of the heads of the metacarpals and on the bases of the phalanges. The DIPJs are least involved in RA [23], but more erosions are detected in sero-negative RA and osteoarthritis. In the hands, the second, third and fifth rays are most commonly affected, and, in the feet, the fifth, third and second show the greatest predilection. There are differences between individual joints in the ease by which ultrasound can detect erosions. In the hands, the second and fifth provide the greatest circumferential views at the level of the MCPJs (Fig. 1c). The fourth is the most difficult to examine, especially on its radial and ulnar aspects. Scheel et al. found that ultrasound was superior to MRI in detecting erosions in the proximal interphalangeal joints but was less efficient at the metacarpo-phalangeal joints [24]. Conversely, in Backhaus and colleagues’ study of patients with negative plain film findings, erosions dominated in the MCP joints. Ultrasound appears to be particularly useful in detecting erosions in patients in the early stages of disease [25]. Erosions detected by US and MRI progress to radiographic detection in the majority of cases [24, 26] within 1 to 2 years. One of the earliest MRI signs of erosion is sub-chondral bone oedema, and there are, as yet, no studies to show that ultrasound is useful in detecting this. Although the contour of the cortex should be carefully examined for clarity and early defects, ultrasound will not appreciate intraosseous abnormalities that are not associated with an overlying surface defect. Erosions in gout are larger, also irregular, and lie further away from the joint. Erosions must be distinguished from other causes of peri-articular bone irregularities. Entheseal new bone formation at the site of tendon and ligament insertions occur in seronegative arthritis and can give a similar appearance. They are particularly prevalent at the DIPJs, where erosive changes related to osteoarthritis are also found. Small joint US following treatment Changes in synovial thickening and vascularisation following treatment can occur rapidly. A reduction in the number of vessels identified within the synovium has been shown by US within 3 days of intravenous corticosteroid administration [27]. Direct interarticular corticosteroid administration has also been shown to reduce the number of synovial vessels, which persist for several years, suggesting that they are a real response to therapy and not just a temporary alteration in flow characteristics induced by pressure changes within the joint [17, 28, 29]. Ultrasound has been used to track changes in total synovial volume by greyscale ultrasound imaging [30], power Doppler signal [31–33] and resistive index [31] in response to systemic anti-TNF therapy. Results show a consistent reduction in all these imaging parameters in the early stages of active treatment, though long-term results are less constant. Terslev and colleagues, in a study of wrist and small joint arthritis in patients with rheumatoid disease treated with Etanercept, noted that much of the initial reduction in synovial volume and vascularity and increasing resistive index was lost at 1 year. Conversely, Fiocco et al., in a study of 27 patients with knee involvement from either rheumatoid or psoriatic arthritis, noted that synovial thickening persisted at 3 months’ follow-up but was reduced after 1 year. A reduction in vascularity of the synovium had a more rapid onset and also remained persistently low at 1 year [32]. The limited evidence would seem to suggest that changes in synovial vascularity can be seen very quickly, independent of the type of treatment used. Changes in synovial volume are slower to respond, and measurable differences may take some time to appear in larger joints. In most studies, these changes appear to persist, suggesting that ultrasound can be used as a marker of early improvement. Furthermore, ultrasound has detected improvements in joints felt to be clinical ‘non-responders’ [30], suggesting an important role for ultrasound in clinical decision making during the early stage of therapy. Most of these studies, however, are unblinded. Evidence that there is a link between early improvement in synovitis detected by US and a reduction in radiographic findings in later years is beginning to emerge [34]. Clinical application of ultrasound in small joint arthritis Ultrasound versus clinical examination The role of ultrasound in the detection of sub-clinical synovitis has been examined in a number of studies, most of which demonstrate increased detection rates of intra-articular abnormalities. With MRI as the gold standard, the sensitivity, specificity, and accuracy of US for the detection of synovitis in the MTPJs were 0.87, 0.74, and 0.79, respectively, and for clinical examination, 0.43, 0.89, and 0.71, respectively [35]. Wakefield et al. examined the role of ultrasound in detecting sub-clinical synovitis in 1,470 joints in 80 patients with oligoarthritis. Of the clinically unaffected joints, 13% had synovitis detected by ultrasound; the majority, 79%, were metatarso-phalangeal joints, and 16% were metacarpo-phalangeal joints. This meant that one-third of the patients with clinical monoarthritis were upgraded to oligoarthritis (>1<6 joints) and a further quarter to polyarthritis (>6 joints) [36]. Szkudlarek and colleagues examined the metatarso-phalangeal joints in patients with rheumatoid arthritis and healthy controls. Joint effusion and synovitis were detected by ultrasound in 102 joints compared with positive findings in only eight joints on clinical examination [35]. In both studies non-specific findings, especially effusion, common in the first and second MTPJs, may have skewed those results. The finding of occult synovial disease using ultrasound has not been limited to rheumatoid arthritis but has also been extended to patients with systemic lupus erythematosis and other rheumatological conditions [37]. Ultrasound versus other imaging techniques Ultrasound has been compared with plain radiography and has shown significantly superior synovitis detection rates. This is unsurprising, as signs of synovitis on plain radiography, including peri-articular osteoporosis, joint space widening and soft tissue swelling, may be difficult to detect and are frequently overlooked. Backhaus et al. demonstrated a fourfold superiority with ultrasound, compared to the plain radiographic findings of peri-articular osteoporosis and soft tissue swelling [23]. In that study, patients with a variety of rheumatological disorders, who had negative findings on plain radiography, had synovitis demonstrated by ultrasound in all cases, whereas MRI was positive in 84%. In the same group, ultrasound detected erosions in 15 patients compared to 26 detected by MRI. More were found in the proximal interphalangeal joints than by MRI, whereas the reverse was true for metacarpo-phalangeal joints, though other authors have not confirmed this [38].The use of low-field MRI, sequence choice and the use of lower resolution dynamic rather than static post-contrast MR images may have contributed to the apparent superiority of ultrasound in detecting synovitis. Furthermore, both joint effusion and synovial thickening were interpreted as ‘synovitis’, which might have also increased detection rates for ultrasound over MRI [23]. Conversely, the analogue ultrasound equipment used would be regarded as relatively low specification by modern standards. Indeed, in a follow-up to that study, published 7 years after the original, ultrasound detected more erosions than MRI did, in 49% of patients (9% at baseline) compared to 32% detected by MRI (27% at baseline) [24]. In the follow-up study the original low-resolution analogue ultrasound equipment was replaced by a more advanced digital system. Although there was also an increase in the number of erosions detected by conventional radiography, from 5 to 33 joints, it is more likely that technological improvements in the ultrasound equipment accounted for the greater detection of erosions and that many had been missed at the baseline study. It is, therefore, imperative that superior resolution equipment is used for rheumatological examination. Other studies have confirmed the superiority of ultrasound over radiography in the detection of erosions [39], independent of the number of projections used [25].Contrast-enhanced ultrasound and MRI have been compared in a small number of studies. Terslev et al. compared Doppler ultrasound with quantification of both the colour fraction and RI with synovial thickness on post-contrast MRI [8]. Dynamic enhanced MRI detected more inflamed MCP joints than ultrasound did, but the reverse was true for the wrist. Szkudlarek and colleagues compared contrast-enhanced ultrasound using the bolus technique with post-contrast MRI in actively inflamed MCPJs [40]. The MRI enhancement rates at 55 s after injection were used, as the authors found this to correlate most closely with histological features of synovial inflammation [41–45]. They concluded that there was good correlation between PDUS and MRI, but noted that the diagnostic impact of contrast enhancement was limited to a relatively small number of patients.Although these studies have attempted to compare ultrasound and MRI contrast enhancement directly, it should be appreciated that there is an essential difference between enhancing synovium on MRI and measurable angioneogenesis on Doppler ultrasound. Enhanced MRI identifies contrast agent in the intravascular, extracellular and joint spaces, the latter depending on the time allowed for diffusion. CEPDUS recognises only new vessel formation within inflamed synovium. It is well recognized that synovial thickening (presumably detectable on enhanced MRI) can be present on ultrasound examination without associated increased vascularity. While it is, therefore, valid to draw some parallels between the two imaging techniques, direct comparison misses vital information on the physiology of the synovium, and differences between them should not be regarded as false negatives or false positives.Klarlund and co-workers compared MRI, plain radiographs and scintigraphy in 55 patients, followed-up for 1 year. MRI detected progression of erosions more often than radiography did, but ultrasound was not used in that study [46]. Low- and high-field MRI have also been directly compared with plain radiography [47]. The different field strengths had equivalent erosion detection rates, and both were superior to radiography. Advantages and disadvantages of ultrasound The current literature suggests that ultrasound is superior to MRI at detecting small quantities of fluid within the joint. There may be improved detection of erosions at the proximal interphalangeal joints and in the wrist and, although ultrasound struggles with some areas of MCP joints, particularly the radial and ulnar aspects of the fourth finger, overall erosion detection by US at the MCPJs is comparable to that by MRI [38]. US demonstrates angioneogenesis in active synovitis directly (Fig. 19), whereas MRI requires injection of contrast medium. US is a dynamic technique; the examination can be tailored to include further involved joints following an up-to-date history with the patient present There are fewer problems with asymptomatic abnormalities, as these can be clinically correlated during the examination. Unlike MR, US is readily suited to guiding intervention, such as small joint injection or synovial biopsy (Figs. 20, 21). There is also increasing evidence that patients prefer ultrasound to MRI [48]. This may be related to claustrophobia but equally to the human interaction that occurs during an ultrasound examination. Fig. 19Long axis-view on the dorsal aspect of the wrist deep to the extensor tendon (asterisks). Intense Doppler signal intensity is detected in the distal portion of the joint. DCR distal carpal row, PCR proximal carpal rowFig. 20Needle introduced into the second metatarso-phalangeal joint (arrow) prior to aspiration and corticosteroid injection. MT metatarsal head, PP proximal phalanxFig. 21A synovial mass (arrowheads) undergoing biopsy. Ultrasound allows accurate placement of a small biopsy needle within the synovial tissue Conversely, ultrasound has difficulty with large deep joints, superficial joints when there are significant deformities, and in the assessment of articular cartilage. US is a relatively time-consuming procedure, especially if multiple joints are examined. The images acquired can be re-read and reclassified by other readers at a later date, but there is no ability to identify abnormalities overlooked and not imaged at the time of the original examination. MR images can be obtained remotely, reviewed, re-read and scored at a later date and preserved for serial comparison. Increasing use of ultrasound video-loops and 3-D ultrasound may assist in this regard in the future. MRI demonstrates single-time shot synovial enhancement in the entire field of view, which can include all the important small joints of the hands and wrists as well as adjacent tendons and ligaments. Although the time taken to review MRI images of the hand and wrist for diagnostic purposes is probably shorter than the time taken to complete an ultrasound examination, a full MRI score such as the rheumatoid arthritis MRI scoring system (RAMRIS), which is a more close approximation to ultrasound, takes considerably longer. Despite this, there is a strong argument that, for research studies of different therapeutic regimens in patients with rheumatoid arthritis, plain films and MRI provide more robust serial assessment and will remain the gold standard for some time. Conclusions In routine rheumatological practice, ultrasound of the small joints of the hands and feet accurately detects occult synovial disease and allows classification of the extent and activity of synovitis as well as superior detection of erosions in comparison with radiography. US can guide intra-articular therapy as well as assess its response and the response to systemic treatment. Future goals for ultrasound trials need to include further validation studies, studies of diagnostic and therapeutic impact and longer term outcomes from clinical and therapeutic decisions based on the ultrasound examinations.

Diabetologia-4-1-2170457

Effect of beta-adrenergic stimulation on whole-body and abdominal subcutaneous adipose tissue lipolysis in lean and obese men

Aims/hypothesis Obesity is characterised by increased triacylglycerol storage in adipose tissue. There is in vitro evidence for a blunted beta-adrenergically mediated lipolytic response in abdominal subcutaneous adipose tissue (SAT) of obese individuals and evidence for this at the whole-body level in vivo. We hypothesised that the beta-adrenergically mediated effect on lipolysis in abdominal SAT is also impaired in vivo in obese humans. Introduction Obesity is characterised by excess fat storage in adipose tissue, in the form of triacylglycerol (TAG). A blunted fat mobilisation, due to decreased adipose tissue lipolysis, might be an important factor contributing to the development or maintenance of the expanded adipose tissue mass in obesity. Fasting lipolysis per unit of lean body mass has been reported to be increased in obesity, whereas a decrease is reported when expressed per unit of fat mass (FM) [1]. The hormonal regulation of lipolysis is still under debate. There is strong evidence from in vitro and in vivo studies for the existence of lipolytic resistance to catecholamines in obese individuals. Blunted whole-body catecholamine-induced lipolysis has been shown in vivo in obese persons [2, 3]. This impaired lipolysis did not improve after weight reduction [4]. Furthermore, a decreased lipolytic response to catecholamines is a feature of childhood-onset obesity [5, 6] and is also present in adipocytes from first-degree relatives of obese persons [7]. These observations suggest that catecholamine resistance of lipolysis may be an important early, and perhaps primary factor, in the development of obesity. In vitro catecholamine resistance of lipolysis was reported in subcutaneous adipocytes of men and women with upper-body obesity, in relation with a decreased cell surface density of β2-adrenoceptors [8]. We hypothesised that beta-adrenergically mediated lipolytic response at the whole-body and abdominal subcutaneous adipose tissue (SAT) level is impaired in vivo in obese persons. If so, this might contribute to the increased fat storage in this adipose tissue depot. To obtain an accurate estimation of lipolysis, state-of-the-art [2H5]glycerol tracer methodology in combination with the measurement of arterio–venous concentration differences across abdominal SAT was used. Obese and lean male control participants were investigated after an overnight fast and during catecholamine stimulation using the non-selective beta-adrenergic agonist isoprenaline. The primary outcome measures of the present study were glycerol rate of appearance (Ra) and glycerol exchange across abdominal SAT. A methodological issue that arises when determining local adipose tissue glycerol release is the possibility that adipose tissue may also take up small amounts of glycerol [9]. A pilot study was performed to determine the time period required to obtain a steady-state in glycerol enrichment in both arterialised and venous blood draining from adipose tissue, since a lack of isotopic equilibration may explain previous discrepant findings on glycerol uptake [10]. Methods Participants Three lean (two women and one man; BMI < 25 kg/m2) participants took part in a pilot experiment, during which [2H5]glycerol enrichment was measured for 6 h to determine the time required for obtaining an isotopic steady-state. Thirteen lean (BMI < 25 kg/m2) and ten obese (BMI ≥ 30 kg/m2) non-smoking normotensive men participated in the actual SAT lipolysis study, during which [2H5]glycerol was infused for a 3 h period. Clinical characteristics of the participants are summarised in Table 1. Body weight and body density were determined after an overnight fast, as previously described [11]. All participants were in good health as assessed by their medical history, free of any medication and spent no more than 3 h/week in organised sports activities. The Medical Ethical Committee of Maastricht University approved the study protocol and all participants gave their written informed consent before participating in the study. Table 1Clinical characteristics of the participants Lean (n = 13)Obese (n = 10)Age (years)43 ± 354 ± 3BMI (kg/m2)23.0 ± 0.531.9 ± 0.6aFM (kg)15.2 ± 0.832.4 ± 1.2aWHR0.91 ± 0.011.01 ± 0.01aSystolic blood pressure (mmHg)126 ± 3137 ± 4Diastolic blood pressure (mmHg)77 ± 285 ± 3HOMA-IR1.8 ± 0.23.4 ± 0.3aValues are mean ± SEMap < 0.05 obese vs leanHOMA-IR, homeostatic model assessment of insulin resistance Experimental protocol Pilot study The time course in [2H5]glycerol enrichment was determined in order to identify when steady-state levels were achieved (n = 3). Glycerol enrichment was measured in arterialised blood and blood draining the abdominal SAT (adipose vein) during primed (3 μmol/kg) constant infusion for 6 h of [2H5]glycerol (0.20 μmol kg−1 min−1). Blood samples were taken simultaneously from the two sites, both at baseline before the start of the tracer infusion (t0 min), and at ten time points during glycerol infusion (t60, t90, t120, t150, t180, t210, t240, t330, t345, t360 min). Whole-body and SAT lipolysis study Glycerol enrichment and exchange across abdominal SAT were investigated during primed (3 μmol/kg) constant infusion of [2H5]glycerol for 3 h (0.20 μmol kg−1 min−1). Following a 120 min baseline period, isoprenaline was infused at a rate of 20 ng [kg fat free mass (FFM)]−1 min−1 for 60 min. During the experiment, heart rate was recorded continuously by means of a three-lead ECG. When heart rate increased by more than 40 beats per min or in the event of ECG irregularities, isoprenaline infusion was stopped (n = 2, one lean and one obese participant). Before the start of the tracer infusion an arterialised blood sample was taken for background enrichment. Blood samples were also taken simultaneously from the arterialised and adipose vein at three baseline time points (t90, t105 and t120 min) and at three time points during the last 30 min of isoprenaline infusion (t150, t165 and t180 min). Adipose tissue blood flow (ATBF) was monitored continuously using the 133Xe washout technique [12]. ATBF results have been published previously in another context [13]. Clinical methods All participants were asked to refrain from drinking alcohol and to perform no strenuous exercise for 24 h before the study. Participants came to the laboratory by car or bus at 08:00 hours after an overnight fast. Three cannulas were inserted before the start of the experiment. Arterialised venous blood was obtained through a 20-gauge cannula inserted retrogradely into a superficial dorsal hand vein. The hand was warmed in a hotbox, which was maintained at 60°C to achieve adequate arterialisation [14]. In the same arm, a second cannula was inserted in a forearm antecubital vein for the infusion of [2H5]glycerol tracer and the non-selective beta-adrenergic agonist isoprenaline at a rate of 20 ng (kg FFM)−1 min−1. At this infusion rate, plasma isoprenaline concentrations are comparable in lean and obese participants [3]. Finally, the veins on the anterior abdominal wall were identified by means of a fibre-optic light source. In order to obtain adipose tissue venous blood, a 10 cm 22-gauge catheter (Central venous catheter kit Seldinger technique; Becton Dickinson, Alphen aan den Rijn, the Netherlands) was introduced anterogradely over a guide wire into one of the superficial veins and threaded towards the groin, so that its tip lay just superior to the inguinal ligament [15]. This method provides the drainage from the adipose tissue of the abdomen, uncontaminated by muscle drainage and with only a minor contribution from skin [15]. The adipose vein was kept patent by continuous saline (9 g/l NaCl) infusion at a rate of 80 ml/h. The participants rested in a supine position for the duration of the study. Analytical methods A small portion of blood was used to measure oxygen saturation (%HbO2) and ensure adequate arterialisation (ABL510; Radiometer, Copenhagen, Denmark). Blood was collected in tubes containing EDTA and immediately centrifuged for 10 min at 1,000g, 4°C. The plasma was removed for enzymatic colorimetric quantification of NEFA (NEFA C kit; Wako, Neuss, Germany), glycerol (Boehringer, Mannheim, Germany) and TAG (Sigma, St Louis, MO, USA) on a centrifugal spectrophotometer (Cobas Fara; Roche Diagnostica, Basel, Switzerland). Plasma glucose concentration (ABX Diagnostics, Montpellier, France) was measured on an automated spectrophotometer (Cobas Mira; Roche Diagnostica). Plasma insulin was measured with a double antibody radioimmunoassay (Linco Research, St Charles, MO, USA). Insulin sensitivity was assessed by the homeostasis model assessment index for insulin resistance, calculated from fasting glucose and insulin [16]. Packed cell volume was measured using a microcapillary system (Hirschmann Laborgeräte, Eberstadt, Germany). Isotope enrichment To determine isotopic enrichment of glycerol, samples first were derivatised. Acetone (1 ml) was added to 150 μl plasma and each tube was vortexed for 2 min and centrifuged for 20 min at 17,500g, 4°C. The supernatant fraction was transferred to a clean tube and dried under nitrogen at 37°C and derivatised by adding 80 μl ethyl acetate (cat. no. 45765; Sigma-Aldrich, Seelze, Germany) and 80 μl heptafluorobutyric acid anhydride (cat. no. 63164; Pierce Biotechnology, Rockford, IL, USA). The tubes were vortexed for 2 min and incubated for 1 h at 70°C. Samples were than rotated end over end for 5 min at 25°C and evaporated under nitrogen at room temperature. Ethyl acetate (70 μl) was added before injection into the GC-MS (Finnigan MAT 252, Bremen, Germany) for measurement of glycerol enrichment. Stable isotope enrichment was analysed by selectively monitoring the mass to charge ratio (m/z) of molecular ions 253 and 257 for glycerol [17]. Calculations The net exchange (flux) of metabolites across abdominal SAT was calculated by multiplying the arterio–venous concentration difference of metabolites by adipose tissue plasma flow. Plasma flow was calculated as ATBF×(1–packed cell volume), with packed cell volume expressed as a fraction. A positive net flux indicates net uptake from plasma, whereas a negative net flux indicates net tissue release. The expected adipose vein enrichment, in case of no glycerol uptake, was calculated as arterialised enrichment multiplied by arterialised glycerol concentration divided by the measured adipose vein enrichment. The Ra of glycerol was calculated according to the following steady-state equation: where TTR is tracer/tracee ratio and F is the isotope infusion rate (μmol kg−1 min−1). The fractional extraction (fract) of glycerol across abdominal SAT was calculated by dividing the arterio–venous concentration difference of [2H5]glycerol by the arterialised [2H5]glycerol concentration. Abdominal SAT total glycerol uptake was calculated as follows: where the units are nmol (100 g tissue)−1 min−1; (glycerolart) is arterialised glycerol concentration (μmol/l); and ATBF is in ml (100 g tissue)−1 min−1. Abdominal SAT total glycerol release was calculated from the formula: Statistical analysis Baseline fasting values and changes (Δbeta-adrenergic stimulation to baseline) were compared between groups (obese vs lean) using Student’s unpaired t test. Statistical calculations were performed with SPSS for Macintosh (version 11.0; SPSS, Chicago, IL, USA). Data are presented as mean ± SEM. A value of p < 0.05 was considered statistically significant. Results Characteristics Obese participants had significantly higher BMI, FM, waist to hip ratio, fasting circulating TAG and insulin concentrations than their lean counterparts (Tables 1 and 2). Table 2Circulating (arterialised) metabolite levels during baseline (fasting) and isoprenaline infusion in lean and obese participants LeanObeseBaseline (n = 13)Isoprenaline (n = 10)Baseline (n = 10)Isoprenaline (n = 7)TAG (μmol/l)701 ± 66648 ± 641,464 ± 190b1,667 ± 217aNEFA (μmol/l)661 ± 41942 ± 53638 ± 421,124 ± 82aGlycerol (μmol/l)102 ± 5118 ± 7106 ± 4147 ± 10aGlycerol Ra (μmol/min)199 ± 12311 ± 28220 ± 15391 ± 30Glycerol Ra/FM [μmol (kg FM)−1 min−1]13.1 ± 0.920.9 ± 1.67.3 ± 0.6b12.9 ± 1.1aGlucose (mmol/l)5.3 ± 0.15.4 ± 0.15.5 ± 0.25.4 ± 0.1Insulin (pmol/l)50 ± 474 ± 694 ± 7b167 ± 16aValues are mean ± SEMbp < 0.05 baseline obese vs lean; ap < 0.05 change (Δ) from baseline obese vs lean using unpaired Student’s t test Tracer/tracee ratio In the pilot experiment, the tracer/tracee ratios (TTR) obtained during a 6 h [2H5]glycerol infusion after an overnight fast were examined (n = 3). Arterialised and adipose vein TTR reached a steady-state after 1 h of infusion (Fig. 1). Mean values are presented, as all participants (n = 3) showed the same pattern. The measured adipose vein enrichment was consistently lower than the expected enrichment, implying uptake of glycerol by adipose tissue. In the actual SAT lipolysis experiment (3 h [2H5]glycerol infusion), TTR also reached a steady-state after 1 h and remained stable during isoprenaline infusion. Data in lean and obese men were comparable (data not shown). Fig. 1Plasma glycerol TTR during 6 h primed constant infusion of [2H5]glycerol (n = 3) in arterialised blood (squares) and blood draining from abdominal SAT (adipose vein; black circles). White circles, expected adipose vein enrichment. The measured adipose vein enrichment was consistently lower than the expected enrichment Circulating metabolites Beta-adrenergic stimulation with isoprenaline increased arterialised TAG concentrations in obese participants, while in lean participants TAG concentrations decreased during isoprenaline infusion (Table 2). Thus, the change in arterialised TAG concentrations from baseline to isoprenaline was different between obese and lean participants (ΔTAG obese vs lean, 100 ± 37 vs −45 ± 22 μmol/l, p < 0.05; Table 2). Furthermore, beta-adrenergic stimulation increased arterialised NEFA and glycerol concentrations in lean and obese participants. The beta-adrenergic mediated increase in arterialised NEFA (ΔNEFA 454 ± 68 vs 271 ± 46 μmol/l, p < 0.05) and glycerol concentration (Δglycerol 40 ± 8 vs 15 ± 5 μmol/l, p < 0.05) was more pronounced in obese than in lean participants (Table 2), suggesting a higher whole-body lipolytic response in the former. Indeed, beta-adrenergic stimulation increased whole-body glycerol Ra in lean and obese participants (Table 2), this increase tending to be higher in the obese group (Δglycerol Ra obese vs lean: 172 ± 19 vs 109 ± 13 μmol/min, p = 0.07; Table 2). Expressed per unit of FM, fasting glycerol Ra was significantly reduced in obese compared with lean participants (p < 0.05; Table 2). Interestingly, the beta-adrenergically mediated increase in glycerol Ra per unit of FM was significantly blunted in obese participants [Δglycerol Ra per unit FM: 5.4 ± 0.9 vs 7.7 ± 1.5 μmol (kg FM)−1 min−1, p < 0.05] (Table 2), suggesting a blunted lipolytic response per unit of FM in obese participants. Finally, beta-adrenergic stimulation increased arterialised insulin concentrations in lean and obese participants (Table 2). This increase in circulating insulin levels was significantly higher in obese than in lean participants (Δinsulin 62 ± 13 vs 25 ± 4 pmol/l, p < 0.05; Table 2). Abdominal SAT lipolysis Glycerol uptake by abdominal SAT was observed in lean and obese participants after an overnight fast (Fig. 2a). Fractional extraction of [2H5]glycerol from the circulation (lean vs obese, 16.6 ± 4.5 vs 13.9 ± 6.7%) and total glycerol uptake expressed relative to total glycerol release were very small (lean vs obese, 9.7 ± 3.4 vs 9.3 ± 2.5% of total release) with no significant difference between lean and obese participants (p = 0.74 and p = 0.92, respectively). Adipose tissue total glycerol uptake increased during beta-adrenergic stimulation in lean and obese participants, but this increase was not significantly different between groups [Δ total glycerol uptake obese vs lean, 4 ± 9 vs 21 ± 5 nmol (100 g tissue)−1 min−1, p = 0.15] (Fig. 2a). The increased total glycerol uptake during beta-adrenergic stimulation appeared to be partly explained by the increase in ATBF (r = 0.633, p < 0.05). Fig. 2Total glycerol uptake (a) and release (b) across abdominal SAT after an overnight fast (black bars) and during beta-adrenergic stimulation (white bars) in obese vs lean participants during 3 h [2H5]glycerol infusion. Values are mean ± SEM. *p < 0.05 for change (Δ) from baseline obese vs lean Fasting net glycerol and NEFA release across abdominal SAT were comparable between lean and obese participants (Table 3). Beta-adrenergic stimulation increased net NEFA and glycerol release across abdominal SAT to a greater extent in lean than in obese participants, although changes were not significantly different between groups (Table 3). In line with these findings, the beta-adrenergically mediated increase in total glycerol release across abdominal SAT was blunted in the obese group [Δ total glycerol release obese vs lean, 140 ± 71 vs 394 ± 112 nmol (100 g tissue)−1 min−1, p < 0.05] (Fig. 2b), suggesting a blunted lipolytic response per unit of abdominal SAT in obese participants. Finally, obese men tended to show an increased net TAG flux across abdominal SAT during beta-adrenergic stimulation [Δ net TAG flux obese vs lean, 75 ± 32 vs 16 ± 11 nmol (100 g tissue)−1 min−1, p = 0.06] (Table 3). Table 3Blood flow and net SAT fluxes during baseline (fasting) and isoprenaline infusion in lean and obese participants LeanObeseBaseline (n = 13)Isoprenaline (n = 10)Baseline (n = 10)Isoprenaline (n = 7)ATBF [ml (100 g tissue)−1 min−1]2.2 ± 0.26.3 ± 1.21.4 ± 0.2a3.6 ± 0.6Net SAT fluxes [nmol (100 g tissue)−1 min−1]TAG25 ± 843 ± 1734 ± 29113 ± 62NEFA−780 ± 160−2,101 ± 371−486 ± 101−1,824 ± 667Glycerol−229 ± 49−640 ± 148−211 ± 51−486 ± 128Glucose53 ± 49−69 ± 151−143 ± 96−677 ± 632Values are mean ± SEMA positive net flux indicates net uptake from plasma, whereas a negative net flux indicates net tissue releaseap < 0.05 baseline obese vs lean using unpaired Student’s t test Discussion The present study was designed to investigate in vivo whole-body and abdominal SAT lipolysis in obese and lean men. To our knowledge, this is the first study to show in vivo that obese participants have a blunted beta-adrenergically mediated lipolytic response per unit of adipose tissue. Methodological considerations A point of discussion with studies on glycerol uptake and release using tracer methodology is the infusion time of the labelled glycerol. In previous studies, the infusion time was relatively short (1–3 h), raising the question of whether equilibration between labelled glycerol and the adipose tissue glycerol pool is complete or not [9, 10]. We investigated glycerol enrichment during a 3 h and 6 h period of [2H5]glycerol infusion. Steady-state levels in labelled [2H5]glycerol were achieved in arterialised and adipose vein enrichment after 1 h and remained constant for the subsequent 5 h. Thus, our data support the use of a relatively short infusion time (1 h) for study of glycerol metabolism. Glycerol uptake The present data show a slight glycerol uptake by abdominal SAT of lean and obese participants. Glycerol uptake was not significantly different between lean and obese participants. Uptake and dilution of [2H5]glycerol across abdominal SAT has been shown previously during 1 h [9] of tracer infusion. In this study, the dilution of the labelled glycerol was consistently greater than expected from the measured net release of glycerol, indicating significant uptake of glycerol by adipose tissue. In line with this study, we observed a two- to fivefold higher glycerol enrichment in arterialised than in venous blood draining adipose tissue. The observed enrichment in venous blood was universally lower than that predicted from the net addition of glycerol to venous blood. This indicates that an exchange must occur between enriched glycerol in the blood and the unenriched non-esterified glycerol pool in adipose tissue. In contrast, some studies were unable to detect significant uptake of glycerol by adipose tissue after 1 h of tracer infusion [10]. The reason for this apparent discrepancy remains to be elucidated. It should be mentioned that glycerol uptake is low in human adipose tissue as is the activity of the enzyme glycerol kinase [18]. This enzyme is responsible for the phosphorylation of glycerol into glycerol 3-phosphate, making it available for re-esterification. Abdominal SAT lipolytic response to beta-adrenergic stimulation A blunted isoprenaline-induced increase in total glycerol release per unit abdominal SAT was observed in obese men, indicating that in vivo beta-adrenergic mediated lipolytic response in abdominal SAT of obese participants is blunted. Our data are consistent with evidence of catecholamine resistance in vitro and in situ in obese individuals [2–4], in children with obesity [5, 6] and also in relatives of obese individuals [7]. Defects in catecholamine signal transduction have been observed at the β2-adrenoceptor level and further downstream or directly involving hormone-sensitive lipase [8, 19–22]. However, from our experiments it is not possible to determine at which level the observed defect is located. Interestingly, catecholamine resistance has been observed in adipose tissue of first-degree relatives of obese participants [7] and persists after weight reduction [4], suggesting that catecholamine resistance may be a primary defect in obesity. Furthermore, plasma insulin concentrations may play an important role in regulating lipolysis [23]. Therefore, we cannot fully rule out the possibility that the blunted lipolytic response per unit adipose tissue mass that we observed is a secondary phenomenon, due to the higher degree of hyperinsulinaemia during beta-adrenergic stimulation in obese compared with lean participants. However, this explanation seems unlikely, since a blunted in situ lipolytic response in abdominal SAT of obese women was still observed when the confounding influence of hyperinsulinaemia had been excluded using a pancreatic hormonal clamp [2]. In contrast to the present study, two in situ microdialysis studies performed in men found that the increase in interstitial glycerol during isoprenaline administration did not differ between lean and obese individuals [24, 25]. A possible explanation for this is that in microdialysis studies interstitial glycerol is used as a measure of lipolysis. Since glycerol is taken up by adipose tissue, interstitial glycerol concentration may not reflect the overall rate of lipolysis. Rather, it may be the net result of TAG and glycerol metabolism, thus reflecting net glycerol turnover [26]. Whole-body beta-adrenergically mediated lipolytic response Whole-body lipolytic response during isoprenaline infusion tended to be higher in obese participants. This was reflected by a higher increase in circulating NEFA and glycerol concentrations during beta-adrenergic stimulation in obese than in lean participants. Expressed per unit of FM, beta-adrenergically mediated lipolysis (glycerol Ra) was significantly lower in obese than in lean men. This suggests that the increased whole-body beta-adrenergically mediated lipolytic response in obese individuals is directly linked to the increased adipose tissue mass, as has been shown before in upper body obese women [2]. Increased release of NEFA into the circulation increases NEFA delivery to the liver, resulting in increased hepatic VLDL-TAG output and hence increased circulating TAG levels during beta-adrenergic stimulation, as was observed in our obese participants. The control of whole-body lipid metabolism is, to a large extent, dependent on the efficient regulation of lipid metabolism in adipose tissue and the liver. Moreover, hepatic VLDL-TAG is a precursor of TAG stored in adipose tissue [27, 28]. Consequently, a greater VLDL-TAG delivery to adipose tissue and greater lipoprotein lipase-mediated hydrolysis might explain the tendency towards increased positive TAG flux across abdominal SAT of obese participants during beta-adrenergic stimulation. Our observation is in agreement with a study by Samra et al. [29] showing an increased rate of action of lipoprotein lipase during epinephrine infusion. These in vivo findings are in contrast with in vitro studies showing that lipoprotein lipase expression and activity are suppressed by epinephrine [30, 31]. Future studies are needed to elucidate whether an increased TAG flux across SAT might contribute to the increased TAG storage in adipose tissue of obese participants. Conclusion The present study demonstrates in vivo that obese men have a blunted beta-adrenergically mediated lipolytic response in abdominal SAT. Therefore, a blunted lipolysis during beta-adrenergic stimulation may be an important factor in the development or maintenance of increased TAG stores and obesity.

Neurochem_Res-4-1-2270371

Changes in Glial Cell Line-derived Neurotrophic Factor Expression in the Rostral and Caudal Stumps of the Transected Adult Rat Spinal Cord

Limited information is available regarding the role of endogenous Glial cell line-derived neurotrophic factor (GDNF) in the spinal cord following transection injury. The present study investigated the possible role of GDNF in injured spinal cords following transection injury (T9–T10) in adult rats. The locomotor function recovery of animals by the BBB (Basso, Beattie, Bresnahan) scale score showed that hindlimb support and stepping function increased gradually from 7 days post operation (dpo) to 21 dpo. However, the locomotion function in the hindlimbs decreased effectively in GDNF-antibody treated rats. GDNF immunoreactivty in neurons in the ventral horn of the rostral stump was stained strongly at 3 and 7 dpo, and in the caudal stump at 14 dpo, while immunostaining in astrocytes was also seen at all time-points after transection injury. Western blot showed that the level of GDNF protein underwent a rapid decrease at 7 dpo in both stumps, and was followed by a partial recovery at a later time-point, when compared with the sham-operated group. GDNF mRNA-positive signals were detected in neurons of the ventral horn, especially in lamina IX. No regenerative fibers from corticospinal tract can be seen in the caudal segment near the injury site using BDA tracing technique. No somatosensory evoked potentials (SEP) could be recorded throughout the experimental period as well. These findings suggested that intrinsic GDNF in the spinal cord could play an essential role in neuroplasticity. The mechanism may be that GDNF is involved in the regulation of local circuitry in transected spinal cords of adult rats. Introduction Spinal cord injury (SCI), a common result of car accidents, high-altitude falls and crashes, and other violent injuries is increasing yearly. It has been reported that there are about 15–40 traumatic SCI cases per million persons annually population worldwide [1]. Once the spinal cord is damaged, it usually results in severe neurologic dysfunction and disability. Over the past several years, many extensive experiments have been done in order to find a way to promote the repair of the injured spinal cord. However, no good therapeutic approaches have yet been found. Recently, it has been shown that delivery of exogenous neurotrophic factors (NTFs) to the injured spinal cord has beneficial effects in improving functional recovery [2], indicating the potential value of NTFs in the treatment of SCI. Glial cell line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor-β (TGF-β) family, was originally purified using an assay based on its ability to maintain the survival and function of embryonic ventral midbrain dopaminergic neurons in vitro [3]. GDNF supports neural survival in several neuronal populations, including midbrain dopamine neurons and motoneurons in the central nervous system. It promotes the survival and the differentiation of many peripheral neurons, such as sympathetic, parasympathetic, and sensory neurons [3–10]. Following injury, GDNF showed a benefit effect to rescue motoneurons degeneration after spinal root avulsion and distal nerve axotomy [11], promotion of axonal regeneration [12] and enhancing regeneration of dorsal roots (DR) into the adult rat spinal cord [13]. In addition, the transplantation of GDNF-producing cells greatly enhances the survival of spinal cord motorneurons [11, 14, 15] and regeneration of several spinal systems, including dorsal column sensory, and regionally projecting propriospinal pathways [16, 17] after SCI. The biological action of GDNF is mediated by a two-component receptor GFRα-1 and protein tyrosine kinase Ret [18–21]. The GFRα-1 receptors expressed on a variety of neurons that project into the spinal cord, including supraspinal neurons, dorsal root ganglia, and local neurons. GDNF mRNAs are widely distributed in a variety of neuronal and non-neuronal tissues of embryos and adults [4, 22–25]. Although exogenous GDNF is available partially to promote propriospinal axonal regeneration and locomotion functional recovery, the role of intrinsic GDNF in injured spinal cords is largely unknown. Therefore, the present study investigated the possible role of GDNF at various time intervals following spinal cord transection injury in adult rats, so as to gain insights into the contribution of endogenous GDNF in spinal neuroplasticity. Materials and methods Characterization of antibodies To localize GDNF protein in the spinal cord, an affinity-purified rabbit polyclonal antibody (D-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used in this study. The specificity of antibody for GDNF was confirmed by Western blots using rat spinal cord homogenates. Preparation of probe for in situ hybridization For detection of GDNF cellular expression in situ, we used Digoxigenin-labeled oligonucleotide probe designed by Primer premier 5.0 package which was complementary to the rats GDNF gene sequence (33 mer, 5′-GCCCTACTTTGTCAC–TCACCAGCCTTCTATTTC-3′). This antisense DNA single-stranded oligonucleotide probe was synthesized by Takara Biotechnology Company. Animal grouping and surgery Sixty-five adult Sprague-Dawley rats of either sex (weighing 200–220 g) were obtained from the Animal Experiment Center of Sichuan University of Medical Sciences. Fifty rats among all animals, designated as sham-operated group and spinal cord transection group that animals were allowed to survive 3, 7, 14 and 21 days post operation (dpo), were used for immunohistochemistry and in situ hybridization (n = 5 in each group), and Western blot analysis (n = 5 in each group) respectively. Another five rats were intraperitoneally injected anti-GDNF solution (2.5 ml/kg) once every 2 days until 21 dpo, designed as antibody neutralization group. Five animals, subjected to cord transaction, were injected with distilled water as the distilled water group. The last five operated rats were used to investigate corticospinal tract sprouting. Every effort was taken to reduce the number of animals and suffering during the experiments. All the experiments using animals were carried out according to the guidelines of the NIH Guide for Care and Use of Laboratory Animal. For the cord transection operation, the rats were anesthetized with 3.6% Chloral Hydrate (1 ml/100 g). A midline incision was made in the back, then the T7–T8 spinous processes and the vertebral laminae were removed to expose the spinal cord at T9–T10 level. The cord was transected between T9 and T10 segments with a pair of microscissors. After surgery, the superficial back muscles and the skin were sutured along the midline. Behavior tests All rats in each group were evaluated using the BBB (Basso, Beattie, Bresnahan) scale score [26]. The BBB score for evaluation of hindlimbs function after transected injury ranges from zero, which corresponds to flaccid paralysis, to 21, which is normal gait. Animals were allowed to walk around freely in a circular field (1.2 m in diameter) for 4 min while movements of the hindlimbs were closely observed. Ranking according to the scoring system described by Basso et al. [26] includes frequency and quality of hindlimb movement as well as forelimb/hindlimb coordination. Measurement of somatosensory evoked potentials (SEP) The right peroneal nerve of the rats was dissected and a stimulation electrode was placed on it while a hole (3–4 mm in diameter) was made on the skull (2 mm to the left of the midline and 2 mm in front of the fonticulus posterior). An electrode was placed on the dura of brain cortex to record the SEP. The reference electrode was inserted at the nose epithelium. A ground electrode was inserted at the tail. The stimulus intensity was set high enough to produce a marked muscle twitch in the hind limb, about 1.1 mA, the amplitude was 0.2 ms and the frequency 3 Hz. The SEP Tracings represented the average of 200 responses. Immunohistochemistry After anesthesia with 3.6% Chloral Hydrate (1 ml/100 g), rats were perfused with 500 ml of cold phosphate-buffered saline (PBS) for 5 min and 500 ml cold 4% paraformaldehyde solution for 30 min. The T9 and T10 spinal cord segments in the sham-operated group, and the cord stumps rostral and caudal to the injury site in the experimental group were harvested. This was followed by a postfix for 6–12 h, then immersed in 0.1 M PBS containing 20% sucrose overnight. Sections of 20 μm thickness were cut on a freezing microtome and used for freefloating GDNF immunostaining, then rinsed with 0.01 M PBS and soaked in PBS containing 3% H2O2 for 30 min at room temperature to quench the endogenous peroxidase activity. They were immersed in PBS containing 5% goat serum and 0.3% TritonX-100 solution at room temperature for 2 h, then incubated at 4°C for 24 h with anti-GDNF (1:1,000, Chemicon) rabbit polyclonal antibody. Sections were incubated with biotinylated goat anti-rabbit IgG antibody (1:100 dilution) for 1.5 h at room temperature, and by reaction with avidin-biotinylated peroxidase complexes (1:250, ABC Elite, Vector Labs). GDNF immunoreactivity was visualized with brown staining using diaminobenzidine (DAB) and H2O2 as substrate for 5 min, and observed with a light microscope. Negative control experiments in which PBS was substituted for the primary antibody were performed to ascertain the specificity of antibody staining. In situ hybridization Sections from sham-operated rats of 25 μm thickness were also cut on a freezing microtome and used for in situ hybridization. Sections were fixed in 4% paraformaldehyde in 0.1 M PBS, pH 7.2 (all treatments were performed at room temperature unless otherwise indicated), and further treated with 0.3% TritonX-100 solution for 10 min and proteinase K (5 μg/ml) at 37°C for 25 min, refixed with 4% paraformaldehyde for 5 min, repeatedly immersed in 0.1 M PBS, then acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) to prevent non-specific binding of the probes. The sections were washed with 2× SSC (pH 7.0) and then prehybridized in a hybridization solution (50% formamide, 10% dextran sulfate, 1× Denhardt’s solution, 0.2 mg/ml Herring sperm DNA, and 10 mM dithiothreitol) without probes at 37°C for 2 h before hybridization, then hybridized in 100 μl hybridization solution containing 1 μl probes at 37°C for 12–16 h in a moist chamber. This was followed by washing in decreasing concentrations of SSC, from 4× SSC (pH 7.0) at 37°C for 20 min, 2× SSC (pH 7.0) at 42°C for 20 min, 1× SSC (pH 7.0) at 48°C for 20 min and ending with 0.5× SSC (pH 7.0) at 50°C for 20 min. Then sections were incubated at 37°C in 1% blocking buffer (Roche) for 1 h, subsequently reacted in 1:1,000 sheep anti-digoxygenin-alkaline phosphatase (AP) antibody in 1% blocking buffer at 4°C overnight. Lastly, AP activity was detected using nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) substrate (Roche). The sections were visualized with blue and purple sedimentation then observed with a light microscope. GFAP/GDNF double-label immunohistochemistry Two-color immunohistochemical staining for simultaneous detection of glial fibrillary acidic protein (GFAP)/GDNF expression was performed as described above. Briefly, sections were stained with anti-GDNF rabbit polyclonal antibody (1:1,000), and using DAB solutions as a substrate. This was followed by second incubation with anti-GFAP rabbit polyclonal antibody (1:1,000, AB5804, Chemicon), and finally, reacted with TMB solutions. Double labeling showed a combination of brown and blue product. Western blotting The spinal cord from the rostral and caudal stumps was obtained. After carefully removing the spinal meninges, the cords were homogenized on ice in a Lysis Buffer containing 0.05 M Tris–HCl (pH 7.4, Amresco), 0.5 M EDTA (Amresco), 30% TritonX-100 (Amresco), NaCl (Amresco), 10% SDS (Sigma) and 1 mM PMSF (Amresco), then centrifuged at 12,000g for 30 min. The supernatant was obtained and stored at −80°C for late use. Protein concentration was assayed with BCA reagent (Sigma, St. Louis, MO, USA). A 20 μl aliquot of the samples was loaded on to each lane and electrophoresed on 12% SDS-polyacrylamide gel (SDS-PAGE) for 2.5 h at a constant voltage of 120 V. Proteins were transferred from the gel to a nitrocellulose membrane for 435 min at 24 V. The membrane was blocked with phosphate-buffered saline containing 0.05% Tween-20 (PBST) with 10% non-fat dry milk overnight at 4°C. The membrane was rinsed with PBST and incubated with the primary antibody for GDNF (1:1,000) at 4°C. The membrane was incubated with a HRP-conjugated goat anti-rabbit IgG (1:5,000; Vector Laboratories, CA) for 2 h at room temperature. The membrane was developed in ECM kit and exposed against X-ray film in a darkroom. Densitometry analysis for the level of GDNF protein was performed by Bio-Gel Imagining system equipped with Genius synaptic gene tool software. β-actin (the primary antibody, 1:1,000, the secondary antibody, 1:2,000; Santa Cruz Biotechnology) was used as an internal control. BDA anterograde tracing At 14 dpo, the animals for this part were anesthetized and fixed in a David Kopf Instruments (Tujunga, CA) stereotaxic head-holder device. Burr holes were made in the dorsal cranium, and biotinylated dextran amine (BDA) (10% BDA solution, Molecular Probes) was microinjected into eight sites at a depth of 0.7 mm from the cortical surface (0.5 μl/site) to cover the hindlimb region. Animals were then sacrificed 2 weeks later to allow sufficient time for axonal transport of BDA in corticospinal tract. The spinal cords were removed and postfixed at 3 days in cold 4% paraformaldehyde in 0.1 M PBS (pH 7.2). Transverse sections (30 μm) of spinal cord at the injury site and neighboring rostral and caudal parts to the injury site were processed for the presence of BDA-labeled axons by incubation in avidin-HRP (Molecular Probes). Lastly, DAB stain was performed to visualize the positive fiber, as brown color staining. GDNF antibody neutralization After 14 dpo, each rat was intraperitoneally injected with 0.5 ml (30 mg/ml, 30 mg of anti-GDNF diluted in 1 ml of distilled water) anti-GDNF solution once every 2 days until 21 dpo. GDNF antibody was the distilled water replace as control in another five rats. The locomotion in hindlimbs by BBB score was evaluated at 3, 7, 14, and 21 dpo. Statistical analysis All data were expressed as the mean ± S.E.M. They were analyzed using One-way ANOVA and LSD-q test by SPSS software package. The statistical significance was defined as P < 0.05. Results Behavior tests The BBB score for locomotor function in sham-operated rats hindlimbs was 21. Compared with the sham-operation group, the BBB score of animals in the group with only the cord transection and distilled water group increased gradually from 7 to 21 dpo. A significant decrease in the BBB score for the GDNF-antibody treated group was observed (P < 0.05) (Table 1). Table 1Mean values of BBB scores in cord transected rats (mean ± S.E.M)Group3 Days7 Days14 Days21 DaysSham-operation group5 ± 0.621 ± 021 ± 021 ± 0Transection group0 ± 00.8 ± 0.32.4 ± 0.73.6 ± 0.5Distilled water group0 ± 00.6 ± 0.72.0 ± 0.43.3 ± 0.3GDNF-antibody treated group0 ± 00.2 ± 0.40.6 ± 0.30.9 ± 0.5BBB score in rats subjected to the sham operation was constant. Following transection injury, the BBB score of animals increased significantly from 7 to 21 dpo (P < 0.05). There had no statistical significance in BBB score between cord transection group and distilled water group (P > 0.05), while a significant decrease of BBB score in GDNF-antibody treated group was observed (P < 0.05) Somatosensory evoked potentials (SEP) Evoked potentials are the electrical signals generated by the nervous system in response to sensory stimuli. Somatosensory evoked potentials consist of a series of waves that reflect sequential activation of neural structures along the somatosensory pathways following electrical stimulation of peripheral nerves. SEP (P1N1P2 type) was recorded in the control rats and shown in Fig. 1. Throughout the experimental period, no SEP could be recorded in rats subjected to cord transection. Fig. 1Somatosensory evoked potential was recorded in normal adult rats (P1N1P2 type). The latency of SEP: P1 (10.3 ± 0.26) ms, N1 (8.81 ± 0.34) ms, P2 (15.5 ± 0.43) ms, amplitude: P1 (2.34 ± 0.02), N1 (16.3 ± 0.14), P2 (−5.06 ± 0.05) The GDNF immunostained intensity changes In the control group, GDNF immunoreactivity was observed in the cytoplasm and nuclei of the neurons from the ventral horn in the gray matter (Fig. 3a). Most of the small and medium-sized neurons in the intermediate zone and dorsal horn were stained faintly. GDNF positive profiles (Fig. 3b) simultaneously labeled with GFAP, were present in the white matter (Fig. 2b). In the negative control group, no specific immunopositive staining was detected (Fig. 2a). Fig. 2In the negative control group, no specific immunopositive staining was detected (a). GDNF positive profiles, simultaneously label-GFAP, were present in the white matter (b) In the rostral stump, strong immunostaining for GDNF was observed in neurons from the ventral horn at 3 and 7 dpo (Fig. 3c, d; Table 2). Moderate to intense immunostained neurons were seen at 14 and 21 dpo, as shown in Figs. 3g, 4a and Table 2. In the white matter, astrocytes showed strong immunoreactivity of GDNF from 3 to 21 dpo (Figs. 3d–f, 4b; Table 2). Fig. 3Under higher magnification (400×), GDNF immunoreactivity (IR) was observed in neurons from the ventral horn of the gray matter in the normal spinal cord (a, arrow). GDNF (IR) in astrocytes was also seen in white matter (b, arrow). In the rostral stump, strong GDNF IR was detected in neurons from the ventral horn at 3 and 7 dpo (c, e, 400×, arrows). Moderate to intense immunostained neurons were labeled at 14 dpo (g, 400×, arrow). Intensely immunostained IR in astrocytes of the white matter were observed from 3 to 14 dpo (d, f, h, 400×, arrows)Table 2GDNF immunostaining intensity in neurons and astrocytesNormal 3 Days7 Days14 Days21  DaysNeurons+++++++++∼+++++∼+++Rostral part Astrocytes ++++++++++++++Caudal part Neurons++∼+++++++++Astrocytes++++++++++++Intensity: + weak, ++ moderate, +++ strongNeurons in the ventral horn of the rostral stump was strongly stained for GDNF at 3 and 7 dpo, and in the caudal stump at 14 dpo. The GDNF positive staining in astrocytes was also stronger at all time-points after transection injury than seen in control groupFig. 4In the rostral stump, moderate to intense immunostained neurons were labeled at 21 dpo (a, 400×, arrow). Intensely immunostained IR in astrocytes of the white matter were also observed at 21 dpo (b, 400×, arrow). In the caudal stump, immunostained products were detected in neurons of the gray matter at 3 and 7 dpo (c, d, 400×, arrows). Strong immunostained IR for GDNF in neurons was observed at 14 dpo (e, 400×, arrow). Under higher magnification, immunoreactive structures of GDNF were localized in both cytoplasm and nuclei at 21 dpo (g, 400×, arrow), GDNF IR in astrocytes of the white matter was intensely immunostained at 14 and 21 dpo (f, h, 400×, arrows) In the gray matter of the caudal stump, moderately immunostained neurons were detected at 3, 7 and 21 dpo, and more so at 14 dpo (Fig. 4c–e, g; Table 2). The subcellular localization for GDNF was observed both in cytoplasm and nuclei at 21 dpo (Fig. 4g). At both 14 and 21 dpo, astrocytes in the white matter strongly stain with GDNF immunoreactivity as shown in Fig. 4f, h and Table 2. Localization of GDNF mRNA in spinal cord of rats Signals of hybridization for GDNF mRNA were markedly detected in the Central canal and lamina III, IV and V, in which small neurons were moderately NBT/BCIP stained (Fig. 5a). Medium-sized neurons were seen in the field of the intermediomedial nucleus and nucleus dorsalis (Fig. 5a). In the ventral horn, GDNF mRNA-positive large neurons were also observed in lamina IX (Fig. 5b). A few scattered glial cells were probe-labeled with light density in white matter (Fig. 5c). Fig. 5In the spinal cord of adult rats, GDNF mRNA-positive product were markedly detected in neurons from the lamina III, IV and V and the central canal (a). Medium-sized neurons were seen in the field of intermediomedial nucleus and Nucleus dorsalis. In the ventral horn, GDNF mRNA-positive large neurons were also observed in lamina IX. Under higher magnification (200×), positive products with blue and purple staining were localized in the cytoplasm (b, arrows), also in white matter scattered glial cells were probe-labeled with light density (c, arrows) Western blotting A single positive band was observed at a molecular weight of about 34,000, which corresponds to the molecular weight of GDNF. In the rostral stump, the level of GDNF protein increased at 3 dpo, when compared with the control group (P < 0.05), then decreased quickly to its lowest level at 7 dpo (P < 0.05) which was less than that of the control level, and then began to increase again at 14 dpo. However, GDNF protein was higher than what was seen in the control group at 21 dpo (Figs. 6a, 7). In the caudal stump, GDNF protein immediately decreased at 3 dpo, reached its lowest level at 7 dpo (P < 0.05), then increased from 14 to 21 dpo, but it did not go back to the control level (Figs. 6b, 7). Fig. 6In the rostral stump, GDNF protein level increased at 3 dpo (P < 0.05), then reached its lowest point at 7 dpo which was below the control group (P < 0.05), then began to recover at 14 dpo, but it was less than that of normal level. This was followed by a continuous increase higher than the control group at 21 dpo (a). In the caudal stump, GDNF protein decreased at 3 dpo, reached its lowest point at 7 dpo (P < 0.05), then started to increase at 14 dpo, but it was lower than the normal level at 21 dpo (b)Fig. 7GDNF levels were significantly different from each other (P < 0.05), except for the comparison between the 21 dpo and normal group, as well as the 21 and 3 dpo group in the rostral stump. The groups were significantly different from each other (P < 0.05), except for the comparison between the 3 and 14 dpo group in the caudal stump BDA tracing BDA-positive axons which stained as brown silkiness were detected in rostral cord sections of the injury site (Fig. 8a), but not in the field of scar tissue (Fig. 8b), and in the caudal segment near the injury site (Fig. 8c). Fig. 8BDA-positive axons staining as brown silkiness were detected in rostral cord sections near the injury site (a), but not in the field of scar tissue (b), and caudal cord sections near the injury site (c) Discussion In the present study, GDNF immunoreactive products in neurons from the gray matter and in astrocytes from the white matter of the spinal cord were observed in the sham-operation group. This indicates that GDNF could be involved in either the physiological function or the maintenance of survival for all kinds of neurons and astrocytes. It has been shown that GDNF mRNAs widely distributed in a variety of neuronal and non-neuronal tissues of embryos and adults [4, 23–25]. In addition, GDNF mRNA positive signals were found in the ventral horns, the field of nucleus dorsalis, and the dorsal horns in the spinal cords of adult rats [2, 16, 27] and in the dorsal root ganglia in newborn rats [23]. Therefore, it raised the possibility that GDNF was synthesized by cells of the spinal cord and the GDNF expression may be changed after lesion, which implies that GDNF plays a potential role in both physiological and pathological conditions. Following cord transection, rats exhibited spinal cord dysfunction throughout the experimental period. There was an absence of SEP in surface recordings, indicating a loss of sensory function in the hindlimbs following transection injury. Comparatively, locomotion function recovery was observed from 7 to 21 dpo, which was parallel to the improvement in BBB score. It showed that locomotor functional plasticity in the hindlimbs of rats did occur following SCI. As corticospinal tract regeneration was not seen in the caudal part near the injury site and scar tissues, it appears impossible to reestablish connectivity between the areas rostral and caudal of the injury site. The possible reason for locomotor function improvement may be related to the modulation of spinal circuit activity from endogenous neurotrophins. In the present study, BBB scores in the GDNF-antibody treated rats was inferior to that of the group not receiving the antibody treatment, indicating intrinsic GDNF may be available for improving locomotor function recovery to some extent in the hindlimbs of rats [15, 28]. It has been reported that GDNF can promote the repair or survival of spinal motoneurons [4, 7, 29–32]. Several other studies have shown that the administration of GDNF could promote axonal regeneration, and enhance locomotion functional recovery [2, 15, 30, 33–36]. GDNF immunoreactivity products in neurons from the ventral horn of the rostral stump were strongly stained at 3 and 7 dpo, and in the caudal stump at 14 dpo, suggesting that the GDNF expression in neurons was up-regulated following injury. This may be related to the locomotion functional recovery of hindlimbs. Glazner et al. [37] showed that strong labeling for GDNFR-α and c-ret mRNA was observed in large neurons from the ventral horn. Our results also supported the mRNA expression for GDNF in neurons of the ventral horn, as well as astrocytes in white matter using in situ hybridization. RET expression was unregulated following peripheral axotomy in alpha-motoneurons [38], and topical application of GDNF 30 min after SCI significantly improved motor function and reduced blood–spinal cord barrier (BSCB) breakdown, edema formation, and cell injury at 5 h has been reported [39]. Therefore, GDNF is available to assist in locomotion functional plasticity by exerting behavioral and anatomic neuroprotection following SCI. In the present study, the expression patterns of endogenous GDNF indicated its possible role in maintaining motorneuron survival because GDNF mRNA-positive products were detected in neurons from the ventral horn by in situ hybridization. Of course, GDNF retrograde axonal transport from a target tissue to neuronal cell bodies [40, 41] is not absolutely excluded. It has been reported that GDNF produced by skeletal muscle is taken up at the nerve terminals and retrogradely transported to motoneurons of the ventral horn by axons [30]. Comparing the results of immunostaining, the GDNF level varied throughout the spinal cord at 3 dpo. In the caudal stump, GDNF showed a significant decrease. This was probably due to the transection injury that triggers a process destructive to ascending and descending tracts conduction and that extends tissue loss. Differently, a rapid up-regulation of GDNF was observed in the rostral stump. This suggested that GDNF had accumulated around the transection site due to interruption of its transport within the axons. Satake et al. [42] reported GDNF transcription began to increase within 30 min after injury and peaked within 3 h after spinal cord contusion injury. Interestingly, the level of GDNF protein reached its lowest point at 7 dpo in both stumps, probably due to the consequences of cell death or apoptosis and/or interruption of the spinal cord pathways. Subsequently, GDNF protein levels gradually started to recover and surpassed the control level in the rostral stumps at 21 dpo, which may be explained by (1) GDNF transport within axons was strengthened, (2) inflammation leads to the increase of GDNF synthesis [36], and (3) induction of de novo synthesis of GDNF by neurons and/or non-neuronal cells. Noticeably, the GDNF positive cells in the white matter were labeled by GFAP simultaneously, which identified the labeled cells as astrocytes. Indeed, an association between reactive astrocytes and regenerating nerve fibers in the white matter of the central nervous system in vivo has been reported [43, 44]. Also, increased GFR-1 in astrocytes of degenerating white matter in adult rat spinal cords after mechanical injury has been shown [2]. These results indicated that GDNF expression in astrocytes may be involved in neuroplasticity following spinal cord transection injury. The present study provides new evidence to understand the role of intrinsic GDNF in the rostral and caudal stumps of injured spinal cords. It suggests that GDNF plays an essential role in the neuroplasticity of the local circuitry, especially in the caudal stump of the transected spinal cords.

Eur_Spine_J-2-2-1602200

Bilateral pedicle stress fracture in the lumbar spine of a sedentary office worker

A case of bilateral pedicle fracture in the lumbar spine of a sedentary office worker is being presented. No such case has been reported in the literature previously. Bilateral pedicle fracture is a rare entity. Few cases have been reported in literature. All the reported cases had some underlying causative factors like degenerative spine disease, previous spinal surgery or stress-related activities, e.g. athletes. Our case is a 36-year-old sedentary office worker with none of the factors mentioned. We present a case of a 36-year-old sedentary worker with long-standing low backache. There were no root tension signs. Plain radiographs were inconclusive. The patient had a CT scan. The CT scan revealed long-standing defects in the pedicles of L2 vertebra with pseudoarthrosis. Infiltration with anaesthetic relieved the symptoms. Our patient was managed conservatively with spine rehabilitation physiotherapy program. Pedicle fracture can develop due to abnormal stresses in the pedicle either because of previous spinal surgery or spondylitic changes in the spine. Bilateral pedicle fracture in the absence of these conditions is extremely rare. Introduction Isolated pedicle fracture in the spine is uncommon. It has been reported in association with previous spine surgery, or in highly active athletic individuals. We report a case of bilateral pedicle fracture in a patient with chronic backache without the history of trauma or any of the above-mentioned conditions. The patient was a sedentary worker with minimal physical demands. Case report We present a case of a 36-year-old female with a history of chronic backache. Her symptoms were of mild to moderate in intensity. Discomfort was mainly related to activity but she was performing her normal duties as an office manager without significant problems. On examination there were no neurological deficit or root tension signs. Plain radiographs were inconclusive. Clinical examination revealed localized deep tenderness at L2; therefore, a CT scan was performed. The CT scan showed bilateral long-standing defects through the pedicles of 2nd lumbar vertebra with pseudoarthrosis and sclerosis (Fig. 1a, b). Infiltration with local anaesthetic, under the X-ray guidance, in the involved area relieved the symptoms. Surgical management was discussed with the patient, which was declined. The patient underwent spine rehabilitation physiotherapy program, which resulted in the improvement of the symptoms. Fig. 1a CT scan of the vertebra showing bilateral pedicle fracture. b CT scan showing bilateral pedicle fracture with pseudoarthrosis Discussion Bilateral pedicle stress fracture in the spine is a rare finding. Traughber and Havlina [10] reported the first case of a bilateral pedicle stress fracture. Cyron et al. [2] demonstrated that the parsintereticularis is thought to be the weakest site in the neural arch, followed by the pedicle. In cases with established unilateral stress fracture, there is a redistribution of forces in the neural arch, which leads to compensatory sclerosis of contralateral bony structures. A compensatory hypertrophy of the contralateral pedicle has been documented [1, 8]. This entity was first described by Wilkinson and Hall [11] in 1974. These authors reported on seven patients whose radiographs were suspicious for a neoplastic process occurring in a pedicle (e.g. osteoid osteoma or osteoblastoma). Further investigation revealed unilateral spondylolysis with sclerosis of the contralateral posterior elements. The incidence of bilateral pedicle stress fracture is unknown in an otherwise normal spine. Gunzburg and Fraser [3] introduced the term pediculolysis in 1991 in a patient with multilevel facet joint osteoarthritis and minimal spondylolisthesis at L4–L5. Pedicle fracture has been reported in cases following posterolateral instrumented spinal fusion [4, 5, 7]. Bilateral pedicle stress fractures or pediculolysis have been documented in an athlete of high physical demand [6]. Stanley and Smith [9] reported a case of pedicle fracture following laminectomy. In our patient the fractures appear to be old with evidence of sclerosis at the fracture margins along with pseudoarthrosis. This type of bilateral pedicle stress fracture has not been documented in a normal spine of a sedentary office worker without a history of major trauma or surgery. Conclusion Pedicle fracture is uncommon but has been reported in association with degenerative spondylolisthesis, unilateral spondylolysis, following spinal surgery but not in an otherwise normal spine. Pedicle stress fracture may show either as a recent fracture or may have signs of established pseudoarthrosis. So far there is no case of bilateral pedicle stress fracture in an otherwise normal spine.

Pediatr_Radiol-3-1-1950217

Pediatric DXA: clinical applications

Normal bone mineral accrual requires adequate dietary intake of calcium, vitamin D and other nutrients; hepatic and renal activation of vitamin D; normal hormone levels (thyroid, parathyroid, reproductive and growth hormones); and neuromuscular functioning with sufficient stress upon the skeleton to induce bone deposition. The presence of genetic or acquired diseases and the therapies that are used to treat them can also impact bone health. Since the introduction of clinical DXA in pediatrics in the early 1990s, there has been considerable investigation into the causes of low bone mineral density (BMD) in children. Pediatricians have also become aware of the role adequate bone mass accrual in childhood has in preventing osteoporotic fractures in late adulthood. Additionally, the availability of medications to improve BMD has increased with the development of bisphosphonates. These factors have led to the increased utilization of DXA in pediatrics. This review summarizes much of the previous research regarding BMD in children and is meant to assist radiologists and clinicians with DXA utilization and interpretation. Introduction Peak bone mineral accrual occurs during early puberty and peak bone mass is achieved in young adulthood. Low BMD can result from a wide variety of childhood diseases or be due to the effects of their treatment. Increasingly, pediatric specialists are requesting evaluation of BMD and radiologists are expected to be knowledgeable in the technique, interpretation and clinical applications of DXA. A review of the technical and interpretive aspects of DXA has been recently presented [1]. The clinical uses of DXA in pediatrics are exceedingly broad. This paper reviews the clinical data regarding pediatric DXA and can be used to assist radiologists and clinicians in DXA utilization and interpretation. Gastrointestinal disorders Gastrointestinal diseases may impact bone health in several ways. Poor calcium intake, as in patients with a milk allergy, and reduced calcium absorption, as in patients with untreated celiac disease, result in low BMD. Early correction of the underlying deficiency allows normal bone mineralization to occur. In addition to poor calcium absorption, inflammatory bowel disease likely impacts bone health through other factors including chronic diarrhea, decreased lean tissue mass, reduced physical activity, increased inflammatory cytokines, and CS therapy. As in all chronic conditions, correction of short stature and delayed maturation will allow better identification of those patients with abnormal DXA findings who will have significant bone mineral deficits. Milk allergy Henderson and Hayes [2] reported a positive correlation between LS and hip BMD and calcium intake in children with significant milk allergy. They found a 5–8% increase in LS Z-scores and a 9–13% increase in hip Z-scores in patients with daily calcium intake above the recommended dietary allowances. These findings were confirmed in children with reduced dairy intake due to a variety of other causes [3, 4]. There was a positive correlation between BMD and calcium intake with normal values in the subgroup with normal dietary calcium intake and decreasing BMD as the daily intake of calcium decreased. LS BMD was maintained, even with severe dairy restrictions when calcium intake was maintained through diet or supplementation. Inflammatory bowel disease Boot et al. [5] found decreased LS and TBBMD in children with Crohn disease or ulcerative colitis. The decreases were statistically significant even when they accounted for the delayed bone age in their patients. Similarly, Ahmed et al. [6] found reduced LS and TBBMD Z-scores in nearly 70% of children with Crohn disease and, to a lesser extent, ulcerative colitis, when compared to healthy age-matched controls. However, when height was taken into account, only 22% of their patients had low BMD. Burnham et al. [7] found decreased height, lean tissue mass and TBBMC in children with Crohn disease when compared to normal controls. However, with correction for height, age, race, Tanner stage and lean tissue mass, no statistically significant differences were found between the study and control groups. The authors also found no correlation between CS treatment and height-corrected BMC. Similar findings were reported by Walther et al. [8]. Thus, in patients with inflammatory bowel disease, the apparent low LS BMD and TBBMC is largely a result of short stature, delayed maturation and decreased lean tissue mass. Celiac disease Kalayci et al. [9] found lower LS BMD in untreated celiac disease patients compared to treated patients. They found that nearly all children with celiac disease whose treatment began before the age of 4 years reached normal BMD compared to only 50% of children whose treatment began after the age of 4 years. They also found that all patients who initially presented with gastrointestinal symptoms had normal BMD after 1 year of a gluten-free diet. In prepubertal children with celiac disease, Barera et al. [10], Tau et al. [11] and Szathmari et al. [12] found an increase in height, weight, BMC and BMD after 1.5 years of a gluten-free diet when compared to baseline measurements. These reports indicate that in patients with celiac disease, normalization of DXA findings can be expected with early diagnosis and treatment. Liver disease The effects of liver dysfunction on bone health are complex and may involve vitamin malabsorption, failure of vitamin D activation, calcium malabsorption, bile salt deficiency and chronic malnutrition. Argao et al. [13] evaluated children with a variety of chronic cholestatic liver diseases and found distal radial BMC to fall quickly after birth and in infancy. The values remained low throughout childhood and reflected the severity of the underlying hepatic dysfunction. In a study of prepubertal children with Alagille syndrome, Olsen et al. [14] found reduced height, weight and TBBMC compared to healthy controls matched for age, gender, ethnicity and physical maturity. Malabsorption of fat-soluble vitamins, particularly vitamin D, significantly reduces LS and TBBMC. D’Antiga et al. [15] found normal LS BMC and BMD in patients with childhood liver failure who were at least 1 year out from orthotopic liver transplantation. Return to normal values appeared to be unaffected by the severity of bone disease or cholestasis prior to transplantation. Thus, liver dysfunction with disordered vitamin D metabolism results in early decreased bone mineral accrual. With successful liver transplantation, normalization of DXA findings can be expected. Renal diseases Chronic kidney disease results in abnormal bone metabolism via disturbances in calcium and phosphate handling, altered vitamin D and parathyroid hormone levels and function, and altered renal clearance of other metabolites. Additional factors that affect the BMD in these patients include malnutrition, metabolic acidosis, anemia and growth hormone abnormalities resulting in growth retardation. As with many chronic medical conditions, short stature will greatly affect BMD and needs to be accounted for in DXA interpretation. Bakr [16] found reduced LS BMD in 60% of pre- and postdialysis patients. The degree of osteopenia correlated with biochemical markers of secondary hyperparathyroidism. However, the DXA BMD values were not corrected for height. Similar results were reported by Pluskiewicz et al. [17] following a 2-year longitudinal study of adolescents with chronic kidney disease. They found decreased or stable LS BMD (uncorrected for height) during a time when an increase would be expected. The most severe decreases in BMD were found in patients who were treated with CS. In contrast, Ahmed et al. [18] found normal TBBMD and TBBMC as well as normal LS BMD in children with chronic kidney disease when the DXA values were corrected for height. Similar findings were reported in children before transplantation [19]. Boot et al. [20] reported normal LS and TBBMD in patients with kidney disease of relatively short duration and in the setting of adequate vitamin D replacement. Two-year follow-up of these patients demonstrated continued maintenance of normal BMD [21]. Several authors found that the height-corrected LS BMD was reduced for up to 2 years after transplantation; this was felt to be due to high-dose CS treatment required to suppress transplant rejection. However, by 3 years after transplantation, LS BMD had recovered to normal values [18, 22–24]. Idiopathic hypercalciuria results in excessive urinary excretion of calcium and has increasingly been identified as a cause of decreased BMD in children [25–28]. The degree of BMD reduction does not appear to correlate with the presence of urolithiasis or urinary uric acid concentration. Studies of children and their mothers with idiopathic hypercalciuria indicate that low LS and hip BMD persist into adulthood [25, 26]. In summary, the various metabolic disturbances resulting from altered renal physiology may compromise normal bone mineralization and remodeling, but children with chronic kidney disease often maintain normal BMD if calcium and vitamin D metabolism are normal. CS treatment, either before transplantation or when used to suppress transplant rejection, appears to contribute to low BMD but short-term follow-up indicates eventual normalization of LS BMD. Regardless of the nature of renal disease, correcting for height is essential for accurate interpretation of DXA results in these patients. Endocrinological diseases Skeletal development is significantly influenced by multiple hormone systems that are essential to normal bone mineral accrual. Both insufficient and excessive hormone levels can have deleterious effects, resulting in inadequate bone formation or disproportionate bone resorption. Pubertal hormones play a critical role in bone mass acquisition with major differences occurring based on sex and skeletal site [29]. Patients with early or delayed puberty will show early or delayed peak bone mass accrual, respectively [30]. Treatment of precocious puberty includes gonadotropin-releasing hormone analogs that could theoretically interfere with normal bone mineral acquisition. Short-term treatment does not interfere with normal BMD increases in these patients [31]. Disorders of growth hormone, insulin and glucocorticoids Accurate DXA evaluation of children with growth hormone deficiency or idiopathic short stature must account for their smaller bones [1]. Children with growth hormone deficiency will present with low areal [32] and volumetric LS [33] BMD when first diagnosed. Children with idiopathic short stature have also been found to have low areal LS and hip BMD prior to therapy [34]. With sustained growth hormone replacement these values have been reported to remain stable [33] or normalize rapidly [34]. Children with growth hormone deficiency have a robust response to replacement therapy with increases in height, lean tissue mass and TBBMC [33]. Increases in BMD occur even after peak height is achieved, and this indicates the need to continue hormone therapy until peak BMD is achieved [35, 36]. Type 1 diabetes mellitus is a known risk factor for osteoporosis, but the underlying mechanisms are not fully determined. Adolescent patients have lower bone mass and bone size despite normal growth and maturation [37]. Studies suggest that children and adolescents with diabetes have reduced bone formation and increased bone resorption resulting in low bone mass and BMD [38, 39]. Hemoglobin A1C level, a measure of glycemic control, negatively correlates with BMD [40] and TBBMC [41]. The negative effects of chronic high-dose CS therapy on bone have been well documented. CS replacement therapy, as in congenital adrenal hyperplasia, has not been found to result in reduced BMD [42, 43]. However, young women with congenital adrenal hyperplasia are at risk of reduced BMD if there is biochemical evidence of androgen suppression due to CS therapy [44]. Patients with either childhood-onset or adult-onset Cushing disease often have severe reductions in BMD due to increased osteoclastic activity and bone resorption with normal or reduced bone deposition [45]. Additionally, concomitant reductions in sex and growth hormones contribute to their low BMD. Adolescents appear to be particularly vulnerable to reduced trabecular bone density as measured at the LS. BMD deficits and biochemical measures of bone turnover showed marked improvement after 2 years remission from hypercortisolism when compared to baseline values, but low LS BMD persisted in the majority of patients [46]. Disorders of reproductive hormones Reproductive hormones play an important role in the acquisition of bone mineral; estrogen is known to have a protective effect against the development of osteoporosis in postmenopausal women. The effects of hormonal birth control use on BMD in adolescent girls have been investigated by several researchers. The trend toward lower estrogen levels in oral contraceptives has been associated with decreased BMD in adolescent girls who use oral contraceptives [47, 48]. Progesterone-mediated contraceptives (Depo-Provera) have been shown to inhibit bone mineral acquisition in the LS and hip in adolescent girls [49–51]. The extent of this reduction appears to correlate with the duration of treatment and can be lessened with supplemental estrogen [51]. Partial or full BMD recovery can occur after cessation of treatment and can be facilitated with increased physical activity and adequate calcium and vitamin D intake. Girls with anorexia nervosa have been found to have decreased BMD at multiple skeletal sites [52–54]. These reductions are thought to be due to a combination of nutritional (decreased calcium and caloric intake), hormonal (decreased estrogen levels, delayed puberty), and mechanical (decreased lean tissue mass) factors. With resumption of normal caloric intake and return of menses, there is biochemical evidence of increased bone turnover; however, BMD levels remain low and Z-scores may continue to fall for at least 1 year [53]. It is not known if full BMD recovery will occur by early adulthood, but adult women who had adolescent-onset anorexia nervosa have more dramatic reductions in BMD than those with onset during adulthood [55, 56]. Estrogen replacement with oral contraceptives and vigorous exercise can protect against BMD loss in anorexia nervosa [57]. Untreated congenital hypothyroidism can result in markedly reduced BMD. Children with adequate long-term thyroid replacement therapy show normal LS and hip BMD and BMC [58, 59] and normal findings continue into young adulthood [60]. Later in childhood, hypothyroidism is usually caused by Hashimoto thyroiditis. LS and distal radial BMD are expected to be normal at diagnosis and to be maintained with thyroid hormone therapy, and normal peak bone mass is to be expected in these patients [61]. Hyperthyroidism in Graves disease markedly decreases LS and hip BMD, but these normalize within 1 year following thyroid ablation and hormone therapy [62]. In summary, hormonal imbalances from any of a large number of endocrine disorders are associated with abnormal DXA findings and reduced BMD. With hormone replacement therapy, improved bone mineral accrual is to be expected in most of the endocrine deficiencies. However, with anorexia nervosa, especially with onset in adolescence, there appears to be a sustained reduction in BMD even after resumption of normal caloric intake and menses. Respiratory diseases Cystic fibrosis In cystic fibrosis (CF), low BMD may be caused by reduced gastrointestinal absorption of calcium and vitamin D, reduced testosterone levels, chronic hypoxia, chronic CS use, and reduced lean tissue mass. Because of the multifactorial nature of reduced BMD in CF, BMD Z-scores often reflect the severity of illness. Clinically stable children with mild CF have been shown to have normal BMD [63–65]. However, adolescents and adults with CF have accelerated bone loss over time reflecting disease progression [66, 67]. Henderson and Madsen [68] and Buntain et al. [66] found progressively decreasing BMD Z-scores with increasing age in CF patients. LS, hip and TBBMD in the prepubertal group were normal. TBBMD was reduced in adolescents; however, all other sites remained normal. In adult patients, BMD was reduced at all sites. In a follow-up study, Haworth et al. [69] reported that patients older than 25 years showed accelerated LS and hip BMD losses. Bhudhikanok et al. [70] found that LS, hip, and TBBMD were low at baseline and documented further bone loss at follow-up. CS use was identified as the most modifiable factor accounting for this bone loss and the authors suggested that bisphosphonates may be beneficial in these patients. The benefits of bisphosphonates in CF were confirmed by Aris et al. [71, 72] and Haworth et al. [73] who found increases in LS and hip BMD following treatment with intravenous or oral bisphosphonates, respectively. Asthma Asthma may inhibit normal bone metabolism due to chronic hypoxia and decreased physical activity. Additionally, asthmatic patients are treated with both oral and inhaled CS to reduce airway inflammation. Patients treated with low or moderate levels of inhaled CS have been shown to have normal BMD Z-scores when compared to CS-naive asthmatic controls but reduced BMD Z-scores when compared to normal controls when height is not taken into account [74–77]. Allen et al. [78] and Harris et al. [79] found significant reductions in BMD in asthmatic children receiving high doses of inhaled CS. Hematological diseases Anemia and hemophilia Chronic anemias can affect bone structure and density through bone marrow hyperplasia (resulting in expansion of the medullary space, trabecular coarsening and cortical thinning), and vasoocclusion (resulting in medullary and diaphyseal infarction). Additional factors for reduced BMD include reduced lean tissue mass, decreased physical activity and hypogonadism, and in the case of thalassemia, endocrine dysfunction. Brinker et al. [80] found low BMD in children with sickle cell anemia. These findings confirmed those of Soliman et al. [81] who reported low LS BMD in prepubertal children with sickle cell anemia. Lal et al. [82] and Buison et al. [83] noted markedly reduced BMD and BMC, which correlated with disease severity in these children. These changes were found to persist into adulthood [84]. Additionally, low calcium intake and low vitamin D levels may contribute to decreased BMD in these patients. Vogiatzi et al. [85] and Benigno et al. [86] evaluated children with thalassemia major who were fully treated with transfusion and chelation therapy and found reduced BMD at diagnosis and showed further reductions at follow-up. Patients with hemophilia are at risk for osteoporosis due to reduced levels of physical activity and sports, particularly those involving running, jumping and axial loading of the skeleton. The severity of hemophilic arthropathy can be quantitated using a clinically derived joint score, and this has been found to correlate with BMD [87]. Low BMD is independent of height and weight but correlates with disease severity and extent. These changes can be expected to persist into adulthood [88]. Oncological diseases Acute lymphocytic leukemia ALL is the most common childhood malignancy, but because of its excellent prognosis, most children with ALL survive into adulthood. Arikoski et al. [89] found reduced BMD in ALL survivors up to 20 years after treatment. Of their 29 patients, 20 had received cranial irradiation and four males had received testicular irradiation. The use of high-dose methotrexate and cranial irradiation were found to be significantly correlated with low BMD. Differing results were presented by Brennan et al. [90] who also reported significantly reduced LS, hip and distal radial BMD in long-term survivors of ALL but found no statistically significant correlation of BMD with cranial irradiation, growth hormone status or height Z-scores. They suggested that the low BMD found in their patients was related to the effects of chemotherapy rather than cranial irradiation. Van der Sluis et al. [91] found normal LS and TBBMD a mean of 10 years after ALL treatment that included high-dose methotrexate and CS but not cranial irradiation. Their patients had all been prepubertal at the time of initial diagnosis and none had signs of significant gonadal or growth hormone dysfunction at the time of follow-up. The authors concluded that the deleterious effects of ALL and its treatment in childhood on BMD may be due to cranial irradiation but, in its absence, BMD will normalize by early adulthood. This conclusion is supported by other researchers [92, 93]. Jarfelt et al. [94] examined bone turnover and growth hormone status with respect to physical activity levels and BMD in adult survivors of childhood ALL. The patients were all prepubertal at the end of treatment and had been treated with high doses of CS and methotrexate. No patient had evidence for gonadal or endocrine dysfunction. The BMD values were normal for the group. Only the level of physical fitness at follow-up correlated positively with BMD. The authors stressed the importance of physical activity in restoring and maintaining normal BMD in survivors of childhood ALL. In summary, children with ALL will have reduced BMD during treatment and shortly thereafter. The recuperative capacity in young children is high and normal BMD should be expected even after high-dose methotrexate and CS therapy. The role of adequate physical activity is being increasingly stressed as an important factor in normal BMD recovery. Children who have survived ALL may be at risk of persistently low BMD in adulthood if they have confounding factors such as gonadal dysfunction or if the course of their disease coincided with the period of expected rapid accrual of bone mineral that normally occurs during puberty. Other malignancies There are only limited data regarding the effects of other childhood malignancies on BMD. Aisenberg et al. [95] and Vassilopoulou-Sellin et al. [96] found reduced femoral neck and TBBMD in young adult survivors of various childhood cancers. Gonadal dysfunction due to pelvic or cranial irradiation was the factor most strongly correlated with reduced BMD. CS treatment did not correlate with low BMD. Nysom et al. [97] reported normal size-adjusted TBBMC in adult survivors of childhood lymphoma and found no relationship between TBBMC and cumulative methotrexate or CS doses. Kelly et al. [98] evaluated adult survivors of various pediatric solid tumors and found reduced BMD in at least one site in half of their patients. Only the total number of chemotherapeutic agents correlated with reduced BMD. Five of six extremities involved with a bone sarcoma showed reduced BMD. Similar results were found in a group of sarcoma survivors reported by Ruza et al. [99]. Interestingly, they found that those diagnosed prior to puberty had more severe BMD reductions later in life. This may be due to the extensive physical disabilities associated with amputations and limb salvage procedures in these patients. Odame et al. [100] found that patients with childhood brain tumors treated with cranial irradiation had reduced LS and TBBMD and these were correlated with reduced physical activity and poorer quality of life. The authors postulated that the higher cranial radiation doses used for the treatment of brain tumors had a profound effect on long-term BMD compared to the relatively lower doses used in the treatment of ALL. In summary, there are multiple factors that affect short- and long-term BMD in these patients/survivors. These include pubertal status at diagnosis, type of malignancy, local (sarcomas, central nervous system tumors) versus systemic (leukemia) disease, initial (malnutrition, immobilization) versus prolonged (amputation) disease-related disability, types of chemotherapy, and radiation ports and dosages that may cause gonadal or growth hormonal dysfunction. Neurological diseases Cerebral palsy Because of decreased muscle development and ambulation, children with cerebral palsy are at increased risk of osteoporosis-associated fractures. Frequently, assessment of the LS and hips with DXA is impossible due to difficulties in positioning patients with contraction deformities, muscular spasm-induced motion artifacts or orthopedic hardware-related artifacts. Because of these limitations, Harcke et al. [101] suggested scanning the distal femur with these patients positioned on their side. Distal femoral BMD was found to be highly correlated with hip BMD and the technique yielded highly reproducible BMD data [102]. Henderson et al. [103] demonstrated reduced LS and hip BMD in children with cerebral palsy. Measures of nutritional and ambulatory status were found to be the best predictors of BMD. In children with moderate to severe disease, Henderson et al. [104] found distal femur and LS BMD to be markedly reduced and both closely correlated with disease severity. Distal femoral BMD was negatively correlated with age, indicating progressive bone mineral deficits as these children grew older. The lack of a strong correlation between LS BMD and fracture risk in the lower extremities emphasizes the need for direct assessment of the distal femur with DXA in these patients. In a prospective longitudinal study of patients with cerebral palsy, Henderson et al. [105] found that low initial LS and distal femur BMD Z-scores were associated with poor growth, nutritional status and motor function, and these contributed to reduced bone mineralization at follow-up. Treatment with bisphosphonates for 1 year resulted in a substantial increase in distal femoral BMD in children with quadriplegic cerebral palsy [106]. The improvement was sustained for at least 6 months after the last treatment. However, Bachrach et al. [107] found that LS BMD returned to baseline values 2 years after bisphosphonate therapy was terminated. Importantly, despite the lack of sustained BMD improvement, no patient had a fracture during the treatment or follow-up periods. In summary, children with CP show decreased bone mineral status that reflects the duration and severity of their disease. Distal femoral measurements better reflect the bone status at the sites more at risk of fracture. Bisphosphonate therapy increases BMD during treatment and it appears to have sustained benefits with reduced fracture rates even after treatment is terminated. Meningomyelocele Quan et al. [108] found reduced forearm BMD in children with meningomyelocele. Patients with a history of fracture had substantially lower forearm BMD than those without a fracture history. The reduced upper extremity BMD in these patients with preserved upper extremity function may indicate that both systemic and local factors affect bone mineralization. Hypercalciuria in nonambulatory patients with meningomyelocele may be such a factor. Quan et al. [109] later evaluated the effect of thiazide treatment for hypercalciuria in these patients and found no change after 1 year in forearm BMD when compared to placebo-treated controls. Valtonen et al. [110] found normal forearm and LS BMD in adult patients with meningomyelocele, but reduced hip BMD in one-third of the patients. There was a trend for low hip BMD in the nonambulatory compared to ambulatory patients but not for the LS. This dissociation of LS and hip BMD values was thought to be due to relatively preserved axial loading on the LS in upright patients with meningomyelocele. The normalization of forearm BMD in these adult patients was attributed to increased upper extremity muscular stress with the use of crutches and manual wheelchairs. Connective tissue diseases Juvenile rheumatoid arthritis and systemic lupus erythematosus Rheumatoid diseases have long been known to affect bone health negatively. Disease extent, severity, subsequent disability and CS therapy negatively impact bone mineralization. Pepmueller et al. [111] and Pereira et al. [112] found decreased regional, LS, and TBBMD in patients with JRA. The decrease was more severe in children with longer disease duration and was similar for oligoarthritis and polyarthritis. Lien et al. [113] found decreased TBBMC and TBBMD in patients with juvenile idiopathic arthritis. Disease duration and severity correlated with TBBM but not CS therapy. Henderson et al. [114] found no statistically significant difference in TBBMD in prepubertal children with mild-to-moderate JRA and no history of CS treatment when compared to normal controls. When a similar study was performed on CS-naive older girls with mild-to-moderate JRA, Henderson et al. [115] found decreased TBBMC. As with other chronic diseases of childhood, it appears that persistent disease activity through puberty results in decreased BMC. Mul et al. [116] found marked reductions in LS BMD and BMC in children with rheumatic diseases treated for at least 1 year with high-dose CS. Bianchi et al. [117] found that long-term methotrexate for JRA did not result in reduced LS or TBBMD. In summary, prepubertal children with JRA of mild or moderate severity without a history of CS treatment will have TBBMD similar to healthy children. With increasing disease severity and duration, especially through puberty, TBBMC will decrease when compared to normal children. These changes are in contrast to the normal DXA findings reported in patients with juvenile systemic lupus erythematosus [118, 119]. Unlike patients with JRA, these patients typically do not have bone and joint involvement and thus are more likely to have preserved BMD. However, factors that may negatively impact bone health in these patients include immobility, limited exposure to sunlight, and CS therapy. Musculoskeletal diseases For normal mineralization to occur, adequate muscle-induced mechanical stresses are required to induce bony remodeling. Insufficient muscle mass and activity result in poor bone accrual and low BMD. Additionally, therapy of primary muscle disorders with CS also inhibits normal bone mineralization. Duchenne muscular dystrophy and dermatomyositis In a longitudinal study of boys with Duchenne muscular dystrophy, Larson and Henderson [120] found that LS and hip BMD correlated with functional mobility level with ambulatory boys having higher LS BMD than nonambulatory boys. Hip BMD was decreased before loss of ambulation and showed progressive reduction over time. Fractures occurred in nearly half of patients, most typically involving the lower extremity and frequently resulting in loss of ambulation. Bianchi et al. [121] found decreased LS and TBBMD in ambulatory boys with Duchenne muscular dystrophy when compared to healthy boys. LS BMD was more severely reduced in the subset of Duchenne muscular dystrophy patients treated with CS. The authors noted greater lower extremity than upper extremity bone mineral loss using regional DXA analysis, especially in the CS-treated patients, and they attributed this to reduced mechanical stress on the lower extremities. Hawker et al. [122] found an increase in LS and TBBMD after 2 years of bisphosphonate treatment, supplemental calcium, and vitamin D. Improvement in TBBMD was inversely related to age at baseline. In summary, patients with Duchenne muscular dystrophy have reduced LS BMD, especially in the setting of CS treatment. The low LS and hip BMD worsen with loss of ambulatory status and are frequently associated with fractures of the lower extremities. Studies of a small number of children with dermatomyositis have found low LS BMD that worsens with ongoing CS therapy [123, 124]. Patients treated with bisphosphonates for osteoporosis-related compression fractures show increases in LS BMD [123]. Osteogenesis imperfecta Osteogenesis imperfecta includes a spectrum of genetic disorders of collagen synthesis resulting in abnormal skeletal and connective tissues. Reduced BMD and other factors result in fragile bones and multiple fractures. Fracture occurrence and DXA findings in these children vary with subtype with few fractures and normal or near-normal DXA findings in type I osteogenesis imperfecta and more frequent fractures and marked reductions in BMD in types III and IV [125, 126]. (Type II typically results in perinatal demise.) Since as many as 40% of patients with osteogenesis imperfecta will have normal BMD and BMC, a normal DXA study does not preclude this diagnosis or distinguish these cases from non-accidental injuries [125]. In recent years, clinical trials using bisphosphonates in osteogenesis imperfecta have yielded impressive results with multiple studies demonstrating increases in LS, hip and TBBMD and improved quality of life [127–130]. Patients with the lowest baseline bone mass experienced the most significant gains with treatment. Improved mobility, ambulation, muscle force and reduced chronic pain and fatigue lead to improved quality of life, and the stimulus of physical activity is known to be beneficial to bone. Increased bone mass in the skull following bisphosphonate therapy in these patients implies that the drug also has a direct effect on bone accrual irrespective of physical activity [129]. The theoretical concern that this therapy might negatively impact linear growth in these children has not been shown to be true [131].

Naturwissenschaften-3-1-2039849

Why do house-hunting ants recruit in both directions?

To perform tasks, organisms often use multiple procedures. Explaining the breadth of such behavioural repertoires is not always straightforward. During house hunting, colonies of Temnothorax albipennis ants use a range of behaviours to organise their emigrations. In particular, the ants use tandem running to recruit naïve ants to potential nest sites. Initially, they use forward tandem runs (FTRs) in which one leader takes a single follower along the route from the old nest to the new one. Later, they use reverse tandem runs (RTRs) in the opposite direction. Tandem runs are used to teach active ants the route between the nests, so that they can be involved quickly in nest evaluation and subsequent recruitment. When a quorum of decision-makers at the new nest is reached, they switch to carrying nestmates. This is three times faster than tandem running. As a rule, having more FTRs early should thus mean faster emigrations, thereby reducing the colony’s vulnerability. So why do ants use RTRs, which are both slow and late? It would seem quicker and simpler for the ants to use more FTRs (and higher quorums) to have enough knowledgeable ants to do all the carrying. In this study, we present the first testable theoretical explanation for the role of RTRs. We set out to find the theoretically fastest emigration strategy for a set of emigration conditions. We conclude that RTRs can have a positive effect on emigration speed if FTRs are limited. In these cases, low quorums together with lots of reverse tandem running give the fastest emigration. Introduction Organisms often employ more than one mechanism to accomplish a task. For instance, animals typically navigate with multiple ‘input channels’. The classic example is homing by the rock dove Columba livia, for which magnetic fields, the sun, landmarks and geophysical processes have all been shown to be used (Wiltschko and Wiltschko 2003). The range of behaviours or mechanisms organisms employ may be puzzling. At times, an apparent simplicity is observed. Defence strategies against predators are a well-studied example here. Acacia trees (Acacia spp.) either have chemical defences to ward off herbivores or have symbiotic relationships with protective ants (Rehr et al. 1973). Hosts parasitised by Eurasian cuckoos Cuculus canorus famously reject cuckoo eggs, but never reject cuckoo chicks (Davies 2000). This simplicity may be the result of evolutionary lag but, more interestingly, may also be caused by one strategy making another maladaptive by reducing predator abundance (Planqué et al. 2002; Britton et al. 2007). Conversely, the portfolio of behaviours may be varied and complex. Different mechanisms may complement one another, and true redundancy is often hard to show (Able and Bingman 1987). Indeed, the existence of a suite of mechanisms against a broad ensemble of predators is readily understandable. One exemplar is the vertebrate immune system (Perelson 2002). Another striking example of a system in which different mechanisms augment and complement one another, but now at a collective level, is house hunting in social insects. This has become one of the model systems to study distributed decision making in animals. When the nest is destroyed, the colony has to decide collectively where to settle next during a time of crisis (Franks et al. 2003a). Individual ants or bees have been shown to combine sophisticated assessments of potential nest sites (Seeley 1977; Seeley and Morse 1978; Mallon and Franks 2000; Franks et al. 2003b) with various recruitment mechanisms to collate information, and thus make collective decisions (Mallon et al. 2001; Pratt et al. 2002; Pratt 2005; Seeley and Visscher 2003, 2004; Visscher 2007). A typical emigration by a colony of Temnothorax albipennis may be described as follows. When the old nest is destroyed, a fraction of ants goes out scouting to find a new home. Upon finding a nest, the nest is assessed (Mallon and Franks 2000) and ants start recruiting other ants to it with a latency that is inversely proportional to the perceived nest quality (Mallon et al. 2001), using a process called forward tandem running (Möglich et al. 1974). During a forward tandem run, a knowledgeable ant teams up with a naïve ant. The leader slowly progresses towards the new nest, each time waiting for the follower to catch up, thereby teaching her the way (Franks and Richardson 2006). Through this slow recruitment process, information on the location of the new nest spreads, and recruiter numbers increase. Once a nest population reaches a certain quorum threshold, the recruiters switch from slow tandem running to much faster social carrying, and transport the remaining passive ants and brood to the new nests (Pratt et al. 2002; Pratt 2005). This description has been the basis of several models (Pratt et al. 2002; Pratt et al. 2005; Marshall et al. 2006; Planqué et al. 2006). However, a behaviour commonly employed by these ants is usually not included (but see Pratt et al. 2005) and has never been analysed. After the quorum, recruiter ants are not only engaged in social carrying, but also regularly perform tandem runs from the new back to the old nest. These so-called reverse tandem runs (Möglich 1978) are often more common than forward tandem runs (Mallon et al. 2001; Pratt et al. 2002), but their function is much less well understood. To maximise fitness, the colony should emigrate as quickly as possible to avoid predation and other hazards. Therefore, during house hunting, a fast build up of recruiters is essential. Why then do ants mix fast carrying with slow reverse tandem running, when they already have forward tandem running at their disposal? In this paper, the role of reverse tandem running is theoretically investigated. In particular, through the use of mathematical models, we explore under what circumstances reverse tandem running can have a positive influence on emigration speed. Materials and methods We present two mathematical models to investigate the possible role of reverse tandem runs in ant colony emigrations. Reverse tandem running does not contribute to the decision-making process of which new nest to choose (Pratt et al. 2002; Franks et al. 2003a). We, therefore, restrict ourselves to emigrations to one new nest only. We model the emigration as follows (Fig. 1). Only a small fraction of the ants in a colony are actively involved during emigrations (Pratt et al. 2002; Langridge 2006). In this paper, we thus divide the colony’s N ants into fractions FN of active ants A, and (1 − F)N passive ants P. This assumption to divide ants into active and passive is a crucial one, without which the models collapse. We will come back to this in “Discussion”. Numbers of scouts and recruiters are denoted S and R, respectively. The rates at which active ants leave the nest and become scouts, and scouts recruiters, are given by μ and k, respectively. Forward tandem running occurs at a rate λ until the quorum Q is met, after which recruiters carry passive ants and brood at a rate φ. To incorporate reverse tandem runs, we need to model which ants follow these tandem runs. Available data from nest-choice experiments (Mallon et al. 2001) suggest two possibilities: Either the reverse tandem runs are followed by ants that have not found the new nest yet, or by ants that have been carried to the new nest. These two options are not necessarily mutually exclusive: the carried ants could have been scouts. We modelled both these two possible interpretations. Model 1 assumes that reverse tandem runs are followed by uncommitted scouts in the arena, and model 2 assumes they are followed by passive ants that were carried to the new nest. Fig. 1Diagrams of the two different models for which reverse tandem runs are hypothesised to increase speed. FTR Recruitment through forward tandem running; RTR recruitment through reverse tandem running. The parameters are explained in Table 1 To capture in detail the influence of reverse tandem runs on emigration dynamics, we need to consider the following points: Both tandem running and social carrying involve a pair of ants from two different classes. Hence, recruitment can only occur if ants of both participating classes are available;Once the quorum has been met, recruiters cannot carry and perform reverse tandem runs simultaneously (we also assume recruiters are not involved in other activities than these two). The interaction between different classes of ants has been modelled using simple interaction terms. We assume that ants of both classes are well mixed in the part of the arena (or nest) where they meet. With populations of ants of size X and Y meeting, the number of ants that on average meet is then proportional to XY/(X + Y). Importantly, the smallest class limits the interaction rate, as is to be expected. We thus also have to specify how much post-quorum time recruiters spend on carrying or reverse tandem running (they are assumed not to spend any time on other behaviours). Before the quorum is met, the rate at which active ants at the old nest, A, become recruiters, R, through tandem running is given by λRA/(A + R). Now let f be the fraction of post-quorum time spent on reverse tandem runs, and the remainder 1 − f spent on social carrying. Then the mean number of scouts becoming recruiters through reverse tandem runs is For carried ants becoming recruiters through reverse tandem running, we have by analogy Similarly, the mean number of passive ants P that become carried ants is given by (1 − f) φRP/(P + R). Recruiter ants should not perform reverse tandem runs when there are no scouts or carried ants left. Therefore, we replace f by in Eqs. 1 and 2, where and min {Cf, f}, respectively. The min operation is for computational reasons only and ensures that decreases continuously but rapidly to zero as S or C decreases, respectively. It has no influence on the models’ predictions. We drop the bar on in the rest of the paper. Forward tandem running only occurs before the quorum is met and carrying and reverse tandem running only after. These are modelled with functions l, c and r, respectively, as follows. and, setting B to A for model 1, and to P for model 2, To aid the reader, we state the full equations for both models. The equations for model 1 are given by with initial conditions (A,S,R,P,C)(0) = (FN − 2ε, ε,ε,(1 − F)N,0). Model 2 is specified by with the same initial conditions as model 1. Models 1 and 2 only differ in the placement of the term fr(λ,R,Q,C). In both models, the ε in the initial conditions is necessary to avoid singularities in the denominators of the interaction functions l, r and c. We have used ε = 0.01 throughout. We also explored a number of other models in which some assumptions were relaxed. These are briefly discussed in the final section of this paper. The main hypothesis we tested on both these models was: Reverse tandem runs speed up the emigration if recruiter numbers failed to increase early in the emigration; this occurs through a combination of the new nest being hard to find and forward tandem running being prohibited. We tested this hypothesis by finding the fastest emigration strategy for given parameter settings and determining whether reverse tandem runs formed part of this optimal strategy. We first fixed the scouting parameter, μ, the fraction of active ants at the old nest, F, and k, the rate at which scouts become recruiters. Then quorum size Q and fraction of post-quorum reverse tandem running time f were varied to optimise emigration speed. Individual runs were performed in Matlab using a standard ode45 solver. Emigrations were termed completed when all passive ants and active ants at the old nest had disappeared. In particular, we set the threshold for emigration completeness at P + A = 0.01. Optimal strategies were found using the Nelder–Mead simplex method (Nelder and Mead 1965). Parameters μ and F were varied on an equidistant 20×20 grid spanning [0.01,0.2] ×[0.05,0.5]. Recruitment latency k was set at either 0.0001 or 0.001. The ranges of these parameters are inspired by experimental estimates in (Pratt et al. 2002; Pratt 2005). Parameter values are summerised in Table 1. Table 1Values or ranges, where applicable, for the parameters used in models 1 and 2 depicted in Fig. 1ParametersDescriptionValue/rangeNColony size250FFraction of active ants[0.05,0.5]QQuorum thresholdn.a.fFraction of post-quorum reverse tandem running timen.a.μRate at which active ants at old nest become scouts (ant − 1 min − 1)[0.01,0.2]λRate at which ants following tandem runs become recruiters (ant − 1 min − 1)0.1φRate at which passive ants are carried to new nest (ant − 1 min − 1)0.2kRate at which scouts independently become recruiters (ant − 1 min − 1){0.0001,0.001}Parameter choices for λ, φ and N were taken from the ranges in Pratt et al. (2005). Results For both models, the optimal emigration strategy included reverse tandem runs for a wide range of parameters, together with low quorum thresholds (Fig. 2). Fixing k whilst varying μ and F, the optimal strategy often contained more reverse than forward tandem runs for a large part of the parameter range (Fig. 2). The fraction of time spent on reverse tandem running f and the quorum threshold Q were negatively correlated. When either the fraction of active ants F decreased or the scouting parameter μ increased, fraction f increased, and the quorum Q decreased. Choosing a higher recruitment latency by decreasing k gave more reverse tandem running and lower quorum thresholds (Fig. 2). Fig. 2Optimal fractions of post-quorum time spent on reverse tandem runs (top figures), and optimal quorum thresholds (bottom figures) for models 1 (left two columns) and 2 (right two columns) for varying fractions of active ants F and scouting probabilities μ and for two values of recruitment latencies k. Other parameter values used are given in Table 1. See text for simulation details. For both values of k, reverse tandem runs are part of the optimal emigration strategy when scouting probability is high or when fraction of active ants is low. Lowering k enhances the use of reverse tandem running Note that, although models 1 and 2 broadly give similar predictions, they differ in the amount of post-quorum time spent on reverse tandem runs. In model 1, this reaches a full 100% in model 1, but never so in model 2. Overall, the models predict that reverse tandem running should be used more than forward tandem running, and the quorum threshold lowered, if the recruitment latency increases by decreasing k (scouting ants wait longer before starting their first recruitment act), in combination with either a decreasing fraction of active ants F, or an increasing scouting parameter μ. For all but very large F, the optimal quorum threshold corresponded to the time when all active ants have left the old nest to go scouting. In the absence of multiple new nests, the decision when to switch from forward tandem running to social carrying is thus best made at the old nest. Recruiters should thus apply the following rule: Continue forward tandem running until there are no ants left to perform them with and then switch to social carrying; if few forward tandem runs have been performed (by the recruiters), combine carrying with reverse tandem runs; otherwise, do not. The numbers of forward tandem runs, reverse tandem runs, the numbers of carried ants in the new nest and total emigration time were computed for each of the optimal strategies of models 1 and 2 (Fig. 3). We note four points. First, for a large parameter range, there are more reverse tandem runs than forward tandem runs. This is broadly consistent with the experimental data from nest choice experiments in Mallon et al. (2001). Numbers there range between 3 and 17 forward tandems, and between 9 and 25 reverse tandems, and reverse tandems were always performed more often than forward tandems. Second, as a validation of our optimisation method, note that the optimal emigration time varies smoothly under parameter changes, as is to be expected for this type of model. Third, for model 1, despite a clear drop in the post-quorum time spent on reverse tandem runs with increasing F (see Fig. 2, top left), the number of reverse tandem runs in fact varies smoothly. Fourth, the number of carried ants that remained in the nest at the end of the emigration is clearly different between models 1 and 2. In model 1, this number is just (1 − F)N, the number of passive ants in the colony. In model 2, however, over half of the colony may end up being recruiters by drawing recruits from the carried class using reverse tandem runs. Fig. 3Numbers of forward and reverse tandem runs, number of carried ants and emigration time, computed for each of the optimal strategies for models 1 (top row) and 2 (bottom row), illustrated in Fig. 2. See that figure for details and parameter choices. Here we have only illustrated k = 0.001 Discussion To maximise their fitness, ants should try to achieve the fastest emigrations to minimise vulnerability (Franks et al. 2003a; Franks et al. 2003b). Therefore, the active ants either have to become scouts, discover a new site and then become recruiters or wait at the old nest until a recruiter leads them to the new nest. Both of these processes may be hampered: When all the active ants go out scouting, recruiter numbers slowly increase if the new nest is hard to find or if those few cannot find any active ant back at the old nest to tandem run with. In terms of the models, this could occur if scouts slowly become recruiters (low value for k), in combination with either a small class of active ants at the old nest (F is small) or all active ants having gone scouting (high value for scouting rate μ). Under either or both of these circumstances, the model predicts that ants should not waste time trying to recruit by forward tandem runs but should do the next best thing and use a low quorum threshold to quickly switch to carrying. The recruiters should then invest a fraction of their time to recruit scouts or carried ants using reverse tandem runs, thus boosting the recruiter population and speeding up the emigration. Conversely, the model also predicts that reverse tandem runs should not be used if either the new nest is easy to find (recruiter numbers then build quickly anyway), or when there are many ants to follow a forward tandem run. These predictions fit quite well with previous experimental work. Ants have been shown to leave their intact old nest if the new nest is sufficiently better, but have lower standards when their nest is destroyed (Dornhaus et al. 2004). In these experiments, reverse tandem runs were mainly observed when the old nest was destroyed, combined with few forward tandem runs. The model offers a simple explanation for this: The greater panic might have caused fewer scouts to remain at the old nest, thereby obstructing early recruitment. Whilst investigating speed–accuracy trade-offs, Franks et al. (2003a) found lower quorums under harsh than under mild conditions. This again fits with the models. On the other hand, the models also predict higher numbers of reverse tandem runs. In the experiments, this difference in reverse tandem running activity between mild and harsher conditions was found to be non-significant (Franks et al. 2003a). Critique on model 2 Although evidence for which ants follow reverse tandem runs is scarce, both models give the same qualitative predictions. Note, however, that the dynamics of the different ant classes during a simulated emigration in model 2 poorly match observed experimental dynamics (see, e.g., Planqué et al. 2006). In many cases, the final number of ants active in an emigration is much greater than the original number of active ants. An example is given in Fig. 4. These recruiter numbers far exceed observed numbers of active ants (available in Table 3 in Pratt et al. 2005). Indeed, simulated emigrations often end with half the colony being recruiters and less than half carried into the new nest (Fig. 3, bottom row, third from left). Fig. 4Examples of temporal dynamics for models 1 and 2. At μ = 0.05, F = 0.1447, we have taken parameters optimal for models 1 and 2, respectively. Notice that, in model 1, the number of recruiters rises to about 35, but in model 2, there are no less than 100 recruiters at the end of the emigration, indicating that recruitment from the carried class (model 2) may give rise to very unrealistic emigration dynamics Reverse tandem activity Figure 2 (top left) shows that recruiters in model 1 should use a sequential strategy if F is small (and μ is large): When the quorum is met, they first spend all of their time on reverse tandem runs until all scouts have become recruiters, and then switch to carrying. In contrast, when F is large, recruiters mix tandem running and carrying. This qualitative difference may be understood as follows. The total number of recruiters is bounded by FN, the number of active ants in the colony. As F decreases, the remaining recruiters take longer to carry all the passive ants. Hence, the time costs for not having the recruiters increases and the time to recruit the remaining scouts decreases (as there are fewer scouts too). Hence, in this situation, recruiters should devote their post-quorum time, first, all on reverse tandem running before starting carrying. When F is large, the reverse argument applies. With less passive ants, there should be less emphasis on additional recruitment by reverse tandem running. One does not have to make many hands if the work was light to start with. Note that this behaviour for F →0 is different for model 2. In this paper, we never observe sequential strategies (Fig. 2, top right), as there is no end to building recruiter numbers but by completing the entire emigration. This argument also explains another difference between these models: The number of reverse tandem runs during an emigration. In model 1, there is a clear maximum for intermediate F, whereas in model 2 the number of reverse tandems strictly decreases with F (Fig. 3, second from left, top and bottom). Nonlinearities in the models and divisions between active and passive ants Contrary to the models in this paper, two previous models of house hunting by T. albipennis ants (Pratt et al. 2002; Planqué et al. 2006) assumed linear terms for tandem running and social carrying. There, these processes occurred at rates only proportional to the number of recruiters. The predictions of the current models proved to be strongly dependent on the assumption of non-linearity of these terms. The corresponding linear models predicted that reverse tandem running should not be used for practically any parameter choices in F and μ or k. Another ingredient in this model shared with the previous (Pratt et al. 2002; Planqué et al. 2006), models of this collective decision-making system is the division between active and passive ants. There is as yet little experimental evidence suggesting this division really exists. All we know at present is that a limited fraction of ants is actively engaged during an emigration. We have thus also explored models in which this division was absent, using both linear and non-linear interaction terms such as those in models 1 and 2 presented in this paper. In none of these models did reverse tandem running contribute to the optimal emigration speed. The division between active and passive ants is thus a crucial ingredient for reverse tandem running to have a positive impact on emigration speed, which should be experimentally validated. Hypothesised explanations Several hypotheses on the potential role of reverse tandem running have been put forward (Pratt et al. 2002). First, the ants might have a “home nest”, which changes during the emigration, thereby reversing the direction of any recruitment events from “home” to another nest (Pratt et al. 2002). If true, this would predict a change of direction when about half of the colony had been displaced. This is not in agreement with the available data (Mallon et al. 2001). Moreover, this hypothesis does not offer a suggestion why tandem running often occurs early in the emigration. In other words, it might explain the direction, but not the occurrence itself. Second, it has been suggested that reverse tandem running may re-allocate recruitment (Pratt et al. 2002). Again, this does not fit the available data from Mallon et al. (2001). Reverse tandem runs were nearly always observed between the best nest and the old nest. The models in this paper do not incorporate choice between nest sites, but we conjecture that early flexible commitment (Planqué et al. 2006) will be more efficient in redirecting ants to better nests than late recruitment. Other experimental results also corroborate that reverse tandem running does not influence the decision-making process (Franks et al. 2003a). The first models in which reverse tandem runs have been explicitly incorporated to analyse their role have yielded clear predictions: under a range of conditions, we expect a negative correlation between levels of early and late recruitment. This finding lends itself well to simple experiments, and we aim to present those in the near future. The build up of recruiter numbers serves two purposes: to decide on a nest and to increase the number of ants actively involved in transport. The decision-making process and the implementation of this decision are thus conflated. This in itself is a side-effect of the distributed nature of this system. Reverse tandem running may thus be a logical extension to overcome this inherent problem. This suggests that such additional backup behaviours could be a common feature of decentralised collective decision-making systems.

Matern_Child_Health_J-2-2-1592152

The History of Preconception Care: Evolving Guidelines and Standards

This article explores the history of the preconception movement in the United States and the current status of professional practice guidelines and standards. Professionals with varying backgrounds (nurses, nurse practitioners, family practice physicians, pediatricians, nurse midwives, obstetricians/gynecologists) are in a position to provide preconception health services; standards and guidelines for numerous professional organizations, therefore, are explored. The professional nursing organization with the most highly developed preconception health standards is the American Academy of Nurse Midwives (ACNM); for physicians, it is the American College of Obstetricians and Gynecologists (ACOG). These guidelines and standards are discussed in detail. Introduction From the earliest recorded time, women have been advised to increase their level of wellness and to avoid hazardous substances before becoming pregnant. Plutarch wrote that the ancient Spartans: “...ordered the maidens to exercise themselves with wrestling, running, throwing the quoit and casting the dart, to the end that the fruit they conceived might, in strong and healthy bodies, take firmer root and find better growth” [1]. In the Old Testament [2], the following passage appears: “And the angel of the Lord appeared unto the woman, and said unto her, ‘Behold now thou art barren, and bearest not: but thou shalt conceive and bear a son. Now, therefore beware, I pray thee, and drink not wine and strong drink, and eat not any unclean thing.’ ” As the modern practice of obstetrics evolved, it became a specialty that separated women’s prepregnancy wellness from prenatal care, but in the last quarter century this has begun to change. The dominant model of prenatal care as the main perinatal prevention strategy has been reexamined, and recognition of the importance of the prepregnancy period has emerged. Today the general public and health care professionals could easily be overwhelmed by the amount of information available which promotes prepregnancy or preconceptional health. A Google search (conducted April 25, 2005) of the term “preconception health” identified 630,000 hits. On the same day a search of the National Library of Medicine’s PubMed found 338 entries for “preconception health” with the earliest published in 1978, and 103 articles for “preconceptional” with the earliest published in 1982; in addition, most of the articles identified through the two search terms were unique. The nation’s approach to women’s health care may well be at the tipping point of redefining the perinatal period to include women’s wellness across the reproductive life span as an appropriate and favored approach to impacting reducing poor pregnancy outcomes. Stimulated by research findings which underscored the limitations of traditional prenatal care on decreasing the incidence of congenital anomalies, leaders in the health community, professional organizations, advocacy groups and federal government began actively encouraging redefinition of the United States’ perinatal prevention paradigm in the early 1980s. Because the usual pathways to impact on perinatal outcomes (prenatal and neonatal care) often start too late to achieve primary prevention, the need to reach women with prevention opportunities before prenatal care was recognized and the concept of preconceptional health promotion emerged. This article explores the history of the preconception movement in the United States and the current status of professional practice guidelines and standards. The decade of the eighties One of the first federal position papers to acknowledge the need to change the nation’s approach to prevention appeared in 1979; while focused on the need to develop a continuum of child health care, it included the concept of prepregnancy care [3]. The programmatic elements in the proposed comprehensive package of integrated services were identified as:Interconceptional carePrenatal carePerinatal careChild Health careServices for handicapped childrenAdolescent services Interest in moving away from categorical care to more integrated and comprehensive services has subsequently become a hallmark of current efforts to redefine the perinatal prevention paradigm. Why the list employed the word “interconceptional” rather than preconceptional is not clear. Since 1979, “interconceptional” care has come to represent efforts to address health status between pregnancies, birth spacing and intendedness of subsequent conceptions; “preconceptional” generally refers to the woman’s health status and risks before the first pregnancy and her health status shortly before any conception. “Periconceptional” usually refers to the time immediately before conception through the period of organogenesis. It is important to note that this nomenclature is inconsistently applied and not universally accepted. In England, for instance, “preconceptional” is referred to as “preconceptual.” By 1985, the concept of preconceptional care and its potential advantages began to gain momentum. In that year, the Institute of Medicine published Preventing Low Birthweight [4] and noted that numerous opportunities exist before pregnancy to reduce the incidence of low birth weight, but that they are too often overlooked in favor of interventions during pregnancy. The Committee emphasized the importance of prepregnancy risk identification, counseling and risk reduction; health education related to pregnancy outcome in general, and to low birthweight, in particular. The Committee supported restructuring the perinatal prevention paradigm by noting:“Much of the literature about preventing low birthweight focuses on the period of pregnancy—how to improve the content of prenatal care, how to motivate women to reduce risky habits while pregnant, how to encourage women to seek out and remain in prenatal care. By contrast, little attention is given to opportunities for prevention before pregnancy. Only casual attention has been given to the proposition that one of the best protections available against low birthweight and other poor pregnancy outcomes is to have a woman actively plan for pregnancy, enter pregnancy in good health with as few risk factors as possibly, and be fully informed about her reproductive and general health.” (p. 119) The IOM Committee advocated that family planning services be positioned as an essential component of effective preconceptional initiatives, thereby supporting the integration of services rather than reinforcing firm boundaries of categorical programs. It also recommended that the content of reproductive health education, particularly in schools and family planning settings, be expanded to introduce concepts of prepregnancy wellness. The third recommendation of the Committee was to develop the notion of preconceptional consultation to identify and reduce risks associated with poor pregnancy outcomes, particularly for women who had already experienced a poor outcome. In 1989 another federally appointed committee, The Expert Panel on the Content of Prenatal Care, gave a strong endorsement to preconceptional health when it recommended that the preconception visit may be the single most important health care visit when viewed in the context of its effect on pregnancy [5]. The Panel went on to suggest that the last family planning visit should be the preconception visit and that the concept of preconception care should become a routine part of prenatal care with accompanying reimbursement and coverage included in all health insurance plans. However, the Panel indicated that preconception care is likely to be most effective when services are provided as part of general preventive care or during primary care visits for medical conditions. This approach has come to be referred to as “opportunistic care” because it takes advantage of the opportunities afforded by visits for other reasons. Specific components of preconception care were identified by the Panel as (1) risk assessment, (2) health promotion and (3) intervention and follow-up. The Panel advocated that the preconception assessment include a holistic range of investigations including: individual and social conditions, adverse health behaviors, medical, psychological and environmental conditions and barriers to family planning and early prenatal care enrollment. In 1983, the American Academy of Pediatrics (AAP) and the American College of Obstetrics and Gynecologists (ACOG) in partnership with the March of Dimes Birth Defects Foundation published the first Guidelines for Perinatal Care [6]. Appendix C of those first guidelines notes that “Preparation for parenthood should begin prior to conception. At the time of conception the couple should be in optimal physical health and emotionally prepared for parenthood” (p. 257). The Guidelines indicated that when pregnancy is contemplated, the preconception visit should be part of a comprehensive gynecologic examination, and the history should include a detailed family history including ethnic background as well as investigation of lifestyle issues, religion, the home and work environments, hobbies, pets, immunizations, medications and dietary habits. The guidelines recommended use of an extensive checklist to systematically explore areas of potential significance. Based on the patient’s history, the “counseling and instruction session” could be focused in a variety of directions including testing for genetically transmitted diseases and the parent’s carrier status, as well as counseling regarding potential teratogenic exposures, exploration and interventions to address medical problems prior to pregnancy, interconceptional care to address the likelihood of recurrence of any complications experienced in previous pregnancies, including congenital anomalies, and the importance of recording menstrual dates to allow the earliest possible initiation of prenatal care. In 1985, ACOG provided a small grant to Moos and Cefalo to develop and test a checklist which came to be known as the preconceptional health appraisal [7, 8]. The appraisal was designed to aid providers in conducting comprehensive screening for preconceptional risks in a time-efficient manner and employed a self-assessment tool with built-in educational feedback. First introduced into local health department family planning clinics in North Carolina, it was adopted over the next several years by numerous state health departments across the country as well as private providers, large health maintenance organizations and several Canadian provinces. The commitment of public health organizations to introduce women to the concepts of preconceptional health during routine family planning visits placed the public sector in the position of being a leading innovator in the preconception movement. One state that demonstrated particular commitment to changing the perinatal prevention paradigm was Wisconsin. By the mid-1980’s, the Wisconsin Association for Perinatal Care was calling for redefinition of perinatal care to include the preconceptional period [9], and it has been unwavering in its commitment to improve pregnancy outcomes through preconceptional health promotion strategies ever since. Concurrent with the above noted activities, additional credence to the benefits of preconceptional health promotion and primary prevention was provided when two nationally recognized obstetrician-gynecologists published books on the topic, one for the professional community and one for the consumer market [7, 10]. The decade of the nineties Healthy People 2000, the national health promotion and disease prevention objectives for the United States, published in 1990, moved preconceptional care into a standard expectation within the health care system when it defined it as a one of its service and protection objectives. The specific objective reads:Increase to at least 60% the proportion of primary care providers who provide age-appropriate preconception care and counseling (p. 199) Supporting rationale for the objective states that “the purpose of preconception care and counseling is to ensure that couples are healthy prior to pregnancy and prepared to assume the responsibilities of parenthood, thereby reducing the risk of poor pregnancy outcomes” (p. 199). The remainder of the rationale not only echoes the position of the earlier 1989 Expert Panel on the Content of Prenatal Care regarding the opportunities of preconception care to address many medical conditions, personal behaviors and environmental conditions [5] but also stresses preconceptional health promotion as an opportunity to educate young people about the risks of sexual activity, including unintended pregnancies, disease-related infertility and cancers [11]. In 1993, the March of Dimes Birth Defects Foundation published Toward Improving the Outcome of Pregnancy: The 90s and Beyond which introduced the concept of “reproductive awareness” as the basic health promotion strategy needed to improve pregnancy outcomes [12]. The document, often referred to as TIOP, reflects the position of the March of Dimes’ Committee on Perinatal Health. Because awareness of reproductive risks, healthy behaviors and family planning options is essential to improving the outcome of pregnancy, the committee declared that “a society-wide change in reproductive awareness is needed in the United States” (p. 12) and called for “a new strategy to reach each woman of childbearing age with reproductive awareness messages at every health encounter”(p. 12). The committee called upon health care providers to move away from the expectation that the prevention of poor pregnancy outcomes is only the professional responsibility of obstetrical providers by noting:“A woman’s health status in relationship to pregnancy usually is not considered until the first prenatal visit, and prenatal care often is segregated from other health care by provider type and by payment mechanism. Yet women of childbearing age have many encounters with the health care system. These include visits to gynecologists, pediatricians, internists, family physicians, nurse midwives and nurse practitioners, in settings such as public health, school health, women’s health, substance abuse treatment, family planning, and sexually transmitted disease clinics and private offices (p. 13).” TIOP goes on to call for professional standards, structures and financing to be set in place to ensure an annual preconception or interconception risk reduction visit for every woman from menarche to menopause and that a prepregnancy planning visit become a standard component of maternity care, routinely available to all women and integrated into the perinatal care delivery system. TIOP also stressed that reproductive awareness is not sex or gender specific and that the need to reach out to males, especially during the preconception and interconception periods, requires thoughtful and innovative strategies. ACOG published its first technical bulletin on preconception care in May, 1995 [13] The bulletin called for thorough and systematic identification of risks, the provision of education individualized to the patient’s needs, and the initiation of any desired interventions. Risk identification was broadly defined to include the areas of medical, reproductive, and family histories, nutritional habits, drug and environmental exposures and social issues and these efforts were to be targeted at improving outcomes for both the mother and the fetus ACOG took the position that rather than ensuring healthy pregnancy outcomes as suggested by other position papers, preconception care allows the woman and her partner, if so desired, to make informed-decisions. It states:“Once information is available, the patient can be informed about the certainty or limitations of available information, what the risks of pregnancy may be, and precautions that may be beneficial. After women have been informed of the increased risks pregnancy may pose to their health or the health of a fetus or both, they can accept the increased risks, choose to modify their risks or opt to avoid childbearing (p. 2).” ACOG cautioned against over promising the benefits of preconceptional care to both providers and patients by specifically noting that preconceptional services do not guarantee good pregnancy outcomes. It noted that placing emphasis only on women who are planning their next conception or women who have been identified as high-risk will result in a significant number of missed opportunities for primary prevention. Women who experience an unintended pregnancy are at least as likely to have risk factors for poor pregnancy outcomes as women who consciously plan the timing of their pregnancy. Therefore, ACOG recommended that routine visits by women who may, at some time, become pregnant are important opportunities to emphasize the importance of preconceptional health and habits and the advantages of planned pregnancies. Finally, ACOG called for a coordinated multispecialty effort directed by the obstetrician-gynecologist as a means to provide a comprehensive framework for preconceptional health care for all women of childbearing potential. The current decade In 2002, the fifth edition of the AAP/ACOG Guidelines for Perinatal Care was published [14]. In the preceding editions, preconception care had moved from an appendix item to the main text in preceding editions. This latest edition, however, reflects a shift away from framing preconceptional care as appropriately targeted toward prospective parents who are contemplating pregnancy to an emphasis on integration of preconceptional health promotion into all health encounters during a woman’s reproductive years. While some efforts to operationalize a redefined perinatal prevention paradigm grew, others dropped back. For example, Healthy People 2010 eliminated preconceptional health as a specific objective [15]. Except for the objective, “Increase the proportion of pregnancies begun with an optimum folic acid level” (target: 80%), the relevance of a woman’s health status at the time of conception to pregnancy outcomes is not referenced in the document, and reliance on prenatal and neonatal care to impact on pregnancy outcomes is reinforced as the preferred strategy to meet the perinatal objectives. Evolving standards and guidelines from professional organizations As the concept of preconception care has evolved in the health care community, several professional organizations have addressed its importance for their members, and some have developed specific guidelines or standards for preconception care. Nursing organizations In nursing, there are several professional organizations whose members care for reproductive age women. The AANP (American Academy of Nurse Practitioners) is the largest national professional organization for nurse practitioners of all specialties, with 15,000 members. While many nurse practitioners might be delivering preconception health services, AANP has not developed specific educational standards specific to this topical area, nor do their generic practice standards include preconception health. According to AANP Executive Director Dr. Judith Dempster (personal communication, April 11, 2005), members with questions about preconception health standards would be referred to specialty organizations such as AWHONN. AWHONN (Association of Women’s Health, Obstetric and Neonatal Nurses) is the official professional organization for women’s health and obstetric nurses, and is comprised of 22,000 nurses whose goals are to promote excellence in nursing practice. While AWHONN does not have specific standards of care for women during the preconception period, they do offer many products to their members which discuss preconception health, including a practice monograph which outlines the importance and components of preconception care [16]. They also publish a Clinical Position Statement which states that AWHONN supports legislation and policies that encourage women of childbearing age to consume 400 micrograms of synthetic folic acid every day [17]. In addition, AWHONN is also a member of the National Council on Folic Acid (NCFA), a partnership of over 80 national organizations and associations, state folic acid councils and government agencies. While not strictly a preconceptional project, AWHONN developed a specific Clinical Position Statement on Smoking and Childbearing, urging nurses to screen women for tobacco use and help women stop smoking [18]. Although nurses initiated this program during pregnancy, it had major interconceptional ramifications for the women involved, and has added to the literature on smoking cessation for women of reproductive age [19]. ACNM, the American College of Nurse Midwives is the professional organization for all nurse midwives in the United States, and represents over 7,000 nurse midwives who are providers of comprehensive women’s health. In their Core Competencies for Basic Midwifery Practice (www.acnm.org/prof/display.cfm?id=137) they have specific educational and practice standards for preconception health for women. These standards are:“V. Components of Midwifery Care: The Primary Health Care of WomenB. Applies knowledge of midwifery practice in the preconception period that includes, but is not limited to, the following:1. Assessment of individual and family readiness for pregnancy, including emotional, psychosocial and sexual factors2. Impact of health, family and genetic history on pregnancy outcomes3. Influence of environmental and occupational factors, health habits, and behavior on pregnancy planning4. Health and laboratory screening to evaluate the potential for a healthy pregnancy” (p. 3) NAPNAP (the National Association of Pediatric Nurse Practitioners) does not publish any guidelines or standards for preconception care. Their members provide services for adolescents, but the organization has not yet addressed preconception care in a structured manner (personal communication Dr. Karen Kelly Thomas, Executive Director NAPNAP, April 13, 2005). They have, however, published at least one article in their official journal on the topic [20]. In terms of general nursing education, the AACN (The American Association of Colleges of Nursing) does not offer specific guidelines for what is to be taught to student nurses about preconception health, but rather sets standards for overall educational goals. Organizations for all professionals The March of Dimes, while not an organization which targets just one type of professional, has been producing numerous materials for health care professionals about preconception health for many years. Materials and products that the March of Dimes produces for providers of care include: their nursing module Preconception Health Promotion: A Focus for Women’s Wellness [21], a Preconception Curriculum consisting of power point slides for Obstetricians, Family Medicine specialists, Pediatricians, and Internists which suggests that every visit to a health care provider should include elements of preconception care, a Preconception Screening and Counseling Tool, grants to the March of Dimes Chapters for preconception services, an educational program called Genetics and Your Practice (CD and online), and a Genetic screening pocket guide. Many of these can be found on their website www.marchofdimes.com, and their Spanish language website www.nacersano.org. They also produce an e-preconception newsletter (Spanish), an education program for Spanish women called Comenzando Bien, and pamphlets for patient education such as Are You Ready?, Think Ahead for a Healthy Baby, Folic Acid brochures, and Pre-Pregnancy Planning Fact Sheet. The CDC, the March of Dimes, and the National Council on Folic Acid (NCFA) have organized a national folic acid promotion effort for the prevention of serious birth defects of the brain and spine (neural tube defects or NTDs). The goal of the effort is to teach all women about the importance of getting enough folic acid every day. The effort aims to reach every woman who could possibly become pregnant, as well as teach health care professionals and community advocacy groups about the importance of folic acid. The CDC, the March of Dimes, and NCFA have created messages, materials, websites, and other tools to reach providers and women with the folic acid message. In 2005, the CDC and March of Dimes collaborated to develop a national summit on preconception care in order to bring together all the stakeholders in the subject; this supplement issue of Maternal Child Health is one of the many consequences of these efforts by CDC/MOD. Physician organizations The American Academy of Pediatrics represents 60,000 pediatricians, many of whom provide adolescent services to young women of reproductive age. They publish, along with the American College of Obstetricians and Gynecologists, Guidelines for Perinatal Care, the document which is generally accepted to be the outline for acceptable care during the perinatal period for women and infants. In the current edition, 5 pages are devoted to preconception care, which is described as “the identification of those conditions that could affect a future pregnancy or fetus, and that may be amenable to intervention (p. 73)” [14]. The Guidelines note that all encounters with women of reproductive age should include counseling about health which could optimize pregnancy outcome. AAP is also a member of the National Organization on Fetal Alcohol Syndrome, which addresses medical school curriculum for fetal alcohol syndrome, and AAP supports numerous state-wide programs to enhance adolescent health before childbearing. In the American Board of Pediatrics study guide for the certifying examining for Adolescent Medicine certification, materials included are prevention of pregnancy, prevention of substance use, and general reproductive health care, all of which contribute to better preconception health (www.aap.org/sections/adol/adol.pdf). The American Academy of Family Physicians (AAFP) represents 94,000 family physicians, residents and medical students in the United States, and sets the professional standards for care delivered by family physicians. While no specific guidelines or standards for preconception health services developed by AAFP could be located, AAFP addresses the issue of preconception care in many articles in their official journal, and in other practice venues such as suggestions to increase use of folic acid [22, 23]. CREOG is the Council on Resident Education in Obstetrics and Gynecology, and is charged with setting the educational standards for obstetricians and gynecologists in training. CREOG’s Educational Objectives are contained in its core curriculum [24]. Regarding preconception care, CREOG’s core curriculum states: “In evaluating patients for preconceptional care, obstetrician gynecologists must assess those factors of the history, physical examination, and diagnostic studies that pregnancy would alter; assess the patient’s access to and compliance with a plan of prenatal care; and consult with or refer her to other experts on specific conditions that may arise during the pregnancy.”(p. 39). Its standards are as follows:“ II. Antepartum Care—Preconceptional Care1. Perform a thorough history, assessing historical and ongoing risks that my affect future pregnancy2. Counsel a patient regarding the impact of pregnancy on maternal medical conditions.3. Counsel a patient regarding the impact of maternal medical conditions on pregnancy4. Counsel a patient regarding appropriate lifestyle modification conducive to favorable pregnancy outcome.5. Counsel a patient regarding appropriate preconception testing.6. Counsel a patient regarding pregnancy associated risks and conditions, such as advanced age, hypertension, diabetes, genetic disorder, prior aneuploid or anomalous fetus/newborn” (p. 49) ACOG, the American College of Obstetricians and Gynecologists represents 46,000 obstetricians and gynecologists in the United States, and in 2005 celebrated its 50th anniversary. ACOG has advocated for preconception care for several decades, and offers the most detailed guidelines for the provision of preconception services. ACOG’s acknowledges that while ideal health prior to pregnancy does not guarantee a perfect or uncomplicated pregnancy, women who are considering pregnancy should undergo comprehensive preconception evaluation, examination, and counseling. However, preconception care is best when it is provided as part of primary health care, rather than as a discrete new category of health care, particularly since almost one-half of all pregnancies in the United States are unintended [25, 26]. Components of preconception care—ACOG In examining multiple documents produced by ACOG in recent years, certain components of preconception care can be delineated. Periodic assessments Preconception counseling should occur at periodic assessments for all reproductively capable women. These assessments may be yearly or as appropriate [27]. Counseling should include discussions about the likelihood of pregnancy (planned or unplanned), counseling on appropriate medical care and behavior to optimize pregnancy outcomes, specific health risks the woman has that may predispose her to an adverse pregnancy outcome if not addressed, and how to either effectively prevent an unplanned pregnancy or to maximize her physical and emotional health prior to a planned or even unplanned event. Even clinicians that do not provide prenatal care should be able to provide preconception counseling and screening to their reproductively capable patients. The patient’s history should be reviewed and updated on an annual basis or more often if appropriate. The history should include a review of any medical conditions and medications the patient may be using. It is important to ask about prescription as well as over the counter drugs, herbs, and supplements. Inquiries about occupational and hobby exposures to chemicals, solvents, or heavy metals should be made. In this manner, potentially teratogenic agents may be identified. The patient’s reproductive history may provide important clues about future pregnancy risks. Recurrent miscarriages should raise suspicion of a possible genetic or chromosomal problem. The neonatal outcome of previous pregnancies should be noted but it is equally as important to ask women about the current health of any children they have as some congenital disorders do not become manifest until several months or even years after birth and may not be considered by the family to represent a congenital defect. Genetic disorders and genetic screening The woman’s family history and ethnicity for genetic disorders and malformations should be obtained. Carrier screening for Tay-Sachs disease is recommended for individuals of Eastern European (Ashkenazi) Jewish, French Canadian and Cajun descent [28]. ACOG recommends offering cystic fibrosis screening to individuals with a family history, reproductive partners of persons with cystic fibrosis, and to couples in whom one or both partners are Caucasian and are planning pregnancy [29]. Carrier screening for Tay-Sachs, Canavan disease, cystic fibrosis, and familial dysautonomia should be offered to Ashkenazi Jewish individuals during preconception screening. These same individuals may be offered or may request screening for Gaucher disease, Niemann-Pick disease type A, Fanconi anemia group C, Bloom syndrome, or mucolipidosis IV as well. Carrier screening should be offered to any individual with a positive family history of one of these disorders; the screening should be for the specific disorder. When only one partner is of Ashkenazi Jewish descent, that individual should be screened first. Except for Tay-Sachs and cystic fibrosis, the carrier frequency and detection rate for the other disorders is unknown. If an individual is found to be a carrier for Tay-Sachs or one of the other aforementioned diseases, their relatives are at risk for carrying the same mutation; carriers are encouraged to inform their relatives of the risk and availability of carrier screening [28]. Other screening tests exist for genetic disorders with an increased incidence in specific ethnic groups, e.g., alpha or beta thalassemia screening in Orientals, beta thalassemia screening for those of Mediterranean descent, and sickle cell disease screening for African-Americans. Women who have had a previous pregnancy complicated by autosomal trisomy, a sex chromosome aneuploidy, or if either partner has a chromosome translocation or inversion or aneuploidy should be given education about their increased risk of recurrence in future pregnancies and a referral for pre-pregnancy genetic counseling should be offered [30]. Immunizations and infectious diseases The woman’s immunization status, history of childhood diseases, and risk for exposure to hepatitis, HIV, or other sexually transmitted infections needs to be updated at least annually. When practical, preconceptional immunization of women to prevent disease in the offspring is preferred to vaccination of pregnant women with certain vaccines [31]. Approximately 30% of US adults have serologic evidence of prior exposure to toxoplasma gondii, a known teratogen. Screening for Toxoplasma gondii is controversial because evidence that treatment prevents congenital disease is lacking. All reproductively capable women should be advised to avoid eating undercooked or raw meat, wear gloves when working with soil, and practice safe-handling techniques when changing cat litter [32]. Congenital cytomegalovirus (CMV) infection occurs in 1% of all live births in the United States and causes major neonatal illness in 5% to 10% of these cases. Women with young children or those who work with young children may be counseled about reducing the risk of CMV through universal precautions, e.g. the use of latex gloves and rigorous hand-washing after handling diapers or after exposure to respiratory secretions. There are currently no other specific recommendations as there is no evidence that screening and/or treatment programs prevent infection [32, 33]. All women should be kept current with age-appropriate vaccines. Reproductive aged women should be asked about previous infection with varicella and offered vaccination if they report no known history of chickenpox. Conception should be delayed until 1 month after the second injection is given. Women who expect to be at least 3 months pregnant during the influenza season (November to April) should be vaccinated. Behavioral/psychosocial factors A woman’s lifestyle and social behaviors should be reviewed and updated frequently. The use of alcohol, smoking, or other substances may have significant effects on pregnancy. Smoking during pregnancy is associated with low birth weight, abruptio placenta, preeclampsia, and preterm labor [34]. A woman’s personal situation has a significant effect on her health and often changes over time. Clinicians should ask all women about intimate partner violence and women should be aware that it may escalate during pregnancy. In addition, a woman’s mental health history may provide clues about recurrence risks either during or after a pregnancy. A woman’s dietary habits should be reviewed. Daily folic acid intake of 0.4 mg should begin at least 1 month before pregnancy and continue through the first trimester. For women who have had a child with a neural tube defect, a higher dose of folic acid (4.0 mg/d) is recommended and has been shown to decrease the recurrence rate of neural tube defects [35]. Women who may become pregnant—planned or unplanned—should be aware of recent FDA warnings to avoid specific types of fish as they may be high in methyl mercury—a known neurotoxin to a developing fetal nervous system. Women should also be questioned about the need for any dietary restrictions they may have required in childhood. Women with PKU in childhood may not currently be adhering to a phenylalanine free diet but hyperphenylananinemia is associated with neurological impairment in a developing fetus. Maternal prepregnancy weight is associated with several adverse outcomes. Women weighing more than >300 lbs (>136 kg) prior to pregnancy have a markedly increased risk for developing gestational diabetes, preeclampsia, and requiring a cesarean delivery compared with women weighing 100–149 lbs (45–67 kg). In one study, even among a subsample of women who did not have any diabetic or hypertensive diseases, excess weight significantly increased the likelihood of macrosomia and NICU treatment [36]. The relationship between maternal obesity and perinatal outcomes is necessary when discussing routine care as well as specific preconception planning. Medical history and conditions While most reproductive aged women are healthy, there are specific medical conditions associated with adverse pregnancy outcomes if untreated or treated poorly. Some medical conditions are contraindications to pregnancy. Hypertension should be under control prior to initiation of a pregnancy and women using angiotensin-converting enzyme inhibitors and angiotensin II receptor antagonists who are considering pregnancy should be switched to other agents and adjusted on these medications prior to conception. Certain cardiac disorders, such as primary pulmonary hypertension, place women at a very high risk for morbidity or mortality during pregnancy [37]. There is an increased risk (3–10%, depending on the parent’s condition) of congenital heart disease among children born to women who have a history of a congenital heard defect [38]. Women with a history of thromboembolism have an increased risk of recurrence during pregnancy. Women with a history of thrombosis should be offered testing for inherited or acquired thrombophilias, particularly if the results of the testing would affect the management of future pregnancies. Testing may be considered in women with a family history of thrombosis or a first degree relative with a specific mutation [39]. It is very important that women receive a thorough work-up and evaluation prior to being diagnosed with a seizure disorder and started on medication. Women diagnosed with seizures of unknown etiology should be told that even if they are on no medications at all, they have a two-fold higher risk for congenital malformations in their offspring than individuals without seizures. Anticonvulsant therapy use during pregnancy, particularly hydantoin or valproic acid, is associated with an increased risk for specific congenital defects. It is important to determine if anticonvulsant medication can be safely discontinued or to initiate therapeutic changes before pregnancy occurs. Women should be told that enzyme-inducing antiepileptic drugs may lower the efficacy of combination oral contraceptives, so an oral contraceptive containing a higher estrogenic concentration may be necessary. For women taking antiepileptic drugs who are considering a pregnancy, folic acid supplementation 5 mg/day is recommended for one month prior to conception and until the end of the first trimester [40]. Women with poorly controlled asthma before pregnancy tend to have increased difficulty with asthma during pregnancy [41]. Severe, poorly controlled asthma may have an adverse effect on fetal outcome as a result of chronic or intermittent maternal hypoxaemia [42]. Women with quiescent autoimmune disease or a distant history of disease should be carefully evaluated and counseled about maternal and fetal risks. Patients should be counseled that the best time to attempt conception is during periods of inactive disease. Women should be made aware of the deleterious effects of hypothyroidism in pregnancy, including an increased incidence of abortion, obstetric complications and fetal abnormalities in untreated women [43, 44]. Women being treated for hypothyroidism will require increased doses of thyroxine early and throughout pregnancy in order to maintain adequate levels; this is especially important during the first trimester [45]. Low maternal thyroxine concentrations may lead to impaired neurodevelopmental outcome in the neonate and child. Routine assessment for the presence of subclinical hypothyroidism is not recommended. ACOG recommends testing of thyroid function only in women with a personal history of thyroid disease or symptoms of thyroid disease [46]. Diabetes mellitus represents a medical condition where preconception control has been proven to translate into improved maternal-fetal outcomes. Patients with poor glucose control during the first 8–10 weeks of pregnancy have a 2–3 fold higher risk of birth defects in their offspring than patients without diabetes. Good glycemic control before conception diminishes the risk of birth defects to a level comparable to that of the normal population [47]. Women should be asked about any ongoing dermatologic therapy she may be receiving. Isotretinoin (Accutane) is used for a variety of skin conditions and is a known teratogen. Some anti-psoriasis treatments (Acetretin X and methotrexate) are known teratogens, while some antiviral medications (podophyllum) are contraindicated in pregnancy. Table 1 includes suggestions for the clinician during routine or periodic health assessments to optimize possible future pregnancy outcomes for all reproductively capable women. Table 1Suggestions for the clinician during routine or periodic health assessments to optimize possible future pregnancy outcomes for all reproductively capable women• Determine if the woman suffers from any undiagnosed or uncontrolled medical problems. If she does, provide recommendations for treatment of these conditions and when it would be best to attempt pregnancy.Make sure the patient is aware of any associations between the medical condition(s) and medications(s) she is taking and their impact on pregnancy outcomes.Ask the woman about her reproductive intentions at every visit, ascertain what her risk of an unplanned pregnancy may be; for women not actively seeking to become pregnant, discuss her current contraceptive method and any concerns or problems she may be having with it.Review the woman’s family history—including new births among family members—annually as things change over time. Discuss any familial conditions that may herald an increased risk of adverse pregnancy outcome for the woman.Discuss the significance that nutrition can have on maternal fetal outcomes,e.g., the impact of 0.4 mg folic acid per day on neural tube defects in women with no family or previous history of a neural tube defect), the need to avoid excessive vitamin usage, especially vitamins A and D, and the additional measures women on restricted diets may need to take to optimize their health and the health of the developing fetus.Review the patient’s social behaviors or lifestyle patterns—such as smoking, alcohol, or other substance use or abuse—that may affect pregnancy adversely and offer treatment options.Ascertain the immunity status of woman to rubella, hepatitis, and varicella. Ensure she is up to date on immunizations. Conclusion The concept of preconception care has gained momentum over the past two decades, and health care providers are increasingly being urged to provide such care by their professional organizations. It is hoped that, with its stated goal of improving birth outcomes, preconception care could help insure that the health status of all reproductively capable women would be optimized. In examining available standards and guidelines for preconception care, the professional nursing organization with the most highly developed preconception health standards is the American Academy of Nurse Midwives, and for physicians is the American College of Obstetricians and Gynecologists [48]. Other professional organizations examined did not have specific care standards for comprehensive preconception health, but have educated their members about one or more aspects of preconception health (smoking cessation, folic acid awareness).

Biogerontology-3-1-2040177

Heritability of a skeletal biomarker of biological aging

Changes in the skeletal system, which include age-related bone and joint remodeling, can potentially be used as a biomarker of biological aging. The aim of the present study was to investigate the extent and mode of inheritance of skeletal biomarker of biological aging—osseographic score (OSS), in a large sample of ethnically homogeneous pedigrees. The investigated cohort comprised 359 Chuvashian families and included 787 men aged 18–89 years (mean 46.9) and 723 women aged 18–90 years (mean 48.5). The TOSS - transformed OSS standardized in 5-year age groups for each sex, was analyzed as a BA index. We evaluated familial correlations and performed segregation analysis. Results of our study suggest the familial aggregations of TOSS variation in the Chuvashian pedigrees. In a segregation analysis we found a significant major gene (MG) effect in the individual’s TOSS with a dominant most parsimonious model (H2 = 0.32). Genetic factors (MG genotypes) explained 47% of the residual OSS variance after age adjustment and after including sex-genotype interaction, they explained 52% of the residual variance. Results of our study also indicated that the inherited difference in the skeletal aging pattern in men lies mostly in the rate of aging, but in women in the age of the onset of the period of visible skeletal changes. Introduction Biological age (BA) estimates the functional status of an individual in reference to his or her chronological peers on the basis of how well he or she functions in comparison with others of the same chronological age (Borkan and Norris 1986). Changes in the skeletal system, which include age-related bone and joint remodeling, can potentially be used as a biomarker of BA. Skeletal aging measures are strongly correlated with the status of the vital health systems (Gabriel et al. 1999; Kiel et al. 2001, Kadam et al. 2004, Kalichman et al. 2006b) and ultimately with survival rates (Johansson et al. 1998; Haara et al. 2003). The rate of degenerative changes in the skeleton may reflect an individual’s biological resistance, immunity, functional or health status in reference to his or her chronological age. Therefore, age-related skeletal changes can possibly be used as an index of BA. Roentgenography is one of the common methods used to assess health status and age-related changes in bones and joints (Plato 1987; Plato et al. 1994). Previously, Kobyliansky et al. (1995) reported an osseographic scoring (OSS) system that uses hand radiographs to assess skeletal aging. OSS is a synthetic measure for assessing skeletal aging based on radiographic features of the hand, combining both osteoporotic and osteoarthritic changes of hand bones and joints. This system was originally suggested by Pavlovsky (1987) and has been used in BA evaluations in population studies (Kobyliansky et al.1995; Livshits et al. 1996; Pavlovsky and Kobyliansky 1997). Importantly, OSS has been shown to highly correlate with chronological age in adults of different ethnic groups (Karasik et al. 1999, Kalichman et al. 2002). Accumulating research shows that single gene changes in yeast, worms, and rodents can significantly alter life span (Kenyon et al. 1993; Kaeberlein et al. 1999; Cheng et al. 2003; McBurney et al. 2003). The observation that the first-degree relatives of human centenarians are both disease resistant and have longer than average life spans implies a genetic component to human longevity (Perls et al. 2002; Atzmon et al. 2004). Current estimates based on twin studies indicate that the human life span is 25% heritable (McGue et al. 1993; Herskind et al. 1996; Gudmundsson et al. 2000; Perls et al. 2002). Genetic factors may also significantly contribute to interindividual differences in BA (Karasik et al. 2004, 2005). In variance component genetic analysis, performed in Karasik’s study (2004), genetic factors explained 57% of the total variance of the BA index based on OSS measurements. The aim of our study was to investigate the extent and mode of inheritance of a skeletal biomarker of biological aging (OSS) in a large sample of ethnically homogeneous pedigrees. Materials and methods Sample The population sampled consisted of native Chuvashians residing in numerous small villages in the Chuvashia and Bashkartostan Autonomies of the Russian Federation. The data were gathered in three field studies during August/September of 1994 (555 individuals), May/June of 1999 (715 individuals), and September 2002 (240 individuals) by investigators from the Department of Anatomy and Anthropology, Sackler Faculty of Medicine, Tel Aviv University (Israel), and the Institute of Anthropology, Moscow University (Russia). The expeditions were part of a Chuvashia Skeletal Aging Study (ChuSAS) project whose main aim was to investigate the different aspects of skeletal aging within the Chuvashia population. All information collected and measurements taken during all expeditions were done by the same team of investigators. The investigated cohort comprised 359 families and included 787 men aged 18–89 years (mean 46.9) and 723 women aged 18–90 years (mean 48.5). The Chuvashian population is characterized by a stable family structure with traditional relationships. For generations the Chuvashians have resided under the same environmental conditions and have not been exposed to an outside genetic flow (El’chinova et al. 2003). In the present study, we collected data from residents of a number of small villages (with a predominant Chuvashian population) located in the Volga region. A rural population is more homogeneous than urban in terms of ethnicity, occupation, and physical activity. The study participants shared similar living, economic, and professional conditions with most individuals employed in agriculture or in other occupations involving physical labor. Data from 80% to 90% of the families (including all family members who were living in the area at the time of the expedition) were obtained. Since almost every individual was related to one of the families, we were able to collect data on up to 90% of the population in each village. Data obtained from self-completed questionnaires and confirmed during the interview included sex, age, age at menarche, age at menopause and occupation. Menopause was defined as >12 months without menstruation. Age at last menstruation was considered as menopausal age. In order to minimize the possible recall bias, the investigators excluded from the analyses all women who had doubts about their age at menarche. The anthropometrical measurements (weight, stature, etc.) were also collected using standard methodology. In the studied sample, there were no individuals with known rheumatoid or psoriatic arthritis or users of steroid medication. Subjects with post-traumatic hand osteoarthritis were excluded from the study. All procedures involved were consensual. The subjects signed an informed consent form, and the entire project was approved by the Helsinki Ethics Committee of Tel-Aviv University. Additional information about the studied population can be found in our previous publications: sex- and age-related variations of the somatotype (Kalichman and Kobyliansky 2006), reproductive indices, such as age at menarche and at menopause (Kalichman et al. 2006 a, 2007a) and sex- and age-related variations of OSS (Kalichman et al. 2007b). Hand radiographs Single plain radiograms of both hands were taken from each study participant in the postero-anterior position with the X-ray source located 60 cm above, using a standard radiographic technique, as described in detail by Livshits et al. (1996). Hands were placed on the same film-containing plate to avoid any film or development variation. It was exposed for 5–10 s at 100–150 mA without intensifying screens at 50 kV. OSS evaluation An individual’s OSS was determined by the occurrence of the following: (a) bone proliferations (spurs), including apiostoses (tufting of distal phalangeal tuberosity), osteophytes, enthesophytes of the juxta-articular area and at midshaft; (b) manifestations of bone porosity, such as resorption of trabeculae, development of lacuna (juxta-articular osteopenia) and erosion of cortex; (c) loci of osteosclerosis, defined as enostosis and sclerotic nuclei; and subchondral sclerosis; and (d) non-traumatic joint deformities, defined as narrowing of joint cavities and periarticular bone corrosion (Kobyliansky et al. 1995; Pavlovsky and Kobyliansky 1997; Karasik et al. 2005; Kalichman et al. 2007b). Phalanges of the 2nd to 5th fingers of the left hand were examined. The first finger (thumb) does not directly project in a standard X-ray and was therefore not utilized. The presence of a given feature, but not its level of development, was documented for each OSS estimate. The overall number of those skeletal aging features was used as an OSS. Each radiograph was read by an experienced skeletal researcher according to the accepted protocol of OSS evaluation. Ten X-rays were read and then re-read by the investigator to estimate the intra-observer reliability of the readings. All discrepancies were reviewed for systematic errors. This exercise continued until the reliability was high (k > 0.8). Afterwards, the investigator read the remaining X-rays, still blinded to patient identifiers. Before reading each batch of X-rays one re-read 5 X-rays, which had been previously read, to “calibrate” one’s readings to a standard. The intra-observer reliability (kappa statistics) was 0.82 (P < 0.01). The intraclass correlation for the OSS was 0.92 (P < 0.01). OSS reflects amount of skeletal changes occurring stochastically during the life-span, and therefore it is characterized by strong increase of trait variance with age (Kalichman et al. 2007b and Fig. 1). It is reasonable to assume that not the deviation of OSS from the age-group mean, but only relative rate of OSS accumulation with age can be inherited. Consequently we constructed a trait using the standardization in age-sex groups to equalize the means and variances in the whole age range for both sexes. The resulting trait, TOSS (transformed OSS) was further analyzed. The biological meaning of this trait is the relative position of individual (higher or lower than average values of OSS) in his/her age-sex group expressed in the units of SD specific for each group. This accurately fit the definition of BA as the functional status of an individual in reference to his or her chronological peers (Borkan and Norris 1986). Fig. 1Mean OSS values and standard deviations (SD) in age groups of 5 years for men and women Statistical and genetic analysis Prior to any other evaluations, the descriptive statistics reflecting the basic characteristics of the studied sample were computed in the whole sample and within age groups of 5 years. At the first stage we computed familial (spouses, parent-offspring and sib-sib) correlations between the corresponding TOSS levels to examine the potential genetic effect on the skeletal biomarker of BA. The segregation analysis, as implemented in the statistical package MAN (Malkin and Ginsburg 2003), was utilized in this study to test a mode of inheritance of the skeletal biomarker of BA—TOSS. Only individuals older than 29 were included in the analysis, since the high prevalence of zero OSS values in younger individuals make the standardization approach unacceptable. More precisely, in two 5-year age groups younger than 30 years the distribution of TOSS significantly deviated from normal (Chi-squareχ2-test for frequencies gave P < 10–6), whereas in all older age groups did not (P > 0.01). Only pedigrees having at least one measured pair of relatives were included in the analysis. The mixed model of inheritance (Morton and MacLean 1974; Elston 1981; Lalouel 1983; Beaty 1997; Ginsburg and Livshits 1999) estimates the effects of a potential major gene and possible multifactorial effects, and is described in detail in numerous publications, including the aforementioned. The parameters that had been estimated in the general model are defined as follows: p—the population frequency of allele A1 (assuming diallelic gene locus); μgs—the average genotypic value of individuals having genotype g and sex s (g = 1, 2, and 3 corresponds to genotypes A1A1, A1A2, and A2A2, respectively); σ2g—the trait variance in individuals having the same major gene genotype g, it estimates the trait variation resulting from the effect of all possible environmental factors and potential minor genes; τg—the transmission probability parameter, which estimates the probability that a parent of genotype g transmits allele A1 to the next generation; ρ, β, and ε are partial correlations between the trait’s residuals adjusted for a major gene effect, in spouses, parents/offspring, and the siblings, respectively. A transmission probability test, described by Ginsburg and Livshits (1999), was used to estimate the significance of the major gene (MG) effect. A best-fitting and most parsimonious Mendelian model was established after dropping all non-significant parameters. Using the procedure implemented in the package MAN (Malkin and Ginsburg 2003) and parameters estimated for the most parsimonious model, we determined the most likely MG genotype combination for each pedigree. We divided our sample into groups according to sex and the determined individual genotype of putative BA-related MG for genotypes having distinguishable genotypic values. To analyze the age dependence of OSS, as a primary (directly measured) BA-related index, in groups of potentially different inherited BA, we used the stochastic model, described in detail by Kobyliansky et al. (1995). We estimated for each group the following aging population parameters: t0—the earliest age at which the first signs of OSS can be found, q—the individual probability of revealing the first signs of OSS changes per chronological time unit, assuming an individual’s age ≥t0, and B—the coefficient characterizing the rate of OSS changes per time unit (per year) after the process of aging has started. Next, we built the age-dependent expectation of the mean OSS level for each group according to sex and predicted MG genotype. Using the likelihood ratio test, we compared the extent of stochastic model approximation, both accounting for the MG genotype group and without it. Eventually we used ANOVA as implemented in STATISTICA 6.0 (StatSoft 2000) to test the differences between parameters of female organism development such as age at menarche and age at menopause in MG genotype groups. Results Figure 1 shows the OSS mean values and SD in age groups of 5 years. Up to age 45, men have higher OSS scores than women. After age 45 OSS in women was higher, compared with men and the sex difference in the OSS scores increases up to the end of the age range. The OSS variance in age groups increases rapidly up to age 50. Familial correlations of TOSS (Table 1) showed no significant correlation for spouses, but parent-offspring and sibling correlations were significant. This indicates the existence of a clear familial aggregation of TOSS variation in the Chuvashian pedigrees, which cannot be explained by pure common environmental effects. We investigated the nature of TOSS inheritance later using segregation analysis. Because of the method by which TOSS was constructed (standardization within each sex-age group), in the segregation model the sex effects were not significant. The other results are presented in Table 2 and support the MG hypothesis regarding trait inheritance. The most parsimonious Mendelian model was dominant. The recessive allele with a population frequency of 0.6 corresponded to relatively slow aging. For the most parsimonious Mendelian model, H2 = 0.315; total proportion of TOSS variance explained by the model was 0.349. Using this model, we predicted the most likely MG genotype combination for each pedigree included in the analysis. Because of the dominant nature of the most parsimonious model, we divided all individuals into two groups with potentially different genotypic values: (1) homozygous on allele A1 and (2) all the rest. Using a stochastic model of aging (Kobyliansky et al. 1995), we constructed the model of age dependence of directly measured OSS for a whole sample and for each predicted genotype group separately with and without considering the effects of sex. Table 3 presents the parameters of four resulting models and a comparison between them. According to the likelihood ratio test (LRT), Model 1, which included only the effect of age, has a significantly worse extent of approximation than both Model 2, which included the effect of genotype-age, and Model 3, which included the effect of sex-age. The extent of approximation was significantly higher for the effect of genotype-age, which explained 47% of the residual OSS variance after only adjustment for age, and lower for the effect of sex-age (5%). Model 4, which includes all age-sex-genotype effects, showed a significantly higher extent of approximation in comparison with other models and explained 52% of the residual OSS variance after adjustment for age. After adjustment for age and sex, the residual parent-offspring correlation of OSS was significant, but became non-significant after adjustment for the sex-age-genotype. Table 1Familial correlations of TOSSRelativesRN of pairsPSpouses0.083330.15Parent-offspring0.127550.002Sib pairs0.311890.0008Table 2Segregation analysis for TOSS (OSS standardized in age groups of 5 years for each sex)ParameterMain modelsRestricted modelsGeneralMendelianEqual τ-sArbitrary τ-sMendelian most parsimoniousEqual τ-s123456P0.6000.6280.2520.5580.598 ± 0.0330.563μ1−0.727−0.724−1.080−0.758−0.729 ± 0.057−0.702μ20.4810.525−0.3290.3800.429 ± 0.0610.362μ30.3620.3850.3830.380!0.429!0.362!σ120.2990.3250.0740.2550.303 ± 0.0450.289σ221.0740.9880.5041.1651.067 ± 0.103 1.181σ320.2050.2321.0490.255!0.303!0.289!ρ0.0850.1040.099[0.000][0.000][0.000]β0.006−0.0290.033[0.000][0.000][0.000]ε0.3250.2280.3110.3270.249 ± 0.0670.327τ10.910[1.000]0.2720.916[1.000]0.614τ20.632[0.500]0.272!0.618[0.500]0.614!τ30.001[0.000]0.272!0.020[0.000]0.614!Log LH−1425.66−1428.27−1435.91−1427.29−1429.61−1440.51χ2−5.22 NS (1)20.5** (1)1.63 NS (1)4.64 NS (4)26.44** (4)NS—P > 0.05; ** P < 0.01Number in parentheses in the χ2—row specifies a comparative columnConstraints denotation: [ ]— parameter is fixed to shown value; !— parameter is constrained to be equal to the parameter above in the tableTable 3Parameters of stochastic aging models estimated for OSS data without grouping (1) and grouped according to predicted genotype (2), sex (3), or both sex and predicted genotype (4)ParameterAllA1A1A1A2 or A2A2Allt021.223 ± 0.93321.705 ± 0.88422.225 ± 1.179q0.118 ± 0.0150.077 ± 0.0100.217 ± 0.044B0.552 ± 0.0040.518 ± 0.0040.600 ± 0.004TM29.68934.61926.838Model 1 (3 d.f.)Model 2 (6 d.f.)R2=0.838R2=0.915Log LH=−3476.8Log LH=−3065.5–χ2= 822.6 (1)Ment020.524 ± 1.23121.573 ± 1.14221.886 ± 1.683q0.127 ± 0.0240.098 ± 0.0140.233 ± 0.071B0.501 ± 0.0050.449 ± 0.0050.556 ± 0.005TM28.40031.74026.182Woment022.078 ± 1.34221.840 ± 1.16922.623 ± 1.601q0.107 ± 0.0190.057 ± 0.0000.187 ± 0.046B0.617 ± 0.0070.629 ± 0.0080.657 ± 0.007TM31.40039.37327.979Model 3 (6 d.f.)Model 4 (12 d.f.)R2=0.846R2=0.923Log LH=−3443.3Log LH=−3000.7χ2= 67.0 (1)χ2= 952.2 (1); 129.6 (2); 885.2(3);A1A1— genotype corresponding to lower values of TOSS in segregation analysis; t0— the minimal age at which initial bone changes occurred in a group; q—the group probability that an individual will first develop involutive bone change after age t0; B—the group rate of aging (bone change) per time unit; TM—the expected mean age in a group in which the first bone change occurred; number in parentheses indicates the comparative model for LRT; all LRT values are significant with P < 10−6 Figure 2 shows a scatterplot of OSS-age for the predicted genotypes in both sexes and the corresponding curves of age-dependent OSS expectation given by the stochastic model. For women in the group of genotype A1A1 (with slow skeletal aging), the age at menarche was significantly higher (P = 0.012) than in the group of genotypes A1A2 or A2A2; the group mean difference was 0.45 year. The difference in the age at menopause was in the same direction, but not significant for the whole sample. Note that our sample includes women strongly influenced by difficult life conditions during World War II. In these women the variance in the age at menopause is increased in comparison with the normal population. For women whose year of birth ≥1937 (the normal part of the population), the age at menopause was higher in group A1A1 than in other group (P = 0.054); the group mean difference was 1.6 year. Fig. 2OSS values for predicted genotype groups: black—A1A1, gray—A1A2 or A2A2 To compare our findings with results of other authors, we quantitatively analyzed the similarity of differently constructed BA indices. The correlation between the BA index proposed by Karasik et al. (2004) and TOSS was 0.82 in the Chuvashian population. The correlations between the BA index, used by Livshits et al. (1996), versus the BA of Karasik et al. (2004) and TOSS were estimated as 0.80 and 0.89, correspondingly. Though the resemblance of the indices may be influenced by population type, the aforementioned BA indices are sufficiently similar. Discussion In the present study, we evaluated the extent and mode of inheritance of a skeletal biomarker of BA. We constructed this biomarker (TOSS) to be maximally fitted according to the BA definition; it reflects the intensity of an individual’s skeletal changes in relation to its coevals of the same sex, and the range of the index does not depend on the age cohort. We previously evaluated the association between OSS, as an index of BA, with morbidity in a general Chuvasha population (Kalichman et al. 2006b). Statistically significant association was found in the present study between age-adjusted OSS and rheumatic morbidity. Morbidities such as ischemic heart disease, pulmonary diseases, traumatic brain injuries and gynecological diseases also showed differences between mean values of OSS in affected vs. non-affected individuals. However, after correction for multiple testing, the statistical significance was insufficient. The association between OSS and diabetes mellitus was close to significant (P = 0.07) and we surmise that with a larger sample of diabetic individuals, a clear-cut association will be confirmed. It is well-established that genetic factors play an important role in an individual’s BA (Karasik et al. 2005). Our finding suggests the familial aggregations of TOSS variation within the Chuvashian pedigrees. The evaluation of heritability of the skeletal biomarker of BA in segregation analysis suggested the existence of a MG effect with a most parsimonious dominant model. A number of BA indices, based on age-related skeletal changes, were previously proposed and analyzed in different populations. Livshits et al. (1996) analyzed the OSS measurement in the Turkmenian population. They used the age-adjusted OSS values as a BA index. This index showed a significant familial parent-offspring correlation of 0.1 ± 0.09, which is approximately the same as for TOSS in our study (0.12). Nevertheless, in their segregation analysis of the age-adjusted OSS, the Mendelian model was rejected. A possible reason is that a simple age adjustment does not equalize the residual variance of OSS, which significantly increases with age. As a result, parents and offspring received a BA index with different amplitude and this difference probably prevented establishing MG inheritance in segregation analysis. Other phenotypes of BA, as well, are characterized by substantial heritability. Thus, functional age (other term for BA) has been assessed by a general linear regression model of many biomarkers in Minnesota twins (27–88 years old). A subset of these biomarkers (mostly physiological variables) identified by a factor analysis had heritability of ∼59% (Finkel et al. 1995). Duggirala et al. (2002) have reported that BA, measured according to Uttley and Crawford (1994), had a substantial genetic component (heritability ranging from 0.27 ± 0.11 in one Mennonite geographical subsample to 0.47 ± 0.18 in the other). Karasik et al. (2004) studied a sample that included 1,402 members of 288 pedigrees from the Framingham Heart Study; the log-transformation was used to reduce the growth of OSS variance with age. A special BA index was constructed by adjusting the individual’s age for log-transformed OSS with cubic polynomial. The log-transformation automatically excludes from the analysis all individuals with zero OSS (note that for ages >30, the zero OSS value is meaningful and denotes a low BA). For the resulting trait, the genetic variance component analysis showed that sex, height, the body mass index, and, in women, menopausal status and estrogen use, jointly explained approximately 6% of the total variance of BA index. Genetic factors explained an additional 57%. The genome search for this BA index revealed a number of potential chromosomal regions in the two-point analysis, but for multipoint, only the region on chromosome 21 remained significant. The variance component analysis, used by Karasik et al. (2004), a priori assumes a polygene mode of inheritance. Our segregation analysis results confirm the findings about a significant genetic effect and furthermore suggest the existence of a MG effect in an individual’s TOSS with a dominant most parsimonious model. The MG hypotheses may be, to some extent, supported by the fact that in the multipoint variance component linkage analysis by Karasik et al. (2004) only one chromosomal region showed a significant LOD score >3 (likelihood ratio greater than 103). We found that genetic factors (MG genotypes) explained 47% of the residual OSS variance after adjustment for age, and after including the sex-genotype interaction, they explained 52% of the residual variance. For TOSS the MG effect was 32%. Additionally, we compared the age dependence of OSS by directly measuring the individual’s skeletal health index in predicted MG genotype groups. Kobyliansky et al. (1995) proposed a two-stage stochastic statistical model of the population aging pattern for OSS. The implied stages are as follows: (1) the period of life between birth and the apparent onset of skeletal changes (t < ti), defined as the latent period, and (2) the period of visible changes beginning after ti (which is not the same in different individuals), when involutive changes occur continuously throughout the remaining life. Applying this model separately in predicted genotype groups, we compared their aging features by means of model parameters (t0, q, B). The earliest age of the first visible changes, t0, was almost the same in all sex-genotype groups, about 22 years. The probability per year of transition from the latent to the visible changes period, q, was for genotype A1A1 about 1/3 of probability for other individuals. This was especially expressed for women and it results in greater values of the average transition age TM (39.3 years in A1A1-group and 31.7 for the others). The rate of aging, B, was also significantly different in the predicted genotype groups, but this difference was expressed more for men. Thus, we can conclude that the inherited difference in the aging pattern lies in men, mostly in the rate of aging, but in women, it is in the age of onset of the period of visible changes. An additional finding was that in the group A1A1 (slow aging), for women, the age at menarche was significantly greater (P = 0.012) than in the group {A1A2 OR A2A2}. For women born after 1937 (those whose maturation period was not influenced by WW II or the subsequent rehabilitation period), the age at menopause was also greater in the group A1A1 (P = 0.054). Thus, there is a positive relation between the rate of sexual development and the rate of aging, characterized by degenerative skeletal changes. The rate of sexual development is positively associated with intensity of normal metabolism, which also may lead to accelerated aging. Early onset of menarche is also known to be linked to other aging conditions such as elevated blood pressure and glucose intolerance, compared with later maturing girls, independent of body composition (Remsberg et al. 2005). The main molecular characteristic of aging is the progressive accumulation of damages in macromolecules that result on macro level in heterogeneity of tissues and degenerative changes. Rattan (2006) pointed out three major sources of this damage: (1) reactive oxygen species (ROS) and free radicals formed due to external inducers of damage (for example ultra-violet rays), and as a consequence of cellular metabolism; (2) nutritional glucose and its metabolites, and their biochemical interactions with ROS; and (3) spontaneous errors in biochemical processes, such as DNA duplication, transcription, post-transcriptional processing, translation, and post-translational. Bessenyei et al. (2004) reviewed data concerning SNPs associated with life span positioned in a variety of genes regulating metabolic processes, apoptosis, stress response (see also Singh et al. 2006) and immune response. Several polymorphisms in ER-alpha (estrogen receptor that are known to play significant role in skeletal development) were reported to be associated with life span (Bessenyei et al. 2004) and with both osteoarthritis (Jin et al. 2004) and osteoporosis (Albagha et al. 2005) related phenotypes. The VDR (vitamin D receptor) gene is also known to be associated with both types of degenerative changes in certain skeletal sites (Uitterlinden et al. 1997; Yue et al. 2005). A great number of genes with significant association signals were cited in recent reviews of Loughlin (2005) (osteoarthritis) and Liu et al. (2006) (osteoporosis). de Boer et al. (2002) described studies of mice with a mutation in XPD, a gene encoding a DNA helicase that functions in both repair and transcription. Mice with mutation were found to exhibit many symptoms of premature aging, including osteoporosis and kyphosis, osteosclerosis, early greying, cachexia, infertility, and reduced life-span. So OSS, the index comprising various accumulated degenerative skeletal changes, despite to different molecular mechanisms, which are involved in bone loss, cartilage loss and other OSS components, may be under genetic control of some polymorphic genes taking part in DNA repair pathways, transcription and translation control and/or some polymorphic genes controlling intensity of normal metabolism, that influence the rate of molecular damage (Kirkwood and Austad 2000). This supports the assumption that OSS as a skeletal biomarker of BA can be used to evaluate the organism’s general aging status.

Cancer_Causes_Control-2-2-1764866

Transient caloric restriction and cancer risk (The Netherlands)

Over the past century, many animal experiments have shown that caloric restriction can reduce the risk of cancer, a finding that proved to be highly reproducible. Many papers have been published on its potential for human health, but until know little evidence is available on its actual effects in humans. In Utrecht, The Netherlands, we have been investigating the effects of the 1944–1945 Dutch famine on breast cancer risk factors and breast cancer risk, and paradoxically the relatively short-term famine seemed to be related to increased breast cancer risk in later life. One of the differences between the famine situation and the large body of evidence from animal experiments is the duration of caloric restriction. Almost all animal experiments investigated sustained caloric restriction and information on the effects of short-term transient caloric restriction is very scarce. A search in the literature identified some animal experiments on short-term transient caloric restriction and these seemed to be at least supportive to the famine findings. Because caloric restriction in humans for preventive health measures would be mostly short-term, it is important to extend animal research on short-term caloric restriction. During the 20th century, a large body of experimental evidence has been accrued signifying that caloric restriction protects against cancer. Rodent data are abundant and consistently show (i) that caloric restriction lowers the incidence of a variety of spontaneous as well as induced or transplanted tumours; (ii) that this effect is directly proportional to the amount of caloric restriction; (iii) that the effects can largely be ascribed to caloric restriction per se, and not merely to the decrease in one or more dietary components; (iv) that caloric restriction initiated in early life as well as in later life is effective in cancer prevention; (v) that caloric restriction prolongs life [1–5]. A pooled quantification of the inhibitory effects of caloric restriction on spontaneous mammary tumour incidence in mice showed that caloric restriction lowered the incidence with 55% [6]. Preliminary reports on energy restriction in non-human primates appear to be consistent with findings in rodents [7, 8]. What about humans? Obviously, this is difficult to investigate, but can lead to important insights in cancer aetiology and to opportunities for prevention. The 1944–1945 Dutch famine, a relatively short severe famine during World War II [9, 10], could shed some light on this issue, and has been used before as an “experiment of history” [11–13]. During the past years, we have conducted several studies on the effects of the 1944–1945 Dutch famine on breast cancer risk factors and breast cancer risk in Utrecht, The Netherlands. For these studies we used data on women who were between 2 and 33 years of age during the famine. Therefore we were not only able to investigate the long-term effects of caloric restriction, but could also relate those effects to specific time-windows in female development. Women were classified to their degree of famine experience on an individual basis, leading to a three-point famine score (‘absent’, ‘moderate’, or ‘severe’ exposure) that enabled dose-response evaluation [14]. In brief, we found that the famine is associated with subsequent reduced reproductive ability in women who were exposed during childhood. This is manifested by decreased chance of childbirth and an earlier mean age at menopause [15, 16]. Strikingly, research on moderate early life diet restriction in Drosophila melanogaster by Tu and Tatar showed also a decrease in adult fecundity [17]. Furthermore, we found that hormone concentrations, measured postmenopausal, seem to be affected by the famine with increases in sex-steroid as well as insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-3 levels (resulting in unaffected IGF-I to IGFBP-3 ratios) [18, 19]. All our observations showed a dose-response to degree of famine exposure, with smaller but still observable associations in those moderately exposed. With regard to breast cancer risk, the associations between the famine and the reproductive system would suggest a protective effect of the famine [20]. However, the associations with hormone concentrations would suggest the contrary [21, 22], with the exception of increased IGFBP-3 that may relate to decreased breast cancer risk [23]. When we actually determined the effect of famine on breast cancer risk in this population, we found that the famine is related to increased risk, again in a dose response manner—a finding that was stronger in women who were children during the 1944–1945 winter [24]. Body mass index at later ages did not confound this relation nor modified it (last data unpublished), suggesting that catch-up growth after the famine did not play an important role. Total cancer risk, exclusive of breast cancer, seemed not to be affected by the famine [25], which does not preclude that famine may have affected risk of specific types of cancer, but the numbers in our study are presently too small for analysis with more detail. Another famine study by Dirx et al. on breast cancer risk could not detect a protection against breast cancer from the famine either, and if anything, showed a moderate increase in risk in women that were exposed at young ages [11]. In addition, Dirx et al. showed that the famine was, non-significantly, associated with increased prostate cancer and decreased colon cancer risk [12, 13]. In these studies, Dirx et al. used place of residence as a measure of famine exposure whereas we used individual exposure data, based on recall. These findings are not in line with the animal experiments consistently showing caloric restriction to prevent cancer. Several differences between the usually adopted caloric deprivation strategy in animal experiments and the 1944–1945 Dutch famine may explain these differences, such as the amount of restricted calories. The exact degree of caloric restriction during the 1944–1945 Dutch famine is difficult to ascertain, but rations dropped to about 30% of desired norms for adults and to about 50% for young children. The relative amounts of carbohydrates, fats and proteins remained more or less balanced, and supplementations to the rations were sometimes clandestinely available. Young children were relatively protected within families and by charity organisations [9, 10]. Although the amount of caloric restriction during this famine is larger than usually adopted in animal experiments, tumour incidence has been shown to decrease proportionally to degree of caloric restriction in animal experimental settings, amounting to an estimated 62% tumour reduction with 53% caloric restriction [2]. Therefore, it is unlikely that differences in the amount of caloric restriction explain the contradictory results of the famine studies and the animal experiments. More notably, during the Dutch famine people were transiently exposed for a relatively short duration of time (6 months) [9, 10]. Therefore, this “experiment of history”—as most current famines due to crop disaster or war—is only similar to experiments in rodents that studied short-term caloric restriction followed by ad libitum feeding, whereas the vast majority of these studies investigated dietary interventions that were sustained throughout the animals entire live. We were able to identify eight animal experiments that investigated transient and mostly short-term caloric restriction, where after animals were allowed to eat at will [26–33]. Two of these experiments studied the effect of caloric restriction for different time periods during and after the chemically induction of breast cancer in female rats [28, 31]. Sustained caloric restriction dramatically decreased tumour incidence: from 50% in the ad libitum group to 20% in the continuously restricted group [31]. It also seemed that caloric restriction during tumour initiation—from 1 week before until 1 week after 7,12-dimethylbenz-[α]anthracene administration—potently reduced mammary tumour development [28]. However, restriction for any period after tumour initiation followed by ad libitum feeding, if anything, showed no substantial effect, and could even lead to increased breast cancer risk [28]. Furthermore, Kritchevsky noticed that when rats were returned from restricted to ad libitum feeding this resulted in hyperphagia, accelerated weight gain, transient mammary hypertrophy, and enhanced tumour growth [31]. Four other experiments investigated the effect of caloric restriction for different periods on, amongst others, spontaneous overall tumour incidence in rats [26, 27, 30], and mice [29]. Again, these studies showed that sustained caloric restriction throughout life lowered cancer risk substantially. However, caloric restriction confined to early life did either not affect cancer risk [30], or actually seemed to increase it [26, 27, 29]. This feeding strategy did not materially increase lifespan, so this cannot explain the observed increase in cancer risk. The study of Cheney et al. in female mice provides some evidence that caloric restriction confined to mid-life may also eventually lead to increased cancer burden [29]. Recently, interest was rekindled with two more studies on prolonged but transient caloric restriction and 1-methyl-1-nitrosaurea induced mammary carcinogenesis in rats [32, 33]. These studies both show that upon refeeding tumour incidence increased. The disparity between our findings in humans after caloric restriction during a short and severe famine and the abundant literature on sustained caloric restriction and cancer risk in rodents may be ascribed to differences in exposure duration, as is corroborated by the abovementioned animal studies on transient caloric restriction that are in line with the famine observations. From a biological point of view, it may not be surprising that a short and transient period of caloric restriction in early life may increase human cancer risk. During caloric restriction, a range of responses can be seen, most of which could be directly beneficial to overall cancer risk but are unlikely to be of importance once the restriction is discontinued. Proliferation of cells is reduced with both increased rates of apoptosis together with decreased DNA synthesis and increased DNA repair, limiting the number of preneoplastic lesions. Oxidative stress is reduced, resulting in decreased reactive oxygen species that can damage DNA. Furthermore, of interest to hormone associated tumours, levels of a number of hormones and growth factors are altered during caloric restriction: glucocorticoids are increased whereas concentrations of IGF-I (and to a lesser extent IGFBP-3 resulting in decreased bioavailability of IGF-I), insulin, prolactin, estrogens and leptin are decreased [3–5, 34–36]. Changes in leptin levels can interfere with sexual maturation [37, 38], a period in development during which the Dutch famine showed the largest impact on breast cancer risk in our study. It could be that the hypothalamo-pituitary axis, which is not matured in girls until a few years after menarche when regular menses are established [39], has erroneously adapted to the period of paucity, leading to inappropriate set-levels of hormones that could relate to hormone associated cancers [18, 19]. Such erroneous adaptations, also described as a “thrifty phenotype” [40], are the subject of the “foetal and infant origins of adult disease” hypothesis by Barker and colleagues [41]. Evidence is mounting that such long-lasting effects indeed exist and may contribute considerably to later health [42]. The potential involvement of the hypothalamo-pituitary axis herein has been recognised [43]. In conclusion, the general notion that caloric restriction prevents cancer needs some amendment, even for rodents. Evidence is strong that during caloric restriction, cancer risk is decreased proportionally to the amount of restriction, and such interventions can be effective whether started in early life or later. However, a short and transient period of restriction followed by a “normal” diet does not show such effects and could actually be detrimental. Thompson et al. made a similar plea. If humans are advised to eat less as a means to prevent cancer, this would probably result in repetitive periods of weight loss followed by periods of weight gain. He warned that such weight cycling may be associated with a modest acceleration of the carcinogenic response [44]. Animal experiments on short-term interventions or weight-cycling diets are scarce and their results yet inconclusive. Such animal experiments are nevertheless highly relevant and need further investigation before any preventive strategy is researched in experimental settings, or actually adopted, in humans, as short-term interventions are the most feasible for the human situation but may bring along considerable hazards. It would be of importance to see if our observations can be replicated in such animal experiments and in human studies making use of other famine episodes, e.g. the 1959–1961 Chinese famine [45]. Other human studies may involve for instance children from countries with poor nutrition that are adopted by families in economically prosperous countries. These children have been exposed to adverse nutritional circumstances at young ages for a clearly demarcated period followed by nutritional abundance (in contrast to immigration studies of for example Japanese women to the United States who gradually adapt, often partly, to a Western lifestyle). Indian girls adopted in Sweden have for example been shown to reach menarche at younger ages compared to Indian standards [46]. It would be intriguing to see whether other physiologic changes occur in these children, e.g. with regard to hormonal levels. Furthermore, studies on cancer risk in patients with anorexia nervosa are also of interest. Two studies have reported on the topic and found a decreased risk of breast cancer [47, 48]. The generalisability of these observations is however troubled as factors underlying this disease may contribute to the decrease in breast cancer risk, so this may not merely be ascribed to a decreased caloric intake per se. Currently, the relation between famine exposure in early life and risk of other types of cancer than that of the breast is largely unknown. It would be valuable to further investigate these relations to see whether associations are different between cancer types, e.g. comparing hormone to non-hormone associated cancer types. This would give further insight into whether general mechanisms in human cancer aetiology are involved or whether adaptation of hormonal axes leading to harmful hormone concentrations is the potential culprit.

Neurosci_Lett-2-1-2271123

The effect of stress on the expression of the amyloid precursor protein in rat brain

The abnormal processing of the amyloid precursor protein (APP) is a pivotal event in the development of the unique pathology that defines Alzheimer's disease (AD). Stress, and the associated increase in corticosteroids, appear to accelerate brain ageing and may increase vulnerability to Alzheimer's disease via altered APP processing. In this study, rats were repeatedly exposed to an unavoidable stressor, an open elevated platform. Previous studies in this laboratory have shown that a single exposure produces a marked increase in plasma corticosterone levels but animals develop tolerance to this effect between 10 and 20 daily sessions. Twenty-four hours after stress, there was an increase in the ratio of the deglycosylated form of APP in the particulate fraction of the brain, which subsequently habituated after 20 days. The levels of soluble APP (APPs) tended to be lower in the stress groups compared to controls except for a significant increase in the hippocampus after 20 days of platform exposure. Since APPs is reported to have neurotrophic properties, this increased release may represent a neuroprotective response to repeated stress. It is possible that the ability to mount this response decreases with age thus increasing the vulnerability to stress-induced AD-related pathology. The role of amyloid precursor protein (APP), or its processed fragments, in normal brain function is not well understood. There is a significant body of evidence to suggest that the membrane-bound protein plays a role in cell–cell interaction, neuronal plasticity and the formation and the consolidation of synaptic connections [9]. Soluble APPα (APPsα) generated following cleavage by the α-secretase enzyme is thought to promote cell survival, neurite outgrowth and synaptogenesis [15]. The administration of APPsα in vivo can reduce the level of neuronal damage following traumatic brain injury [23] and increase the number of EGF-responsive progenitor cells [6]. Lower levels of APPs in rats were associated with poor performance in the watermaze [1] whereas intracerebroventricular administration of sAPP enhanced the performance of mice in various learning tasks [17]. However if membrane-bound APP is cleaved by the β- and γ-secretase protease enzymes it generates the Aβ peptide that aggregates to form the neuritic plaques which define Alzheimer's disease pathology [9]. For a majority of AD cases it is not known what causes the shift to the amyloidogenic pathway although there is some preclinical and clinical evidence to suggest that stress may exacerbate the progression of AD and may be associated with the pathological hallmarks of the condition. In animals, exposure to isolation stress accelerated the age-dependent deposition of amyloid plaques in the Tg2576 mouse model of AD [11]. Chronic immobilisation stress had a similar effect on both memory impairment and amyloid deposition within the hippocampus and cortex of the APPV7171-CT100 mouse model [13]. These effects may be a direct consequence of the corticosteroids released during the response to stress as prolonged exposure to high levels of these hormones appears to accelerate age-related decrements in neuronal morphology and function [19]. Studies on patients with AD have shown that hypercortisolism is associated with greater cognitive deficits [25] and that increased plasma cortisol is associated with greater decline in both clinical and cognitive measures of dementia severity [10]. The aim of this study was to determine whether exposure to acute and repeated stress affected the levels of the two primary forms of APP in the normal rat brain and whether this was regionally selective. Male Sprague Dawley rats (Harlan, UK) weighing between 250 and 300 g were maintained on a 12:12 h light:dark cycle with lights on at 6.00 a.m. and access to food and water ad libitum. After 1 week they were divided into 4 treatment groups (n = 5 rats per group): a 20 days stress group, a 10 days stress group, an acute stress group (1 day) and a control group. Stress exposure involved placing the rats on an open elevated platform for 60 min. Acutely this procedure reliably increases the levels of corticosteroid hormones in the blood [2] but this response habituates after between 10 and 20 days exposure [18,22]. Control animals were not exposed to the stressor but were taken to the same laboratory and handled in an equivalent manner. All experimental procedures were subjected to local ethical review and covered by a U.K. Home Office project licence (PPL 60/2845). Rats were killed 24 h after the last stress exposure or handling session to measure the effect of stress-induced genomic changes on APP. Brains were removed and the frontal cortex, parietal cortex, striatum and hippocampus were dissected on ice and stored at −80 °C prior to analysis. The pathological changes that characterise Alzheimer's disease first appear in the cortical regions of the brain and the hippocampus. The striatum was included as an area also rich in cholinergic neurones that remains relatively free of pathology until advanced stages of the disease. The brain samples were then homogenised (10% w/v) in homogenising buffer (1 mM EDTA, 1 mM EGTA, 0.32 M sucrose, 1 mM Tris, pH 8.0 containing protease inhibitor cocktail) at 4 °C. Homogenates were centrifuged at 100,000 × g for 60 min at 4 °C and the resulting supernatant, containing the soluble brain fraction, was aliquoted into 1 ml fractions and stored at −20 °C. The pellet was resuspended in 1% (v/v) Triton X-100 and re-centrifuged at 100,000 × g for 60 min at 4 °C. The supernatant, containing the membrane, mitochondrial and nuclear fractions, was stored at −20 °C in 1.0 ml aliquots while the triton-insoluble pellet was discarded. The total protein concentration of each sample was determined spectrophotometrically using the method of Bradford [4] and APP expression determined in 25 ug protein samples by Western blot analysis using a monoclonal primary antibody (MAB348 clone 22C11 Calibochem 1:1500) [16]. This recognises an N-terminal epitope (AA66-81) that is common to both APP and the amyloid precursor-like protein 2 (APLP2). The autoradiographs were scanned and band densities calculated using the SCION image (v3) software package. In order to determine whether there were any regional differences in APP expression between the groups the data were analysed by repeated measures analysis of variance using stress exposure as the between subject factor (GROUP) and brain region as the within subject factor (REGION). Post-hoc analysis of significant effects was carried out using one-way ANOVA or Dunnett t-tests where appropriate. In the soluble brain fractions APPs was detected as a single band with a molecular weight of approximately 128 kDa (Fig. 1). The global analysis revealed that there was a significant effect of GROUP (F[3, 16] = 3.79, p < 0.05) and a significant interaction between GROUP and REGION (F[9, 48] = 3.58, p < 0.005) suggesting that stress had a regionally selective effect on the level of APPs. Subsequent analysis of this interaction revealed that this was due to the significant increase in APPs in the hippocampal region only following 20 days of stress (p < 0.05). In the particulate brain fractions, APP was detected as a doublet with molecular weights of approximately 128 and 121 kDa (Fig. 2A). Global analysis of total APP revealed a significant effect of GROUP (F[3, 16] = 8.02, p < 0.005) but no interaction between GROUP and REGION suggesting that stress had influenced the level of APP but that this was not a regionally selective effect. Post-hoc testing confirmed that all stress groups expressed a significantly greater level of APP protein in the particulate brain fractions compared to controls (p < 0.05). Therefore, although the pattern appeared to be different in the parietal cortex compared to other regions, this effect was not large enough to influence the overall statistical analysis. The 121 kDa molecular weight protein band has previously been demonstrated to represent the deglycosylated form of the protein [16]. When this band was expressed as a percentage of total APP (Fig. 2B) global analysis confirmed a significant effect of GROUP (F[3, 16] = 11.6, p < 0.001) but again no interaction between GROUP and REGION suggesting that stress had affected the APP band ratio but not in a regionally selective way. Post-hoc testing confirmed that, overall, only the acute and 10 days stress group had a significantly increased proportion of deglycosylated APP relative to control (p < 0.05). Close inspection of the data (Fig. 2B) suggests that the APP band ratio was still increased above control levels in the frontal cortex after 20 days stress but this effect did not significantly influence the global analysis. Although stress exposure appears to have altered the processing of APP with changes in the glycosylation patterns of the particulate form and the levels of APPs, it is not clear whether this is due to changes in the release of corticosterone over this time period [18,22] or if it is due to the action of other neurotransmitters, such as acetylcholine, that are altered following stress [14]. Other studies have shown that directly treating rats with the glucocorticoid agonist dexamethasone can also increase particulate APP levels in the rat cortex, cerebellum and brain stem without significant changes in APPs [5]. However, hippocampal APP expression was not specifically reported in that study. In this study, stress significantly elevated the proportion of deglycosylated APP in particulate brain samples for up to 10 days but after 20 days there was no significant difference from control. This follows the previously published pattern of habituation of the plasma corticosterone response in rats treated with exactly the same protocol [18,22]. Corticosteroids have also been shown to control the process of protein glycosylation and in the hippocampus sialyltranferase activity appears to be highly regulated by aldosterone [8]. The expression of the mineralocorticoid receptor, which has a relatively high affinity for aldosterone, is dynamically altered in the dorsal hippocampus during 20 days exposure to the elevated platform [18]. The stress-induced increase in the particulate deglycosylated form of the protein may therefore be a consequence of corticosteroid receptor changes. Although statistically, there was no significant difference between the ratio of deglycosylated to total APP in the hippocampus compared to other brain regions, this was the area that demonstrated the greatest change following acute exposure and was the only one where the ratio fell below control levels after 20 days of stress (Fig. 2B). The hippocampus was also the only area where the amount of APPs increased following repeated stress. The hippocampus is involved in terminating the corticosteroid response to stress [20] and may also be important in habituating to repeated stress as previous studies have shown that 20 days exposure to the elevated platform produces a selective increase in GR immunoreactivity in the dorsal hippocampus [18]. The increase in APPs most likely represents an increase in soluble APPsα that has been shown to exhibit neurotrophic properties. Since the hippocampus it also an area of the brain thought to be crucial for the formation of certain types of memory, the changes in APPs may have consequences for cognitive function. Previous studies have shown that stress can impair memory [21] or under certain circumstances can enhance it [3]. In particular, repeated exposure to the elevated platform has been shown to improve spatial memory using a watermaze task in young rats but only if they have been previously exposed to the apparatus [26]. The increase in deglycosylated APP seen following one or 10 days stress exposure may indicate an increased risk of Aβ formation as the deglycosylated form of the protein is less likely to be trafficked to the cell membrane [16] for cleavage by α-secretase but rather may be retained in the endoplasmic reticulum/golgi. Since β-secretase appears to be localised to the golgi and endosomal compartments of the cell [24] the deglycosylated APP is more likely to undergo amyloidogenic processing by β- and γ-secretase pathway. Recent studies have shown that glucocorticoid treatment of either mouse neuronal cells or the 3XTg-AD mouse increases the formation of Aβ [12]. This appears to be due to an increase in the expression levels of both APP and β-secretase. The formation of Aβ peptide oligomers may also disrupt cognitive function, even prior to nerve cell death [7]. The increase in APPs within the hippocampus over the same period as habituation to a repeated stressor could therefore represent a compensatory response in relatively young adult rats. Further studies will be required to determine the exact mechanisms responsible for the changes in APP processing and whether an age-induced impairment in this neuroadaptation increases the risk of the stimulation of the AD-associated protein processing pathways.

Qual_Life_Res-3-1-2062490

Response shift due to diagnosis and primary treatment of localized prostate cancer: a then-test and a vignette study

Aim Whether a prostate cancer diagnosis induces response shift has not been established so far. Therefore, we assessed response shift in men who were diagnosed with localized prostate cancer. Introduction Response shift, defined as an adaptation to changing health [1], is a beneficial process for patients because it can help in adapting to a new situation. However, it may complicate the correct interpretation of change in health-related quality of life (QoL) scores over time in intervention studies, and therefore needs to be understood. Response shift refers to a change in the meaning of QoL over time [2] and can result from a change in one’s internal standards of measurement (i.e. recalibration), a change in the importance attributed to the domains constituting QoL (i.e. change in values or reprioritization), or a change in the definition of the concept of QoL (i.e. reconceptualization) [3, 4]. These three forms of response shift are illustrated in the following example. Imagine a woman X. When asked to rate her QoL she thought of her (50-h a week) job, her partner and playing volleyball with friends, and rated her QoL as very good. Unfortunately she fell ill. For some months she was not able to work. Her partner and relatives supported her a great deal, which she appreciated enormously. Gradually she recovered and started working again for 20 h a week, but was no longer able to play volleyball. She rated her new QoL again as very good, but this time the main aspects of her QoL consisted of her partner, her family and work. Her ratings did not change, i.e. twice ‘very good’, but in fact three forms of response shift had occurred. ‘Work’ changed from a 50-h a week job to 20 h a week (recalibration), her partner became more important (reprioritization), and her concept of QoL has changed: no more sports, but relatives instead (reconceptualization). This paper focuses on the assessment of response shift induced by a prostate cancer diagnosis. Schwartz and colleagues systematically addressed the state-of-the-art in the assessment and interpretation of response shift [5]. In a meta-analysis, following Cochrane guidelines, the magnitude and clinical relevance of response shifts across 19 longitudinal studies were evaluated. Most studies addressed global QoL and specific QoL domains such as fatigue, well-being, and pain, usually by conducting the then-test. Effect sizes, defined as the mean difference between tests divided by the standard deviation (SD) of the first assessment, were computed by the authors. These were generally small according to Cohen’s criteria [6], with the largest effect sizes found for fatigue, followed by global QoL, physical role limitation, physical well-being, and pain. Effect sizes varied in direction, which complicated their interpretation. Schwartz et al. concluded with recommendations for future response shift publications, such as explaining the meaning of the study results in terms of recalibration, reprioritization, and reconceptualization [5]. Response shift is more likely to occur when an intense and pervasive change in health is experienced [7]. A cancer diagnosis may have a large impact on a person’s experienced health. Our group previously described the process of being diagnosed with prostate cancer through a screening process consisting of a Prostate Specific Antigen (PSA) test and, if indicated, a biopsy. Typically, localized prostate cancer diagnosed through screening is not associated with any physical symptoms. Men’s mental health and the valuation of their own health decreased significantly after they received their diagnosis, and we concluded that being diagnosed with prostate cancer was a deeply felt change in health [8]. We started the present study because we expected that a prostate cancer diagnosis induces response shift. We hypothesized that the pre-diagnosis health state would be rated more positively in retrospect (i.e. if assessed after diagnosis) than at the reference point itself (i.e. pre-diagnosis). Collecting data on QoL before a cancer diagnosis is usually not feasible, since it is unknown who will develop cancer and when, so that the inclusion of a very large cohort would be required. However, the context of the European Randomized study for Screening on Prostate Cancer (ERSPC) [9] enabled us to include a cohort of men shortly before they were screened and subsequently diagnosed. We aimed at assessing the magnitude and direction of response shift effects after diagnosis and again after primary treatment. We employed two methods: the common then-test and a novel approach including rating of vignettes related to side effects of prostate cancer treatment (urinary, bowel, and erectile dysfunction). Patients and methods Ethics approval and informed consent The Ethics Committee of the Erasmus MC approved the research protocol. All participants gave additional written informed consent to be interviewed for the study. Parent study Inclusion of the ERSPC participants was initiated in 1994 among all male inhabitants of the Rotterdam region aged between 55 and 74 years. The only exclusion criterion was a previous prostate cancer diagnosis. Details on study recruitment for the ERSPC have been reported earlier [9]. Respondents Randomly selected participants from the parent study were approached. All men who were due for the second (n = 2,798) or third screening round (n = 2,024) between January 2003 and May 2004 were sent a short questionnaire on health (see below) by mail. Men who were diagnosed through the screening process were interviewed twice by one of the authors (IK); one month post-diagnosis (but before treatment) and again 7 months post-diagnosis. Assessing response shift To assess the magnitude and direction of response shift effects two methods were used: the then-test, and vignettes (a novel method in response shift research). For the resulting study scheme, see Fig. 1.The then-test is a retrospective evaluation of an earlier assessment (retrospective pre-test-post-test design). At post-test respondents are asked to remember how they were doing at the reference point and to retrospectively rate their level of functioning or QoL at that time. The then-test was originally developed to measure recalibration. The method assumes that respondents will use their post-test internal standards when providing a re-evaluation or ‘then-test’ rating of their health at the reference point [5]. The comparison between the then-test and the post-test is thus assumed not to be confounded by recalibration and can be considered as an indication of true change [2]. The comparison of the mean pre-test, which is the assessment that was completed at the reference point, and then-test scores would reflect an estimate of the magnitude and direction of response shift [2]. Because respondents in our study were included before diagnosis, they could provide then-test scores relating to their health before diagnosis and to their health between diagnosis and treatment. Before completing the then-test, respondents were explicitly reminded about the period the then-test was referring to: e.g. the time when the respondents had not yet been diagnosed with prostate cancer and were unaware of having prostate cancer. Respondents were then asked to re-assess their health at that time. Three then-tests were conducted: two referencing pre-diagnosis health (measured at 1 month post-diagnosis and 7 months post-diagnosis) and one referencing 1-month post diagnosis health (measured at 7-months post-diagnosis), see Figs. 2, 3, 4. The respondents completed generic QoL measures, i.e. the Short-Form 36 (SF-36) mental health and vitality, and the EQ-5D VAS for self-rated health, as a pre-test, post-test and then-test. The SF-36 consists of eight scales on physical and mental domains of health. We used the scales on mental health (five items on being nervous, down, peaceful, depressed and happy) and vitality (four items on being full of life, having a lot of energy, being worn out and tired). Higher scores (0–100) indicate better mental health and vitality [10]. The EuroQol (EQ) 5D valuation of own health is a visual analog scale on current overall health, anchored at the lower end (0) by ‘worst imaginable health state’ and at the upper end (100) by ‘best imaginable health state’ [11].As a novel method to assess response shift we used vignettes that each described a health state relating to side effects of therapy for localized prostate cancer, i.e. urinary, bowel or erectile dysfunction. The vignettes contained items of the EQ-5D self-classifier complemented with items on dysfunction, for instance, ‘Mr. A has no problems in walking about, has no problems washing or dressing himself, experiences urinary leakage daily, has no pain or discomfort, is not anxious or depressed’. Respondents were asked to indicate how good or bad they evaluated these health states on visual analog scales anchored at the lower end (0) by ‘very bad’ and at the upper end (10) by ‘very good’. We used the vignettes to explore reprioritization. We hypothesized that men would value the health states as less detrimental after diagnosis than before. After diagnosis they knew they might experience these dysfunctions themselves in the context of prostate cancer treatment.Additionally, information on respondents’ age and on the Gleason score (a clinical criterion for histological grading of the aggressiveness of the tumour) were obtained through the screening office. Fig. 1Study schemeFig. 2Original and then-test scores of the EuroQol valuation of own health by prostate cancer patients (n = 52). If we measure only EQ-VAS preceding diagnosis and at 1-month post-diagnosis, the difference between these scores is regarded the ‘observed change’. However, if the retrospective pre-diagnosis assessment provides a more valid comparison with the post-diagnosis assessment, the ‘true change’ is reflected by the difference between the retrospective pre-diagnosis assessment and the post-diagnosis assessment. The difference between the pre-diagnosis assessment and the retrospective pre-diagnosis assessment provides an indication of the size and direction of the ‘response shift’ induced by the diagnosis. Similar explanations are valid for the other data points in the figureFig. 3Original and then-test scores of the SF-36 mental health by prostate cancer patients (n = 52). ‘Observed change’, ‘True change’ and ‘Response shift’ refer to the differences in SF-36 mental health scores between the assessment at 2 months before diagnosis and post- and then-test at 1 month after diagnosis (for further explanation, see caption at Fig. 2.)Fig. 4Original and then-test scores of the SF-36 vitality by prostate cancer patients (n = 52). ‘Observed change’, ‘True change’ and ‘Response shift’ refer to the differences in SF-36 vitality scores between the assessment at 2 months before diagnosis and post- and thentestthen-test at 1 month after diagnosis (for further explanation, see caption at Fig. 2.) Statistical analysis Procedures concerning imputation of missing responses in the SF-36 items were conducted according to the guidelines of the SF-36 Health Survey Manual [12]. Differences between assessments were tested with paired-samples t-tests. P-values ≤ 0.05 were considered statistically significant. The type I error rate, i.e. the ratio of significant findings to the number of comparisons, was calculated. To assess the magnitude of the differences between the assessments we used Cohen’s effect sizes, defined as the mean difference between tests divided by the SD of the first assessment, and interpreted as follows: 0.2 < d < 0.5 indicates a small, 0.5 ≤ d < 0.8 a moderate, and d ≥ 0.8 a large effect size [6]. The minimal important difference (MID), which is the smallest change in a patient-reported outcome that is perceived by patients as beneficial or that would result in a change of treatment, was operationalized as a difference of at least half a SD [13]. Non-response bias was analysed by testing differences between the respondents and the non-respondents with unpaired t-tests. Results Out of the 3,892 men who completed the initial questionnaire before screening on prostate cancer, 82 were subsequently diagnosed. Of these, 52 (response 63%) consented to participate in two additional telephone interviews at 1 and at 7 months post-diagnosis. All 52 respondents participated in the first interview, which took place before treatment had been initiated. Due to personal circumstances one respondent later refused the second telephone interview. Average age at screening was 67.3 years (SD 4.4), ranging from 60 to 74 years. The Gleason score was favourable in 42 of the 52 patients, i.e. below seven (Table 1). In all respondents but one, treatment had been initiated at 7 months post-diagnosis, i.e. radical prostatectomy (n = 25), brachytherapy (n = 12), active surveillance (n = 10), external radiotherapy (n = 3), or hormonal treatment (n = 1), see Table 1. Table 1Gleason scores and treatment modality of the respondents (n = 52)Gleason score < 7 n = 42Gleason score ≥ 7 n = 10Total n = 52Radical prostatectomy18725External radiotherapy123Brachytherapy1313Active surveillance99Hormonal treatment 11No treatment choice yet11 Original scores, i.e. scores relating to the respondents’ health at the time of the assessment and interviews, and then-test scores relating to the two reference points are given in Table 2. For example, ‘85.2’ in the upper right corner of Table 2 reflects the ‘EQ valuation of own health’ score of the then-test measured at 7 months post-diagnosis referencing 2 months pre-diagnosis. Mental and self-rated health scores worsened significantly from 2 months preceding diagnosis to 1 month post-diagnosis. The average mental health score, for instance, was 83.2 at 2 months pre-diagnosis, and 75.8 at 1-month post-diagnosis; a decrease of 7.4 that exceeds the MID. At 7 months post-diagnosis mental and own health scores had increased again, but not to their original level. Table 2Mean health scores (standard deviation) of the respondents (n = 52) before and after diagnosis, original and thentests scoresReferring toOriginal scores (n = 52)P-value*Thentests1 month post-diagnosis (n = 52)P-value: thentest vs. originalEffect size: thentest vs original 7 months post-diagnosis (n = 51)P-value: thentest vs. originalEffect size: thentest vs. original2 months pre-diagnosisEuroQol own health80.2 (11.7)83.2 (9.0)0.058−0.2685.2 (7.6)0.002a−0.43SF-36 mental health83.2 (11.6)84.5 (11.0)0.304−0.1083.2 (10.9)1.000.01SF-36 vitality75.3 (15.6)79.6 (12.0)0.008−0.2879.4 (12.4)0.046−0.261 month post-diagnosis, before initiation of treatmentEuroQol own health74.5 (15.1)0.01074.1 (14.5)0.6180.03SF-36 mental health75.8 (16.8)0.001a72.8 (18.3)0.0420.17SF-36 vitality74.7 (14.2)0.77172.6 (13.7)0.1110.157 months post-diagnosis, after intiation of treatment in 80% of respondentsEuroQol own health77.6 (13.7)0.196SF-36 mental health80.4 (13.8)0.066SF-36 vitality73.1 (17.7)0.213*P-value of difference with previous original score (P ≤ 0.05 are considered significant)aChange exceeds the minimal important difference (operationalised as ½ standard deviation) Original scores of pre-diagnosis health were lower, indicating worse health than on the then-test scores. For example, the original pre-diagnosis mental health score was 83.2 on average, but the then-test score measured at 1 month post-diagnosis was 84.5, indicating a more positive judgement of pre-diagnosis mental health in retrospect. Original scores of health between diagnosis and treatment, on the other hand, were higher, indicating better health than on the then-test scores. The original vitality score, for instance, was 74.7 at 1 month post-diagnosis, but the then-test score measured at 7 months post-diagnosis was 72.6. This means that vitality between diagnosis and treatment was judged worse when measured in retrospect than when measured at the reference point itself. Original and then-test scores are presented in Figs. 2–4, including estimates of the response shift effects, i.e. the difference between mean pre-test and then-test scores, and estimates of ‘true’ change, i.e. the difference between the mean post-test and then-test scores. Effect sizes of the differences between then-test and original scores were small (Table 2). The vignettes describing urinary, bowel and erectile dysfunction states were rated significantly higher (i.e. better) by respondents at 1 month post-diagnosis than at 2 months pre-diagnosis (P-values 0.038, 0.011, and <0.001, respectively). The valuation of erectile dysfunction showed the largest increase; i.e. from 5.3 to 6.7 on a 0–10 scale, with a moderate effect size of 0.57 (Table 3). This implies that respondents considered especially erectile dysfunction less detrimental after diagnosis with prostate cancer than before diagnosis. The differences between pre- and post-diagnosis valuations of the vignettes exceeded the MID in 4 out of 6 cases (Table 3). Table 3Average valuation by VAS (SD) of prostate cancer specific vignettes, scale 0-10, P-values ≤ 0.05 were considered significantHealth state descriptionPre-diagnosis (n = 52)1 month post-diagnosis (n = 52)P-value pre-diagnosis vs. 1 month post-diagnosisEffect size pre-diagnosis vs. 1 month post-diagnosis7 months post diagnosis (n = 51)P-value pre-diagnosis vs. 7 months post-diagnosisEffect size pre-diagnosis vs. 7 months post-diagnosisDaily urinary leakage5.6 (2.1)6.3 (1.5)a0.038−0.325.9 (1.5)0.348−0.14Daily bowel cramps5.3 (2.0)6.0 (1.6) 0.011−0.416.2 (1.4)a0.012−0.39Serious erectile dysfunction5.3 (2.2)6.7 (1.8)a<0.001−0.576.5 (1.8)a0.005−0.47achange compared to previous score exceeds minimal important difference The results of the then-test were significant in 4 out of 9 comparisons, the results of the vignettes in 5 out of 6. The overall type I error rate, which is the ratio of significant findings to the number of comparisons, was 0.6 (9 out of 15). Non-response analysis The baseline average age in men who were diagnosed with prostate cancer but did not respond to the questionnaire (n = 30) was 66.7 (SD 4.3, range 59–73) years. Respondents and non-respondents did not differ significantly in age or other health measures (data not shown). Discussion Men diagnosed with prostate cancer evaluated their pre-diagnosis health in retrospect as better than at the reference point itself. Post-diagnosis–pre-treatment health was rated worse in retrospect than at the reference point. This suggests that ‘true’ changes in health between the first assessment before diagnosis and the second one at 1 month post-diagnosis were larger than the original scores disclosed, and that response shifts were induced by first, the diagnosis, and second, subsequent treatment. The sizes of the response shifts induced by the diagnosis were larger than those induced by the treatment. The negligible to small effect sizes indicated that only some recalibration occurred. The directions of the effect sizes were interpretable and consistent with our hypotheses. Additionally, men evaluated vignettes relating to side effects of prostate cancer treatment as less detrimental after they were diagnosed than before diagnosis. We interpreted this change as a reprioritization of respondents who became aware after being diagnosed with prostate cancer that they were at risk of experiencing these health states themselves as a consequence of being treated for prostate cancer. In this new context dysfunctional health states were evaluated as less bad than before. The effect sizes were moderate for erectile dysfunction and small in the two other ones, indicating that reprioritization also occurred. The directions of the effect sizes were interpretable and consistent with our hypotheses. The overall type I error rate was 0.6, which indicated that the statistical significance is very unlikely to be caused by chance. This is an additional indication that our findings reflect real differences. We conclude that the results of the then-tests and the ratings of the vignettes both indicate the presence of a response shift and adaptation of the patients to their new situation. In the meta-analysis of Schwartz et al., the largest effect sizes on response shift (although still small) were found for the dimensions global QoL and fatigue [5]. These dimensions, represented in our study by EQ-5D on own health and the SF-36 vitality scale respectively, also resulted in small effect sizes. An important criticism of the then-test approach is its susceptibility to recall bias. Respondents are supposed to be able to remember their previous health at the reference point, which is extremely difficult in case of a chronic disease with no obvious trend towards better or worse health [14]. However, in a study on response shift in cancer patients undergoing various forms of treatment, there was evidence that recall bias was absent [2]. We assume that in the case of a deeply felt change in health (such as being diagnosed with cancer or the initiation of cancer therapy) recall will not cause memory difficulties for most respondents. Therefore, in our study we expect that recall bias did not have a major influence on the results. The then-test results in a retrospective judgement that subsequently is used to construct ‘real change’ since the reference point. This approach assumes that the information that was acquired after the original judgment was made leads to more accurate estimates of QoL than the original judgment itself. This assumption is, however, not always true; for example in the case that the newly acquired information is not correct [14]. The valuation of disease-specific vignettes (the second method used to assess response shift) has to our knowledge not been described before. It resulted in a moderate effect size considering the vignette on erectile dysfunction. The directions of the effect sizes were consistent with our hypotheses. Our results showed that response shift can be studied by using vignettes. We consider the valuation of vignettes as a useful addition to the already available collection of tools to assess response shift. Apart from this theoretical value, the results of the vignettes may also have implications for clinical practice. In case of a diagnosis of localized prostate cancer several treatment options are available. Since there is no consensus about which of these treatments has the best outcome in terms of survival and QoL, considerations of patient preferences regarding mode of treatment and side effects are an essential element in shared decision making on the choice of therapy. To elicit a patient’s preferences and his individual trade-offs between benefits and side effects of various modes of treatment, vignettes can be useful [15]. However, our study showed that patient preferences may change in the course of the diagnostic and treatment process, which illustrates how difficult it is for a patient to imagine the consequences of an intervention in advance. This finding confirms the point made by Cowen et al. to recommend the use of individual utilities (“actually prefer”) instead of population’s utilities (“should prefer”) to optimise the choice of treatment for patients with prostate cancer [16]. We recommend further investigation of the vignettes method. The fact that being diagnosed with prostate cancer was found to induce response shift may be seen as an indication that men regard a prostate cancer diagnosis as a major life event, and is additional evidence for earlier findings [8]. In another study, men with metastic or locally advanced prostate cancer completed assessments on prostate symptoms shortly after diagnosis, and 3 and 6 months thereafter. The second and third assessments included then-tests. The presence of a response shift was suggested in patients and their spouses [17, 18]. The authors remarked that retrospective and prospective assessments cannot be used interchangeably. Lepore and Eton tested two conceptual models of response shift among men newly diagnosed with prostate cancer to explain the frequently observed lack of association between health problems and QoL in cancer patients. No support was found for the suppressor model, according to which health change leads to response shift, which in turn leads to a change in QoL. Some evidence was found for the buffering model, according to which response shift effects moderate the negative association between health problems and QoL. Two aspects of response shift, recalibration and reprioritization, were assessed by then-tests and a measure of primary life goal changes, respectively. They were found to moderate the relation between negative changes in physical health and changes in QoL [19]. Indications of response shift were also found in an earlier study on men treated for localized prostate cancer. Men stated, for instance, that they accepted the side effects of treatment because ‘If they hadn’t intervened, that operation, maybe I wouldn’t be here anymore’ [20]. The present study has several strengths and limitations. The study design is one of its strengths; the unique context of the ERSPC enabled the inclusion of respondents before they (or anyone else) were aware that they had prostate cancer, which is usually unfeasible. To our knowledge this is the first study to measure response shift in men who were diagnosed with cancer. An additional strength is the compliance of the respondents; 51 of the 52 respondents completed the 7-month assessment. For the then-test we selected measures that are considered subjective (i.e. SF-36 mental health and vitality, and EQ-5D of own health) but no objective items, which can be considered a drawback of the study. Furthermore, we acknowledge that offering questionnaires in two different modes (self-administered questionnaires before diagnosis vs. telephone interviews afterwards) may have been less than optimal. This design was chosen based on practical considerations, because assessments by telephone in 3,892 screen participants was not feasible, and self-administered questionnaires at 1 month after diagnosis undesirable since we wanted these assessments to be completed before the initiation of treatment. The unavailability of information on marital status and education is also a drawback. Another potential limitation of our study is that the interval between the initiation of treatment and the assessment at 7-months post-diagnosis was not the same for all respondents; it is possible that response shift may vary according to the length of time that elapsed since treatment. However, information on this interval had been of limited use. The most common therapies for localized prostate cancer nowadays are surgery, radiotherapy, and active surveillance. These therapies differ greatly (by nature) in duration, the onset of side effects and their course over time. It may be that particular groups may be more prone to response shift than others, e.g. depending on age or prognosis. We plan to address this issue further, preferably in a larger sample than used in the current study. Conclusions Using two complementary techniques we found that a diagnosis of prostate cancer induces response shift. From a methodology point of view, the vignette-method needs to be explored further.

Mar_Drugs-5-4-2365694

Evaluation of Harmful Algal Bloom Outreach Activities

With an apparent increase of harmful algal blooms (HABs) worldwide, healthcare providers, public health personnel and coastal managers are struggling to provide scientifically-based appropriately-targeted HAB outreach and education. Since 1998, the Florida Poison Information Center-Miami, with its 24 hour/365 day/year free Aquatic Toxins Hotline (1–888–232–8635) available in several languages, has received over 25,000 HAB-related calls. As part of HAB surveillance, all possible cases of HAB-related illness among callers are reported to the Florida Health Department. This pilot study evaluated an automated call processing menu system that allows callers to access bilingual HAB information, and to speak directly with a trained Poison Information Specialist. The majority (68%) of callers reported satisfaction with the information, and many provided specific suggestions for improvement. This pilot study, the first known evaluation of use and satisfaction with HAB educational outreach materials, demonstrated that the automated system provided useful HAB-related information for the majority of callers, and decreased the routine informational call workload for the Poison Information Specialists, allowing them to focus on callers needing immediate assistance and their healthcare providers. These results will lead to improvement of this valuable HAB outreach, education and surveillance tool. Formal evaluation is recommended for future HAB outreach and educational materials. 1. Introduction Harmful algal blooms (HABs) are caused by blooms of algae known as phytoplankton; these include organisms such as dinoflagellates, diatoms and cyanobacteria [1–6]. HABs can occur in all aquatic environments. In marine environments, they are also known as “red tides” because some of these organisms can change the color of the water to red or brown. HABs may cause harm to the environment and other organisms in two ways. First, through the severe overgrowth of the HAB organisms that depletes oxygen in the local environment, and second when the HAB organisms produce extremely potent natural toxins. Phytoplankton are the base of the marine food web; thus, the toxins they produce can bioconcentrate in organisms higher up in the food chain. Several human illnesses are caused by the ingestion of seafood contaminated with the natural toxins produced by the HAB organisms [1–6]. In addition to exposure through seafood ingestion, environmental exposures can occur through skin contact with contaminated water or by inhalation when the HAB organisms are broken up by waves and their toxins become aerosolized. For example, human exposure to aerosols containing brevetoxins from the Florida red tide dinoflagellate, Karenia brevis, has been associated with reports of respiratory distress, particularly but not exclusively in persons with asthma, as well as increased emergency room admissions for pneumonia, bronchitis and asthma among coastal residents during active Florida red tides [7–12]. With the increasing number of persons interacting with the coastal areas (both freshwater and marine) and with the apparent increase of HABs around the world, healthcare providers, public health personnel, and coastal managers are struggling to provide scientifically-based information [13–17]. There is a paucity of appropriately targeted outreach and educational materials for healthcare providers and persons with possible exposure to the marine and freshwater toxins, as well as coastal resource managers, the media, and the general public. A variety of educational outreach materials and services have been created, but there has been almost no formal evaluation to determine whether these materials are reaching their target audiences and meeting these audiences’ expectations. 1.1. Florida Poison Information Centers The Florida Poison Information Center/Miami (FPIC/Miami) is one of three poison centers created in the State of Florida in 1989 by an act of the Florida Legislature (FS 395.038), and it has rapidly grown into a cost-effective model of a poison center system. The FPIC/Miami is located at the University of Miami Miller School of Medicine and Jackson Health Systems, and directly serves the residents of Southeastern Florida (i.e. Miami-Dade, Broward, Monroe, Palm Beach, Lee and Collier counties) and beyond with toll-free 24 hour/day services in several languages. For example, in 2006 the FPIC/Miami responded to over 57,340 calls; of these calls, 35,741 (62%) were for known or suspected exposures to toxic substances. Approximately 80% of the patients calling from home were managed in the home, without the need for an emergency department visit. The FPIC/Miami placed 18,833 (33%) follow-up calls to continually assess this home management. The FPIC/Miami toxicologists provided over 1,705 consultations with Florida’s physicians to assist in the care of hospitalized patients. In an attempt to prevent poisonings, the FPIC/Miami educators coordinated 669 outreach and education programs for the lay public, health professional, and the media. They also distributed over 198,213 informational brochures, handouts and telephone stickers, reaching over 72,000 persons. Since 1998, with funding from the Florida Department of Health, the National Center for Environmental Health (NCEH) of the Centers for Disease Control and Prevention (CDC), and the University of Miami National Institute of Environmental Health Sciences (NIEHS) Marine and Freshwater Biomedical Sciences Center and the National Science Foundation (NSF)- NIEHS Oceans and Human Health Center, the FPIC-Miami established a toll-free hotline (1–888–232–8635) dedicated to providing information concerning aquatic health issues. All calls are answered by Poison Information Specialists, who are highly trained physicians and nurses. All calls are reported as a form of passive surveillance of HAB-related illness and information requests to the Aquatic Toxins Program (http://www.doh.state.fl.us/ENVIRONMENT/community/aquatic/) and to the Foodborne Illness Program of the Florida Department of Health. Recently, the Hotline was expanded to include additional options for callers. Using an automated call processing menu system, the “Aquatic Toxins Hotline,” now allows callers to access information in English or Spanish about the possible health effects and locations of the Florida red tide (including the National Atmospheric and Oceanographic Administration (NOAA) Harmful Algae Bloom (HAB) Bulletin http://coastwatch.noaa.gov/hab/bulletins_ns.htm), information on ciguatera fish poisoning, blue green algae (cyanobacteria) and resources for learning about general marine toxin issues, as well as still giving the callers the opportunity to select to speak directly to a Poison Information Specialist if they wish. These materials were reviewed and developed with cooperation between the FPIC-Miami, the University of Miami, the Florida Department of Health, the CDC, Mote Marine Laboratory, and the Florida Fish and Wildlife Commission. Concurrent with this initiative, a number of academic, public health and community partners (including Solutions to Avoid Red Tide or START; http://www.start1.com/) collaborated to create a series of informational signs advertising the Aquatic Toxins Hotline as a source of information for Florida red tide. After focus groups with public health personnel, beach managers, hotel and restaurant owners, and tourist boards, these signs were designed to be posted on and near beaches, and targeted at beach users including tourists (see Figure 1). This Pilot Study evaluated whether the new automated system was used by callers and whether the callers considered the information to be useful to them, as well as to collect caller recommendations for the improvement of this service. The goal of this pilot study was to evaluate whether the Florida Aquatic Toxins Hotline was serving the self-identified needs of current callers. 2. Methods The FPIC-Miami receives the telephone numbers of all callers to the Aquatic Toxins Hotline, whether the caller speaks with a Poison Information Specialist or just the automated system. In this evaluation, telephone numbers from the calls made to the Aquatic Toxins Hotline from September 2006 through January 2007 were re-contacted. Of note, throughout this study time period, there was an active Florida red tide on the West Coast of Florida. This evaluation aimed to determine: Basic Caller demographics What information/services callers were seeking Which menu selections were the most commonly chosen Which menu options, in the caller’s opinion, were the most helpful If callers were satisfied with the service provided by the Aquatic Toxins Hotline If callers would call again What would, in the callers’ opinion, make the Aquatic Toxins Hotline service more useful. Trained interviewers contacted the Aquatic Toxins Hotline callers by phone in English and Spanish to explain the study, obtained verbal consent for participation and conducted a short scripted interview (available on request from the corresponding author). Specifically, the interviewers asked a brief series of questions about using the Aquatic Toxins Hotline and about the caller’s perception of the system’s usefulness. Study participants also had an opportunity to make suggestions to improve the system. No personal identifying information was collected. To maximize success in contacting callers, the interviewers called each telephone number three times (once during a week day, once during a week night, and once during the weekend) in an attempt to re-contact the specific person who had called the Aquatic Toxins Hotline. This study was approved by both the University of Miami and Florida Department of Health Human Subject Committees. The data were collected into a Study Access Database. The data were analyzed using SAS version 9.1. The data were analyzed by frequencies as a descriptive process. 3. Results The Aquatic Toxins Hotline has experienced increasing use over the past few years, particularly during periods of active Florida red tides. Since its inception in 1998, there have been 25,000 calls to the Hotline, with 7,000 occurring between 2001 and 2006; during 2006 alone, 2,415 calls were made to the Aquatic Toxins Hotline. During the study period from Sept 2/06 through Jan 31/07, there were 1,163 calls made to the hotline, with 315 (27%) answered by a Poison Information Specialist; of these calls, 53% were for Florida red tide, 6% for ciguatera, 17% for jellyfish, 2% each for PSP/pufferfish, scombroid, and stingrays, and 0.6% blue green algae. This means that only 27% of the callers selected to speak with a Poison Information Specialist from the automated HAB Menu in English or Spanish, while the vast majority (73%) only accessed the automated HAB menu. The Interviewers attempted to contact each of the callers during the study period as described above. Even though the follow-up calls were made within two months of the original contact, only a small percentage (10%) of the callers was able to be re-contacted. Inability to re-contact these individuals was due to a variety of factors such as: no answer despite repeated calls, answering machines, and no real contact number (i.e. original call made from hotel, hospital, etc). Nevertheless, once contacted, all callers (100%) agreed to participate. A total of 118 callers were successfully re-contacted and agreed to participate. Of these, only 89 callers reported that they recalled making a call to the Florida Aquatic Toxins Hotline, and thus these 89 callers were considered the participants in this Pilot Study. Of note, both the actual number of respondents and the percentages are reported below because not all subjects answered all the questions. The majority of the 89 participants were women (41 [61%]), self-described as white (82 [100%]) and non Hispanic (66 [94%]) whose primary language was English (80 [96%]) and with a mean age and standard deviation of 55.5 +/− 12.2 years (see Table 1). The participants were predominantly from Florida (50 [77%]), with the majority from the West coast of Florida (46 [60%]) particularly in Sarasota, the area of recurrent Florida red tide. There were 18 (23%) callers from other parts of the US, including 1 from the US Virgin Islands (data not shown). The majority of the participants had heard about the Aquatic Toxins Hotline from a website (35 [39%]), newspaper or magazine article (16 [18%]), a friend or personal recommendation (13 [15%]), or a sign (7 [8%]). Many of the participants (38 [48%]) called with general information questions; of note, none of the participants reported calling because they or someone they cared for was sick or believed they had been exposed to a toxin. Overall, the majority of participants (59 [68%]) reported that they received an answer to the question that prompted their call to the Florida Aquatic Toxins Hotline (see Table 2). Furthermore, 59 (68%) reported that the Florida Aquatic Toxins Hotline was easy to use, with only 17 (20%) reporting otherwise. Finally, 69 (80%) of the participants reported that they would be likely to use the Florida Aquatic Toxins Hotline again, and 68 (79%) stated that they would recommend the Hotline to others. With regards to the specific information supplied by the automated system, 34 (39%) of participants reported speaking with a Poison Information Specialist; among these participants, 23 (74%) were satisfied and only two (7%) were dissatisfied (see Table 3). Among those participants who used the automated HAB menu, the largest number (46 [54%]) reported listening to the NOAA HAB Bulletin which gives current and forecasted information on the location of the Florida red tides; among these participants, 30 (70%) were satisfied and seven (17%) were dissatisfied. The second largest group (37 [44%]) reported listening to general information about Florida red tides; among these participants, 26 (70%) expressed satisfaction, and 8 (22%) were dissatisfied. Participants also reported listening to information about other marine toxin issues (28 [33%]) with 20 (71%) expressing satisfaction; Ciguatera fish poisoning (11 [13%]) with 10 (90%) expressing satisfaction; and blue green algae (cyanobacteria) (10 [12%] with 9 (90%) expressing satisfaction. At the end of the questionnaire, the participants were asked if there was anything in particular they thought should be done to improve the usefulness of the Florida Freshwater and Marine Health Hotline as an open ended question. The majority of the comments can be summarized as: a) wanting more up-to-date information (particularly geographic location of the Florida red tides) on even a daily basis; and b) wanting to speak with a person directly or listening to an automated system. Specifically, the participants recommended that the Hotline provide the geographic location of the Florida red tides on a map; provide more information to tourists in hotels; and make sure that the Poison Information Specialists were up-to-date with HAB and Florida red tide information. 4. Discussion This study was the first known systematic evaluation of the use of and satisfaction with educational outreach materials for HABs, specifically evaluating callers to an automated Florida Aquatic Toxins Hotline of the South Florida Poison Information Center. The majority (68%) of participants reported that they were satisfied with the information provided by the Aquatic Toxins Hotline; 80% that they would use the Hotline again; and 79% that they would recommend the Hotline to others. Furthermore, even when evaluating the specific services offered by the Aquatic Toxins Hotline automated system (including speaking directly with a Poison Information Specialist), the high level of satisfaction persisted. Of interest, only 8% of participants reporting hearing about the Aquatic Toxins Hotline from signs, with the rest learning about the hotline from a website (39%), newspaper or magazine article (18%), or friend (15%). Our evaluation demonstrated that the primary advantage of the new automated system is that it provided useful HAB information for the large majority of the callers, without requiring contact with a Poison Information Specialist. By decreasing the workload of purely informational calls for the Poison Information Specialists, the Hotline allows the Specialists to focus on those persons who need immediate assistance. 4.1. Limitations As seen in previous studies using the Poison Information data [18,19], only a small percentage (10%) of the callers were able to be re-contacted. This lack of participation in telephone surveys is part of an unfortunate and growing nationwide trend recognized by the evaluation research community [20,21]. Furthermore, the majority of the study participants (77%) gave zip codes of addresses within Florida; since the follow-up calls were made two months from the original call, it would appear that the majority of the participants were either part-time seasonal (i.e., “snow-birds”) or permanent residents. Based on the review of the zip codes and area codes of all Hotline callers, many were likely tourists, and thus were “lost to follow-up” when they could not be reached two months after their original call to the Hotline [20,21]. In addition, these participants were not entirely representative of the overall Hotline caller population since a greater proportion (39%) reported speaking with a Poison Information Specialist than was reported (27%) for the entire population of callers to the Florida Aquatic Toxins Hotline during the study period. However, based on the limited demographic information, the participants interviewed were similar to those contacted in other studies which have used the overall South Florida Poison Information Center Caller Database, i.e., predominantly white non Hispanic older females [18,19]. We did not identify many Hispanic callers in this study. Of note, most of the relevant marine HAB activity is concentrated on the Gulf Coast, while the majority of Florida’s Hispanic population is concentrated on the east coast of Florida. Finally, there were missing data (e.g. 22 out of 89 persons did not report their gender). 5. Discussion and Recommendations This Pilot Evaluation Study demonstrated that the additional outreach provided by the Hotline was successful in getting information to people who wanted it. It also suggested that there needs to be increased outreach and education efforts using a range of media to inform additional target populations about the Aquatic Toxins Hotline, such as residents and tourists, and their healthcare providers. For example, it has been shown in the case of ciguatera fish poisoning, that even in an endemic area such as Miami (Florida), the majority of healthcare providers do not recognize, nor (more important from the point of view of HAB surveillance) do they report cases of ciguatera to the Health Department even though it is a reportable illness throughout the US [22]. Therefore, additional outreach and education not only to the possible victims of HAB-related illness, but also for their healthcare providers is needed. Recommendations for improving the Aquatic Toxins Hotline automated HAB system include the following: 1) Existing automated materials need to be revised to incorporate an ongoing evaluation component, with a focus on tourist not just resident callers, and in coordination with ongoing efforts in providing geographic location [23]; 2) Existing materials will need constant updating; and 3) Ongoing training should be provided for the Poison Information Specialists (as well as other healthcare providers) concerning the possible exposures and health effects from the aquatic toxins as new HAB research data and resources are made available [13, 23–29]. In addition, as different HAB educational outreach materials and services are created, these materials and services need to be evaluated before and after implementation in the target populations to improve their quality and utility. Based on the participation rate in this study, rapid follow up is necessary to ensure an effective evaluation. This will require the incorporation of additional resources and collaboration with investigators with expertise in outreach, education and evaluation in future HAB activities.

Eur_J_Clin_Pharmacol-3-1-2071959

Adherence to HAART therapy measured by electronic monitoring in newly diagnosed HIV patients in Botswana

Aims This pilot study was designed to evaluate the feasibility and benefits of electronic adherence monitoring of antiretroviral medications in HIV patients who recently started Highly Active Anti Retroviral Therapy (HAART) in Francistown, Botswana and to compare this with self-reporting. Introduction Botswana is one of the countries worst affected by the HIV pandemic with a prevalence of approximately 17% of the entire population [1]. An estimated 38.5% of those aged 15–49 years are HIV-positive, and it is estimated that one in every eight children is born with HIV [2]. Since 2002 the nation has embarked on the provision of antiretroviral drugs for all its eligible citizens by implementing the MASA program.(The national antiretroviral therapy program was given the name MASA, the Setswana world for “dawn”). The use of potent antiretroviral combinations has provided unprecedented opportunities for effectively treating HIV disease by suppressing viral replication and has led to dramatic decline in HIV mortality [3, 4]. Adherence to antiviral regimens in HIV infected patients is essential for adequate suppression of viral replication. When adherence falls below a certain level, intermittent viremia will occur, and this may increase the chance of the development of resistant strains possibly followed by therapy failure [5–7]. In contrast, nonadherence to the prescribed antiretroviral regimens is associated with a rapid selection of resistant HIV strains resulting in treatment failure [8, 9]. The required high level of antiretroviral drug adherence in a poor resource setting remains therefore a serious concern. Assessment of adherence in HIV patients such as in this pilot-study may also provide tools to allow feedback and education on an individual health care provider–patient base. For this reason patients in the Botswana Infectious Disease Care Centers (IDCC) treatment program are urged at each visit to the IDCC facility to comply with the prescribed ART regimen. This occurs in three stages: (1) in group instruction sessions together with other HIV patients and individual counselling by a trained nurse or pharmacist, (2) by their individual health care provider (physician), and (3) by the pharmacist. In addition, patients are usually accompanied by a close family member who is asked to assist or remind patients of the pill intake (adherence partner). Reliable information about the actual tablet intake is a prerequisite for any form of management or modification of the adherence to therapy. It has been recognised that many of the traditional methods of assessing adherence such as pill counts, diaries, or self-reports are unreliable. Electronic monitoring enables the recording of the time points of pill bottle openings. This method also has drawbacks and may underestimate adherence [10] and, of course, it does not provide evidence of actual ingestion of the drug [11]. Despite these drawbacks it has been so far the closest to a gold standard for adherence measuring [12], although other methods remain of value. A new method to measure adherence to prescribed medication regimen is the use of electronic monitoring [10, 13, 14]. Such systems commonly rely upon a microprocessor located in the cap of the medication container, where time and date of each opening are recorded. Each cap opening and closing is assumed to reflect a single medication-taking event. The data stored in the microprocessor are transferred to a computer database and uploaded for analysis [11]. Other methods like self-reporting, pharmacy records, and pill counts tend to overestimate patient adherence by anywhere from 20–30% [15–21]. This study was designed as a pilot study to evaluate electronic adherence monitoring in an HIV infected patient group that was put on antiretroviral medication for the first time. A secondary objective was to compare the adherence measured by electronic monitoring with that of self-reporting by means of a medication diary. Methods Patients Thirty consecutive patients were recruited into the study during the period 13–30 October 2005. All were patients with an AIDS-defining illnesses and/or CD4 cell counts< 200 cells/mm3 (uL) who were offered HAART according to the Botswana Guidelines on Antiretroviral Treatment [2]. All patients were ARV-naïve. Design This was a trial in which treatment-naïve patients were monitored with regard to adherence to prescribed anti-HIV medication. Patients were not informed about the use, blinded of the electronic monitoring system, and were only asked to return their pill-bottles at each visit to the pharmacy when they returned for a refill and a consultation. Patients were also supplied with a self-reporting form. The study did not involve study related interventions and the subjects were not required to change behaviour in any way. The subjects were informed that the medication was supplied in special containers that had to be returned to the clinic but not about the monitoring system to prevent bias. The study was approved by the hospital management and the chief physician of the department of internal medicine. Treatment regimens The treatment regimen used consisted of 2-nucleoside reverse transcriptase inhibitors (NRTIs) plus 1 nonnucleoside reverse transcriptase inhibitor (NNRTI). Patients started the recommended first-line treatment with three different agents. These were zidovudine and lamivudine in a combination tablet Combivir (CBV) plus efavirenz (EFV) or nevirapine (NVP). Male and female patients who were not anaemic were prescribed Combivir medication whereas anaemic patients (Hb < 7.5 mmol) were given stavudine (d4T/Zerit) and lamivudine medication. The stavudine dose was adjusted according to bodyweight (30 or 40 mg). All patients except females in the reproductive age category were given once daily EFV. All females of the former category were prescribed NVP at a 200 mg once daily dose for 14 days which, after assessing the liver function parameters, was increased to 200 mg twice daily. The continuation of NVP or EFV was dependent on the absence of significant rise in hepatic enzymes (AST and ALT). Approximately 90% of those who started the NVP or EFV treatment are able to continue this medication. Patients returned to the IDCC after a month for a medical check and refill of their prescription unless clinical events dictated earlier visits to the clinic. The treatment starters were booked to see the doctor after the first 2 weeks of therapy. After seeing the doctor, the self recorded medication card and the electronic monitors were collected, and the data stored in the microprocessor were transferred to a database in the computer. Following this, each bottle was refilled and provided with a new label with medication instructions. Most patients received a refill for a period of 1 month, some however for a shorter period. The potential side effects were discussed with each patient. The results of the first analysis of the electronic monitors were not used in any counselling. The electronic monitors were MEMS IV Track Cap devices (Aardex, Zug, Switzerland) with a MEMS IV Communicator for read-out of the results. Study endpoints The primary study endpoint was the adherence level measured (over a minimal period of 6 weeks) by the percentage of days on which the patients took a correct dosing over the monitored period. Adherence was also expressed as the number of pill-intakes recorded on a self-reporting forms designed to reflect the intended schedule and timing of treatment. In the event a patient opened his/her bottle more than was prescribed (surplus opening), it was assumed that the patient correctly took the prescribed pills. However, occasions on which a patient opened less than the prescribed dose frequency were considered as adherence failures. Sequence and duration of trial period Each patient was immediately given counselling and made familiar with the ART treatment. During the counselling session emphasis was given with regard to the need of strict adherence of the prescribed medication and to methods to prevent disease transmission. They were also informed about the self-reporting form and given a pen to mark taking a treatment with a cross. ART medication was started thereafter and the adherence to the pill intake schedule was monitored by means of using electronic monitors and self-recording for a period of 6 weeks. At the start of the treatment, electronic monitors containing medication for a period of 2 weeks were provided. After an evaluation by the doctor at day 14 of treatment the electronic monitors and self-reporting form were collected and the data in the microprocessor were entered in the database. The electronic monitors were subsequently refilled with medication for the next period of 1 month. A new self-reporting form was also given. Patients were given 2 (in case of Lamivudine/Zidovudine + NVP or EVF) or 3 electronic monitors (in case D4T, 3TC, and NVP or EFV). Patients recruited in the study were asked to return the electronic caps and the self-reporting form on each occasion of a visit to the IDCC. A self-reporting form was issued at the start of the study. This form contains rows where the patient had to mark with x each time they took the pill at the correct time. As some people in Botswana could not read or write, this form was kept very basic. A pencil was given to every patient who participated in the study. Results A total of 30 HIV infected adults were enrolled in the study. In five patients full data could not be obtained because of various reasons. This leaves an evaluable group of 25 (9 male, 16 female; average age 35.6 years, range 22–55 years). Twenty patients completed the 6-week monitoring period and the mean follow-up period was 49 days (range 42–72 days). The reasons for lack of follow-up in the five patients varied, but in three patients it was due to mortality. In these three cases, as the relatives or nursing staff did not know about the value of the medication bottle (due to the blinding of the patients), the bottles were not returned. One patient was admitted to hospital where the nursing staff discarded the pill bottles. One patient failed to return for follow-up. Full follow-up was not obtained in another five patients for various reasons. This included technical failure of the compliance monitor in two cases and a change in the return date of the subjects who then received a refill in a normal container. Adherence assessed from the dosing histories compiled by electronic monitoring are shown in Table 1. Table 1Adherence (%) of the patients (n = 25) by MEMS caps and self-reportingPatient numberTCFollow-up (days)MEMSSelf reportLamivudine /zidovudine (bd)EFV (od)Lamivudine (bd)NVP (od/bd)d4T (bd)Drop out/non-retrievel11111001001001003227095100911003214100No data10010034151981009810054−−−−−−−−1614471100719573457370928196814410010010010092449810010098102141001001001003111448810088981224393100939313144988998100141−−−−−−−−2152449310098931621562100100623172−−−−−−−−2–1182448410084861924598100981002019100No data1001003211−−−−−−−−122172751007594232442110021292424293100939525266971001009726143100100100100271−−−−−−−−42814994No data969629156100100301472010020100avg4285988998928796Treatment codes (TC) are: 1 Lamivudine/zidovudine, Efavirenz (EFV); 2 Lamivudine/Zidovudine, Nevirapine (NVP); 3 Lamivudine, Nevirapine and stavudine (d4T); and 4 Lamivudine and Efavirenz. MEMS indicates the data combined for all different treatments. Patient numbers are not consecutive because patients who died have been omitted. Reasons for drop out and nonretrieval: 1 death; 2 MEMS thrown away; 3 missed by investigator (patients showed up on another date than the investigator expected); and 4 lost to follow-up Assuming that surplus opening of the bottles was associated with correct medication intake, the mean adherence level was 85% (SD = 23%, range 20–100%). When surplus openings were calculated as incorrect, the adherence decreased to 70% (SD=23%, range 14–100%) (data not shown). Seven patients (23%) had an adherence under 90%, a level at which virological failure increases substantially [10]. Examples of the medication records obtained from the electronic monitoring device are shown in Fig. 1 for a patient with good adherence and a patient with low adherence to the regimen. Fig. 1Examples of data of from patients with good and respectively poor adherence Adherence assessed by means of self-reporting The mean adherence assessed by means of self-reporting of medication intake was 98% with two (6%) patients recording adherence level lower than 90%. Adherence by self-reporting differed significantly from the adherence measured by the MEMS monitors method (p < 0.05, paired t-test). Three patients did not hand in their diary. Discussion In this pilot study we assessed the use of MEMS monitors to study the adherence to antiretroviral medication prescribed for HIV patients living in a low resource health care system. We demonstrated that assessment of adherence with this technique is feasible and may provide useful results. There was an approximately 30% drop-out rate of the recruited patients due to inability to recover data or early mortality. This may seem unacceptable in a well-resourced health care system. It is a reality in many countries where patients present with much more advanced disease, when there are sometimes great difficulties coming to the hospital, and patients can often not be reached by telephone or mail as they do not regularly have a postal address. It is likely that some association exists between failure to return to the hospital and adherence to the drug regimen and the current patient set therefore may reflect an overestimation of adherence. Self-reporting of medication intake has been shown to be less reliable than the MEMS monitors [22, 23]. Therefore, it is not surprising that patients recorded adherence levels which were considerably higher compared with those assessed by the presumably more objective electronic monitors. Clearly, none of the methods used to measure adherence record actual intake of medication, and there are even indications that at least in some instances self-reporting is a more accurate record of adherence [24]. Despite these findings we consider the MEMS monitors more appropriate for a developing country with a larger potential for illiteracy. Patients knowledge about the monitoring of compliance will likely affect the absolute level of compliance [25], but there are no reasons why the relative ranking of compliance amongst patient groups is affected, and the device can still be used to improve adherence. In this study we chose not to inform the patients about the use of the monitoring device. One of the critics often cited about the electronic monitor is that the opening of the bottle does not prove ingestion. However, it has been shown recently that projected plasma concentrations based on electronically compiled dosing histories correlates very well with directly measured concentrations [26]. It has also been shown that t in HIV patients electronically compiled dosing histories strongly correlates with viral suppression or the occurrence of virological failure [27]. It is therefore assumed that this method is a fair reflection of medication intake [28]. In this small study performed in HIV patients, some subjects opened their bottles more often than needed (surplus opening). Such surplus opening was not considered as nonadherence (in that case our reported adherence level would be lower). If surplus opening had led to extra intake of medication, it would not have contributed the endpoints of virological failure or the induction of resistance. We assume, therefore, that such surplus opening of the pill bottles had occurred for other reasons. Surplus openings occurred especially in the group of patients that had to take a once daily regimen together with twice daily regimens. These extra openings occurred often at the same time as when the prescribed twice-daily regimen was taken. This may have occurred because the patients were unsure about which bottle belonged to which medication. This may indicate that clearer labelling is essential, especially in an illiterate society. Under-opening most likely reflects nonadherence, although it cannot be excluded that in some cases patients removed several tablets at the same time. However, such behaviour is highly likely to increase the chance of erroneous medication intake and was for this reason registered as nonadherence. Several studies have indicated that medication adherence lower than approximately 90% increases the chance of virological failure and the development of resistant virus strains [12, 28, 29]. The result of this pilot study indicates that even after careful counseling and guidance, a significant number of patients did not manage to adhere sufficiently to the twice-daily pill intake regimen. This observation study was too short to allow an analysis of the clinical impact of the observed low adherence in such patients on the development of virological failure. Additionally, reliable measurement of viral load was impossible in the hospital. However, our data did help to identify some patients with low adherence at the start of treatment and allowed a diversion of scarce resources for extra counseling for such patients. The outcome of monitoring pill intake by the electronic monitors may therefore assist in timely counseling of patients with regard to their medication intake and persistence with the prescribed regimen. Adherence measurements by means of using electronic monitors together with self and adherence partner recording of pill intake are likely to be useful in a much needed larger, long-term and more comprehensive adherence study in a low-resource health-care-system setting. Such a study can contribute to directed efforts to optimise the treatment of HIV-infected patients worldwide.

FEBS_Lett-1-5-1964784

The RhoA transcriptional program in pre-T cells

The GTPase RhoA is essential for the development of pre-T cells in the thymus. To investigate the mechanisms used by RhoA to control thymocyte development we have used Affymetrix gene profiling to identify RhoA regulated genes in T cell progenitors. The data show that RhoA plays a specific and essential role in pre-T cells because it is required for the expression of transcription factors of the Egr-1 and AP-1 families that have critical functions in thymocyte development. Loss of RhoA function in T cell progenitors causes a developmental block that pheno-copies the consequence of losing pre-TCR expression in Recombinase gene 2 (Rag2) null mice. Transcriptional profiling reveals both common and unique gene targets for RhoA and the pre-TCR indicating that RhoA participates in the pre-TCR induced transcriptional program but also mediates pre-TCR independent gene transcription. 1 Introduction The T cell antigen receptor (TCR) complex comprises variable α/β subunits that recognise peptide/major histocompatibility (MHC) complexes and invariant signal transduction subunits of the CD3 antigen. A key stage in T cell development in the thymus is the selection of cells that have successfully rearranged their TCR-β locus. This occurs in T cell precursors, which do not express of the MHC receptors, CD4 and CD8 (double negative (DN) thymocytes) [1]. The DN stages of intrathymic differentiation can be followed by the sequential pattern of expression of CD44 and CD25. T cell progenitors enter the thymus as CD44+CD25− cells (termed DN1); these then express CD25 (DN2) and begin to rearrange T-cell receptor β loci. Cells then lose CD44 expression and continue β chain rearrangements to completion (DN3). Cells that successfully rearrange their TCR-β locus will express a functional receptor complex known as the pre-TCR comprising a TCR-β chain, the p-Tα subunit and the signalling subunits of the CD3 antigen [2–4]. When the pre-TCR is expressed at the cell membrane it promotes cell survival and entry into the cell cycle [5]; cells downregulate CD25 and transit to the DN4 pre-T cell subset. DN4 T cells undergo proliferative expansion and differentiate to CD4+CD8+ double positive (DP) cells. These cells then undergo TCR α-chain gene rearrangements and upon expression of a functional α/β TCR complex are subjected to positive and negative selection to generate CD4+ or CD8+ single positive (SP) thymocytes [6–8]. Normal pre-T cell development requires the coordination of a complex program of gene transcription by signal transduction networks mediated by the pre-TCR complex and cytokine and stromal signals [9,5]. Mice deficient for the recombinase activating (RAG) genes which are unable to rearrange TCR-β subunits and express a pre-TCR are unable to progress beyond the DN3 stage of thymocyte development [10]. Similarly, the absence of pre-Tα or CD3 subunits blocks thymocyte development at the DN3 stage [11,3]. The transition of thymocytes beyond the pre-T cell stage of thymocyte differentiation is also dependent on signal transduction networks mediated by tyrosine and serine kinases [3,12,13]. In this context, crucial responses are mediated by Rho family guanine nucleotide binding proteins such as RhoA, Rac-1 and CDC42 [14–17]. The importance of RhoA for thymocyte development has been demonstrated by studies of transgenic mice that express Clostridium botulinum C3-transferase under the control of T cell specific promoters such as the p56lck and CD2 promoters [17,15,18]. This toxin selectively ADP-ribosylates RhoA within its effector-binding domain and abolishes its biological function. Transgenic mice that express C3-transferase under the control of the p56Lck promoter have a small thymus and severely reduced numbers of peripheral T cells [15,17]. This phenotype is caused by survival defects in DN2 and DN3 thymocytes that lack Rho function [15]. During embryogenesis no cells progress beyond the DN1 stage but in adult mice a few T cell progenitors survive and develop to DN4s and beyond [15]. This ‘leakiness’ either reflects selection of cells that compensate for loss of Rho function in DN2/3 thymocytes or reflects heterogeneous and asynchronous expression of the lck promoter in adult DN thymocytes. The few DP thymocytes and mature T cells found in adult Lck-C3 transgenic mice have numerous defects including reduced survival, proliferation, integrin mediated cell adhesion and defective cell motility [15,19]. A complementary strategy to probe Rho function in the thymus used the CD2 locus control region (LCR) to target C3 transferase to T cell progenitors (CD2–C3 mice) [18]. The inhibition of Rho function at the DN2/3 stage in CD2–C3 mice is not leaky and causes T cells to become blocked in differentiation at the DN3 stage of thymus development. CD2–C3 mice thus have a thymic phenotype indistinguishable from the phenotype of Recombinase gene null mice or mice lacking key structural or signaling components of preTCR complex [10,18,20,21]: thymocyte development is blocked at the pre-T cell/DN3 stage [18]. The basis for the failure of pre-T cell differentiation in CD2–C3 transferase mice is not known. One way to address this issue is to use microarray gene expression profiling to determine the impact of losing Rho function on transcriptional responses in pre-T cells. Previous studies in transformed cell lines have identified a role for RhoA in regulating activating protein-1 (AP1) family of transcription factors [22–24] but transcriptional targets for RhoA signal transduction in primary non-transformed cells have not been explored. The present data show that RhoA regulates expression of genes encoding members of the Fos/Jun and early growth response (Egr) family of transcription factors but also has an impact on expression of genes regulating diverse biological functions including serine/threonine kinases, protein phosphatases, enzymes that regulate protein biosynthesis and proteins that regulate nuclear structure and function. 2 Materials and methods 2.1 Mice Mice were bred and maintained under specific pathogen-free conditions in the transgenic animal unit. RAG2−/− and C3 transgenic mice, which selectively express the bacterial toxin C3-transferase under the control of CD2 promoter and locus control region in the thymus, have been described in detail elsewhere [10,18]. 2.2 Flow cytometric analysis Fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), and biotin-conjugated antibodies were obtained from Pharmingen (San Diego, CA). Tricolour (PE/Cy5 and APC/Alexa750) conjugated antibodies and fluorophore-streptavidin conjugates were from Caltag (Burlingame, CA). Thymocytes were stained for cell surface markers and analysed on a FACS Calibur (Becton Dickinson Immunocytometry Systems Franklin Lakes CA). Data were analysed using CellQuest BDIS or FlowJo (Treestar Inc, Ashland OR) software. CD4−CD8− double negative thymocyte subsets were analysed for CD44 and CD25 expression following lineage exclusion of mature DP and SP cells as well as non-T cell lineage cells using a cocktail of biotinylated antibodies (CD4, CD8, CD3 B220, Mac-1, NK, Gr-1, and γδ) revealed with streptavidin-tricolour and costained with CD25-FITC, CD44-PE and Thy1.2-APC. Intracellular phospho-S6 and TCR-β intracellular staining was carried out on thymocytes pre stained to identify DN3 and DN4 subpopulations. Cells were subsequently fixed in 1% paraformaldehyde for 10 min at room temperature, washed in PBS and permeabilized with saponin buffer (0.5% saponin, 5% FBS, 10 mM HEPES [pH 7.4] in PBS) for 10 min at room temperature. Permeabilized cells were incubated with PE-conjugated TCRβ antibody and phospho S6 antibody for 45 min at room temperature, washed in saponin buffer and subsequently stained with FITC conjugated donkey anti rabbit IgG (Jackson Immunoresearch, West Grove PA). Cells were analysed on an LSR Flow Cytometer (BDIS). 2.3 RNA extraction Thymocytes were isolated from 4- to 6-week-old RAG2−/−, CD2–C3 and C57BL/6 control mice. DN3 pre-T cells were purified firstly by removing CD4, CD8 DP cells using MACS CD4+ and -MACS CD8+ T cell isolation kits (Miltenyi Biotec, Ltd.) followed by magnetic autoMACS separation. CD4/CD8 depleted thymocytes were then labelled with Thy1.2-APC, PE conjugated CD4, CD8, CD44 and TCRγδ antibodies, FITC conjugated CD25 antibodies and the DN3 subpopulation (Thy1+CD4−/CD8−/CD44−/CD25+) was purified by cell sorting on a FACS Vantage (BDIS) using Cell Quest software (BDIS). All sorted cells were analysed by flow cytometry and only used for experiments if the purity was 95–98%. Total RNA was extracted from sorted cells using the Qiagen RNeasy kit according to manufacturers instructions. Total RNA was quantified using the RNA 6000 Nano LabChip® kit and analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies). Total RNA from up to 10 thymi were pooled for microarray analysis and quantitative PCR. 2.4 Microarray Microarray analysis was carried out by the Finnish DNA Microarray Centre at the Centre for Biotechnology, Turku, Finland. For Affymetrix sample preparation, 100–1000 ng of total RNA was used as the starting material to synthesise target cRNA using the GeneChip® Eukaryotic Small Sample Target Labeling Assay Version II according to manufacturers’ instructions (Affymetrix, Santa Clara, CA). The cRNA target sample was hybridised to the Affymetrix Mouse Genome 430 2.0 Array (45,101 probes sets and expressed sequence tag (EST) clusters). Data was analysed with Affymetrix Gene Chip Operating Software (GCOS) version 1.1 and filtered according to recommendations of the manufacturer. Comparison analysis was used to compare the expression profiles from two arrays – results were obtained by analyzing the experimental array in comparison to the baseline array. The global method of scaling was used for normalizing hybridization intensity between arrays. The statistical algorithm used by GCOS defined the expression status of each gene-specific probe set as not changed, increased, marginally increased, decreased or marginally decreased. Genes that were absent or unchanged between comparisons were excluded from the results. Comparison analysis defines a gene as up-regulated if the signal log ratio between the baseline sample and the experimental sample is larger than 1 (2-fold) and the experimental sample is present. Similarly changed genes were further analyzed/classified using the gene ontology tool (www.Affymetrix.com) and represented by biological function. Gene lists were complied and sorted by genes that were either commonly or uniquely regulated in CD2-C3 and RAG2−/− thymocytes. 2.5 Real-time RT-PCR analysis Purified total RNA (200 ng) was reverse transcribed using the iScript™ cDNA synthesis kit (BioRad). Real time RT-PCR was performed in a 96-well plate using iQ™ SYBR Green based detection (BioRad) on a BioRad iCycler in 20 μl reaction volume containing 1 μl cDNA (20 ng), 0.8 μl 10μM sense and antisense primers, 10 μl iQ™ SYBR Green supermix, and 4 μl nuclease free water. Each reaction was performed in duplicate and each experiment repeated in triplicate. 18S rRNA levels were used to normalise RNA concentrations between samples and the relative mRNA levels were calculated using the equation:where E is the efficiency of PCR, ct is the threshold cycle, u is the mRNA of interest, r is the reference gene (18S rRNA), s is the sample and c is the control sample. Primers used for RT-PCR were designed using Beacon Designer 2 software. Primer sequences are: Egr1 forward 5′-ACAGAAGGACAAGAAAGCAGAC-3, reverse 5-CCAGGAGAGGAGTAGGAAGTG-3′, Fos forward 5′-CTACTGTGTTCCTGGCAATAGC-3′, reverse 5′-AACATTGACGCTGAAGGACTAC-3′, Egr3 forward 5′-TGACCAACGAGAAGCCCAATC-3′, reverse 5′-GCTAATGATGTTGTCCTGGCAC-3′, Jun forward 5′-CGCCTCGTTCCTCCAGTC-3′ reverse 5′-ACGTGAGAAGGTCCGAGTTC-3′, JunB forward 5′-CTTCTACGACGATGCCCTCAAC-3′, reverse 5′-GTTCAAGGTCATGCTCTGTTTTAGG-3′, Nur77 forward 5′-CCTGTTGCTAGAGTCTGCCTTC-3′, reverse 5′-CAATCCAACACCAAAGCCACG-3′, 18S RNA forward 5′-GTAACCCGTTGAACCCCATT-3′, reverse 5′-CCATCCAATCGTAGTA-3′. 3 Results and discussion Mice expressing the RhoA inhibitor Clostridium Botulinum C3 transferase under the control of the CD2 locus control region (LCR) have been described previously [18]. Loss of RhoA function in CD2-C3 mice results in loss of CD4/CD8 double positive thymocytes (Fig. 1A) with a block in thymocyte development at the DN3 stage (Fig. 1B). Rag2−/− mice also block thymus development at the DN3 stage (Figs. 1A and B) due to failed expression of the pre-TCR complex. Despite this superficial similarity, loss of Rho function does not prevent TCR beta locus rearrangements although there is a reduced frequency of DN3 cells expressing intracellular TCR-β subunits (Fig. 1C). Ectopic expression of transgenic TCR complexes cannot reverse the developmental block in CD2–C3 thymocytes demonstrating that failed expression of TCR complexes does not explain the developmental defects caused by loss of Rho function [18]. Rho thus appears necessary for pre-TCR function although these results do not discriminate between a direct role for RhoA in pre-TCR signalling or a role for this GTPase in the cytokine/stromal signalling pathways that synergise with pre-TCR signals to control pre-T cell differentiation [25–27]. CD2–C3 transferase mice provide a good model system to probe the immediate transcriptional consequences of losing RhoA function in pre-T cells since the CD2-LCR initiates expression of transgenes in T cell progenitors as they transit from the DN2 to DN3 stage of thymocyte development [18]. Accordingly, it is possible to isolate DN3 thymocytes ex vivo that have only just switched on expression of C3 transferase and prepare RNA for DNA microarray analysis. The Affymetrix Mouse Genome 430 2.0 array, representing 39 000 murine gene transcripts, was used to transcriptionally profile DN3 pre-T cells purified from the thymi of CD3–C3 transgenic mice. In these experiments DN3 thymocytes from wild type control mice were transcriptionally profiled and used for comparisons. The microarray data were analyzed with the Affymetrix Gene Chip Operating Software (GCOS, version 1.1) by comparing the gene expression profiles of CD2–C3 pre-T cells to the profile of normal cells to reveal changes above or below wild type levels. DN3 pre-T cells expressed approximately 18 000 genes and there were only small changes in the transcriptional profile of CD2–C3 DN3 thymocytes compared to control wild type DN3s. We focused our analysis on ⩾2-fold changes, which were further analyzed using the Affymetrix gene ontology tool. A full list of the genes regulated 2-fold or greater by loss of RhoA function in DN3 thymocytes is shown in Supplementary Fig. 1. There were approximately 18 000 genes expressed in wild type DN3 thymocytes: loss of RhoA function caused a ⩾2-fold decrease in expression of 383 genes and a ⩾2-fold increase in expression of 190 genes. RhoA regulated genes in DN3 thymocytes encode proteins with diverse biological functions including serine/threonine kinases, protein phosphatases, enzymes that regulate protein biosynthesis. This point is illustrated in Fig. 2A, which shows the genes whose expression is regulated more than 10-fold. The largest decrease was in expression of the gene encoding FosL2, a Fos family transcription factor (630-fold decrease) but other large changes included a subunit of eukaryotic translation initiation factor 2, Protein Kinase C beta and the membrane receptor CD69. There was also a loss of expression of genes encoding proteins that regulate nuclear structure and function such as genes encoding histones that are involved in nucleosome assembly. In a similar category there was reduced expression of mRNA encoding special AT-rich sequence binding protein 1 (SATB1) in CD2-C3 DN3s (Supplementary Fig. 1). SATB1 acts as a scaffold for chromatin modifiers and is known to regulate higher order chromatin structure and to regulate gene expression by acting as a “docking site” for several chromatin remodeling enzymes [28–30]. Of 55 genes whose expression was decreased ⩾5-fold, 23 encoded transcription factors; of 384 genes decreased ⩾2-fold, 63 were transcription factors (Fig. 2B). Transcription factors downregulated in CD2-C3 DN3 thymocytes included Kruppel family transcription factors KLF4, KLF6 and KLF9. There was also loss of expression of members of the immediate early gene family Egr1 and Egr3, AP-1 family members Fos, FosL2, FosB, Jun, JunB and downregulated expression of genes encoding Ets1 and Nurr77. To validate the microarray analysis, quantitative real-time PCR was used to compare expression of a selection of these different transcription factors in wild type versus CD2-C3 thymocytes (Fig. 2C). The experiments confirmed the reduction in Fos, Jun, JunB, Egr1 and Egr3 and NURR77 in CD2–C3 DN3s compared to wild type controls. A number of gene targets for Egr transcription factors have been described [31] and in this context, RhoA inactivation caused loss of expression of multiple members of the MAP kinase phosphatase family DUSP 1, 2, 4, 6, 16. The latter are induced in negative feedback mechanisms that modulate MAP kinase activity and have been described as targets for Egr family transcription factors [31–33]. The loss of DUSP expression is thus almost certainly a secondary consequence of failed expression of Egr molecules. Egr1 and Egr3 are well characterised pre-TCR induced genes [34–38]. To verify this we compared the transcriptional profile of CD2-C3 DN3s with Recombinase gene 2−/− (RAG2−/−) DN3s which lack expression of the pre-TCR complex because they fail to rearrange their TCR beta locus. Of the 23 transcription factors downregulated ⩾5-fold in CD2–C3 thymocytes 18 were also downregulated in Rag2−/− DN3s (Fig. 2D) and these included Egr1, Egr3, Fos, Fosl2, FosB, Ets1, Nurr77. These microarray analyses were confirmed by quantitative real-time PCR analysis of wild type versus Rag2−/− thymocytes (data not shown). The common loss of expression of members of the Egr family in CD2–C3 and Rag2−/− pre-T cells is thus consistent with a RhoA requirement for the pre-TCR induced gene transcription. However, comparisons of the total number of genes regulated uniquely in CD2–C3 and Rag2−/− DN3s (Fig. 3) (VENN diagrams) indicate that there are a number of gene changes in CD2–C3 cells that are not seen in Rag2−/− DN3s. The full list of genes that were commonly and uniquely regulated 2-fold and greater in CD2 C3 versus Rag2−/− pre-T cells is shown in Supplementary Fig. 2. In terms of ⩾ 2-fold, gene repression, CD2-C3 and Rag2−/− DN3s had 180 gene targets in common with 202 genes being downregulated uniquely in CD2–C3 pre-T cells and 120 genes downregulated uniquely in Rag2−/− cells. In terms of ⩾2-fold gene increases, CD2–C3 and Rag2−/− DN3s had 65 gene targets in common, 129 genes that were increased uniquely in CD2-C3 DN3s and 182 increased uniquely in Rag2−/− cells (Fig. 3). The common transcriptional changes caused by loss of the pre-TCR in Rag2−/− DN3s or loss of Rho function in CD2–C3 mice indicates that Rho function is necessary for some pre-TCR induced transcriptional responses. However, there are genes downregulated in DN3s lacking RhoA function that are unchanged in Rag2−/− DN3s (Supplementary Fig. 3) indicating that RhoA is not just required to mediate the preTCR induced gene program but may also participate in cytokine/stromal cell-initiated signalling pathways that control pre-T cell differentiation. The genes uniquely lost in CD3–C3 DN3 thymocytes include serine kinases (Protein Kinase C beta and Map3k14); transcription factors (SpiB and Dlx1) and regulators of protein biosynthesis (Eif2s3y) (Supplementary Fig. 3). One gene of interest lost in CD2–C3 DN3s but not Rag2 null DN3s was Hes-1 which is induced by Notch receptor-ligand interactions. Notch signalling is required throughout DN to DP stage of thymocyte development to support T cell metabolism and survival [39–42]. A RhoA requirement for Notch signalling has not been described but it is known that RhoA is necessary for integrin mediated cell adhesion and thymocyte cell migration [19]. Accordingly, decreased Hes-1 expression could reflect that pre-T cells lacking Rho function cannot make normal contacts with stromal cells that express the Notch ligands [43,42,44,45]. There were also unique transcriptional changes seen in Rag2−/− cells that were not found in the CD2–C3 DN3s (Supplementary Fig. 4) which indicates that the pre-TCR can regulate a program of gene transcription via RhoA independent pathways The unique changes in Rag2−/− cells not surprisingly included loss of expression of rearranged TCR-β subunits. One other gene downregulated in Rag2−/− DN3s but not CD2–C3 DN3s was RPS6, which encodes the ribosomal S6 subunit. In this respect, we recently described high basal levels of S6 phosphorylation in ex vivo β selected DN3s and no detectable S6 phosphorylation in ex vivo Rag2−/− DN3s [13]. The data in Fig. 4 quantify S6 phosphorylation in CD2–C3 DN3s compared to wild type cells. Normal DN3s are heterogeneous for phosphoS6 with the majority of cells being phosphoS6low but a significant percentage of cells are phosphoS6high. The DN3 thymocyte subpopulation can be subdivided into cells that have not yet completed TCR-β locus rearrangements and those that express a functional TCR-β subunit that allows surface expression and signalling of the pre-TCR complex. Analysis of TCRβ expression by intracellular staining revealed that DN3s that express icTCR-β chains are generally phosphoS6high whereas DN3s that are icTCR-β null are uniformly phosphoS6low. In CD2–C3 DN3s there were also high levels of phosphoS6 in TCR-β high cells. The presence of normal S6 phosphorylation in β selected DN3s lacking Rho function is a further indication that Rho is necessary for a subset of responses in DN3 thymocytes but does not globally block signalling. One important question is whether the transcriptional changes in CD2–C3 DN3s explain why loss of RhoA function results in failed thymocyte differentiation? In this respect, microarray analysis of Rag2−/− pre-T cells revealed decreased expression of TCR-β subunits as a major defect (Supplementary Fig. 4) and failed expression of the TCR-β subunit is indeed responsible for the DN3 developmental block in Rag2−/− mice. In CD2–C3 DN3s there were several gene defects that would explain why RhoA function is essential for pre-T cell development. For example, RhoA is required for expression of Egr3 and Ets1 and loss of either of these transcription factors inhibits pre-T cell proliferation and is suboptimal for transition through early pre-TCR-dependent stages of thymocyte development [46,36]. Loss of individual Egr family or AP-1 transcription factors does not completely abrogate thymocyte differentiation but this reflects that there is considerable redundancy in AP-1 complexes due to the ability of different family members to pair as dimers [47–49]. The simultaneous elimination of Egr1, Egr3, Ets1 and multiple Fos/Jun family members in CD2–C3 DN3s would circumvent the possibility of redundancy [50,51]. This comprehensive loss of AP-1 activity in pre-T cell lacking RhoA function plus the decreased expression of other transcription factors would rapidly cause global changes in the transcriptional program of a cell. Moreover, the transcription factor defects would be exacerbated by the decreased expression of chromatin modifying enzymes such as SATB1. In summary, loss of Rho function or failed expression of the pre-TCR complex in Rag2−/− null mice block T cell development at a common stage. The present data show that the genetic consequences of loss of Rho function versus loss of pre-TCR expression for T cell progenitors are not identical. The present results also show that loss of Rho function in pre-T cells results in downregulation of genes encoding members of the Fos/Jun and Early growth response (Egr) family of transcription factors. The collective loss of these transcription factors and the resultant secondary genetic changes explains why loss of RhoA function prevents pre-T cell development.

Behav_Brain_Res-1-5-2148464

Dorsal hippocampal N-methyl-d-aspartate receptors underlie spatial working memory performance during non-matching to place testing on the T-maze

Previous lesion studies have suggested a functional dissociation along the septotemporal axis of the hippocampus. Whereas the dorsal hippocampus has been implicated in spatial memory processes, the ventral hippocampus may play a role in anxiety. However, these lesion studies are potentially confounded by demyelination of fibres passing through the lesion site, and the possibility of secondary, downstream changes in associated brain structures as a consequence of their chronic denervation following the lesion. In the present study, we have used the microinfusion of muscimol to temporarily inactivate either the dorsal or ventral hippocampus in order to re-examine the contribution of the hippocampal sub-regions to spatial memory. Microinfusion studies spare fibres of passage and offer fewer opportunities for compensatory changes because the effects are transient and short-lasting. Rats were infused prior to spatial working memory testing on a non-matching to place T-maze alternation task. Spatial working memory was impaired by dorsal but not ventral hippocampal inactivation. In a second experiment, infusion of the NMDAR antagonist, D-AP5, into dorsal hippocampus also impaired spatial working memory performance, suggesting that NMDAR function within the dorsal hippocampus makes an essential contribution to this aspect of hippocampal information processing. 1 Introduction A role for the hippocampus in certain kinds of memory is well established. In rodents, the hippocampus has been particularly associated with spatial learning [1]. Complete hippocampal lesions have repeatedly been shown to produce robust and lasting impairments on spatial memory tasks [2,3]. More recently, however, these spatial memory functions have been ascribed specifically to the dorsal (or septal) portion of the rat hippocampus [4,5]. For example, selective cytotoxic lesions restricted to dorsal hippocampus produce spatial learning impairments on tasks like the Morris watermaze [6, 7, see also 8]. In contrast, lesions of the ventral hippocampus are without effect on spatial memory tasks, although conversely, they, but not dorsal hippocampal lesions, are associated with reduced anxiety [9–11]. This double dissociation suggests that different hippocampal sub-regions may mediate different aspects of hippocampal function [5]. Thus far, the conclusion that spatial learning is primarily a function of dorsal hippocampus has largely been based on the results of such cytotoxic lesion studies. However, this approach is potentially confounded in that any behavioural sequelae may be the result of indirect effects of the lesion procedure on other brain areas, rather than as a direct consequence of cell loss in the target region. For example, although the cytotoxic lesion approach is a clear improvement over more traditional techniques (e.g. aspiration, electrolytic or radiofrequency-generated lesions), in terms of sparing fibres of passage [12], there nevertheless remains some evidence of demyelination of nerve fibres passing through the lesion site even with this approach [13,14]. Furthermore, it is also possible that any behavioural effects of the lesion could reflect a secondary, downstream change in an associated brain structure as a consequence of the chronic denervation of that structure following the lesion [15]. It is therefore important to re-examine the dorsal/ventral dissociation using alternative experimental approaches. For example, it is possible to produce a temporary, reversible inactivation of a specific brain region by microinfusing the GABAA agonist muscimol [16], which avoids many of the potential confounds associated with the lesion approach. Microinfusion studies offer fewer opportunities than lesion studies for compensatory changes because the effects of the manipulation are transient and short-lasting. They also permit within-subjects comparisons. Somewhat surprisingly, the effects of dorsal and ventral hippocampal inactivation on spatial working memory performance have never been fully assessed. In the present study, we therefore compared the effects of dorsal and ventral hippocampal infusions of muscimol on spatial learning using a spatial working memory, non-matching to position (discrete trial, rewarded alternation) paradigm on the elevated T-maze [17]. This task is especially sensitive to hippocampal dysfunction (see effect sizes in [7], p. 1184), and is dramatically impaired by dorsal but not ventral hippocampal lesions [7,9,18]. We therefore compared dose-response functions for muscimol infusions into both hippocampal sub-regions (Experiment 1). In a second experiment, the importance of N-methyl-d-aspartate receptor (NMDAR) function, specifically in the dorsal hippocampal subregion, for spatial working memory performance was assessed. The role of NMDARs in spatial working memory is well established [19,20]. Intracerebroventricular (i.c.v.) infusion of the NMDAR antagonist, d(−)-2-amino-5-phosphonopentanoic acid (AP5) resulted in a delay-related impairment in choice accuracy on the same spatial, non-matching to place T-maze task [21]. However, the i.c.v. route of drug delivery results in NMDAR blockade in numerous brain regions, and not just the hippocampus [22]. In view of the proposal that it is the dorsal subregion of the hippocampus that is crucial for spatial working memory performance, we therefore also examined the effects on spatial non-matching to position of direct infusions of AP5 specifically into dorsal hippocampus (Experiment 2). 2 Methods 2.1 Subjects Thirty male Lister-hooded rats (Harlan Olac, Bicester, UK; 250–363 g at the time of surgery) took part in Experiment 1. Nine male Lister-hooded rats (270–370 g at the time of surgery) took part in Experiment 2. All rats were experimentally naïve and maintained on a 12 h light/dark cycle (lights on at 7:00 a.m., with testing during the light phase). During the experiment, the animals were maintained at ∼85% of their free-feeding weight but had access to water ad libitum. Rats were housed in groups of 2–3 prior to surgery but were singly housed after cannulae implantation. The experiments were conducted in accordance with the United Kingdom Animals Scientific Procedures Act (1986); under project license number PPL 30/1505. 2.2 Apparatus In Experiment 1, rewarded alternation took place on an elevated (∼1 m above the floor), low-walled wooden T-maze consisting of a start arm (80 cm long; 10 cm wide) joined to two identical goal arms (60 cm × 10 cm), with each arm surrounded by a 1 cm ridge. In Experiment 2, rewarded alternation took place on an elevated Y-maze (∼80 cm above the floor). Each of the three arms (50 cm × 9 cm) was surrounded by a 0.5 cm ridge and extended from a hexagonal central platform (14 cm diameter). The T- and Y-mazes contained stainless-steel food wells at the far end of each goal arm and each maze was located in a well-lit testing room containing prominent extra-maze cues such as wall posters. 2.3 Procedure In both experiments, rats were trained pre-operatively. Habituation to the maze took place over a five-day period. For the first three days rats were placed on the maze in pairs and left to explore and collect food rewards for 10 min. During days four and five, the rats were placed individually on the maze and allowed to explore freely for five minutes. By this stage all the rats ate from the food wells at the ends of the arms. Training on the rewarded alternation task then followed. Each trial in rewarded alternation had two runs: in the first, one of the goal arms was blocked, allowing the rat to enter only the other goal arm, whereupon it received 1 food pellet (45 mg Rodent Diet Formula A/I, Noyes, Lancaster, NH). During the second run, the block was removed and the rat was placed on the maze and given a free choice of either arm. Rats received two pellets for choosing the previously unvisited arm (i.e. for alternating). Choosing the arm previously visited in the sample run yielded no reward. The time between the sample and the choice runs was approximately 10 s (Experiments 1 and 2a) or 30 s (Experiment 2b). Left/right allocations for the sample and choice runs were pseudo-randomised over ten trials per day, with no more than three consecutive sample runs to the same side. The inter-trial interval was ∼3–4 min. Prior to surgery, each rat was trained on the task until they achieved a criterion of at least 80% correct alternation over two consecutive days. In Experiment 1, allocation to surgery groups (dorsal or ventral cannulation) was based on pre-operative performance. Rats were anaesthetised with Avertin (0.29 g/kg; i.p.) and placed in a stereotaxic frame with the head level between bregma and lambda. An incision of the scalp was made along the midline, followed by a craniotomy. Stainless-steel guide cannulae (23-gauge, 12 mm) were implanted bilaterally into either the dorsal (n = 14; AP: −3.6 (from bregma); M–L: ±2.8 (from bregma); D–V: −0.8 (from brain surface)) or ventral HPC (n = 16; AP: −5.2 (from bregma); M–L: ±4.9 (from bregma); D–V: −3.6 (from brain surface)). In Experiment 2, cannulae were only implanted into the dorsal HPC (n = 9; coordinates as above). Cannulae were secured with three skull screws and acrylic dental cement. Microinjectors (31-gauge, 14 mm) were 2 mm longer than the guide cannulae giving a targeted D–V coordinate (from brain surface) of either −2.8 mm (dorsal) or −5.6 mm (ventral). Stylets were inserted into the guide cannulae to prevent infection and blockages. Rats were allowed a minimum of five days post-surgical recovery after which they received training in the absence of any infusions. Rats failing to score 80% correct alternation on these sessions were given additional training. 2.4 Drugs In Experiment 1, muscimol (C4H6N2O2(1/2 H2O); Tocris, Bristol, UK) was initially dissolved in normal saline (0.9% NaCl) at a concentration of 1 mg/ml. This stock was then diluted to create individual aliquots at each of four concentrations (0.6 mg/ml, 0.3 mg/ml, 0.15 mg/ml, and 0.075 mg/ml), which were then frozen. Saline served as the vehicle. In Experiment 2, muscimol (0.3 mg/ml) and AP5 (d(−)-2-amino-5-phosphonopentanoic acid; 5.9 mg/ml) were dissolved in PBS (pH 7.4), which served as vehicle. On infusion days, the aliquots were defrosted and used to back-fill the microinjectors, which were connected via polyethylene tubing to a pair of Hamilton syringes (10 μl) driven by a microsyringe pump (SP250i, World Precision Instruments, England). Each rat was restrained firmly in a towel, the stylets were removed, and the microinjectors inserted through the guide cannulae into the hippocampus. Drug or vehicle (0.5 μl per side) was infused into the dorsal or ventral hippocampus at a rate of 1 μl/min (i.e. for 30 s), and the microinjectors were kept in place for an additional 60 s to allow the drug to diffuse. Infusions into each hemisphere were made simultaneously and were given 15 min prior to testing. In Experiment 1, rats received five infusions in total (saline and four doses of muscimol: 0.3 μg/side, 0.15 μg/side, 0.075 μg/side, 0.0375 μg/side). In Experiment 2a, rats received three infusions (PBS, AP5: 2.95 μg/side, muscimol: 0.15 μg/side) and in Experiment 2b, the same rats received a further 4 infusions (PBS × 2, AP5: 2.95 μg/side × 2), making seven infusions in total. Infusions were given in a counterbalanced, pseudo-random order with at least 48 h between each infusion. In addition, performance was assessed again 24 h after each infusion and rats failing to score 80% or more on these drug-free sessions received extra training before the next infusion was administered. 2.5 Histology On completion of the experiment, rats were perfused transcardially and their brains were removed, sectioned, and stained with cresyl violet for verification of the injection sites. 3 Results 3.1 Experiment 1 In Experiment 1, of the 30 rats that began the experiment, 23 rats completed all testing and had accurate bilateral cannulae placements and were included in the final analyses (10 dorsal, 13 ventral; Fig. 1). The intra-hippocampal implantation of guide cannulae did not result in any post-surgical deficits in rewarded alternation in either dorsal (mean score = 94%) or ventral (mean score = 90%) cannulated rats. Indeed post-operative performance was slightly better than pre-operative performance in both groups. Student's t-tests carried out for each group confirmed that there were no differences between pre-operative and (uninfused) post-operative performance (dorsal: t(9) = −0.483; p > 0.2; ventral: t(12) = −1.15; p > 0.2). The amount of alternation following saline infusion into either dorsal or ventral hippocampus was similar and comparable to uninfused performance (dorsal = 88%; ventral = 85%). In contrast, bilateral infusion of muscimol into the dorsal hippocampus produced a decrease in alternation levels (see Fig. 2). These data were analyzed using a repeated-measures ANOVA [model: cannula placement2 × (drug treatment5 × S23)]. The ANOVA revealed a significant effect of cannula placement (F1,21 = 14.44; p < 0.001) and drug treatment (F4,84 = 9.01; p < 0.001) but the interaction was not quite significant (F4,84 = 2.05; p = 0.09). Analysis of simple main effects and subsequent pairwise comparisons (using Sidak's method, see [23]) found an effect of cannula placement at three doses of muscimol: 0.0375 μg/side (F1,21 = 8.94; p < 0.01), 0.075 μg/side (F1,21 = 6.94; p < 0.05) and 0.3 μg/side (F1,21 = 4.45; p < 0.05) with the dorsal group exhibiting greater impairment than the ventral group. The groups did not differ following saline infusion (p > 0.2) or when the muscimol dose was 0.15 μg/side (p > 0.1). Simple main effects analysis also revealed an effect of drug within the dorsal cannula placement group (F4,18 = 6.86; p < 0.005), with all doses of muscimol leading to performance impairment compared to saline infusion. In contrast, there was no effect of drug within the ventral placement group (F4,18 = 2.7; NS). In other words, although there was some suggestion that performance was decreasing as the dose of muscimol increased (see Fig. 2), no individual dose of muscimol into the ventral hippocampus significantly impaired performance compared to saline infusion. On alternate days, the rats received 10 trials of rewarded alternation in the absence of any infusions to investigate the possibility of permanent or progressive damage to the targeted regions. Importantly, this data showed that rats from both groups alternated on more than 85% of trials 24 h after receiving infusions. Post-infusion performance of dorsal and ventral cannulated rats was investigated with a repeated-measures ANOVA [model: cannula placement2 × (block5 × S23)]. The ANOVA found no effect of cannula placement (F1,21 = 2.37; NS), or block (F4,84 = 0.49; NS), and there was no interaction (F4,84 = 1.99; NS). This result demonstrates that the T-maze impairments identified in the previous analysis were reversible and only evident while the drug was actually present in the hippocampus. 3.2 Experiment 2 In Experiment 2, all nine rats had accurate bilateral dorsal hippocampal cannulae placements (Fig. 1). Experiment 2 was divided into two stages. Procedurally, Experiment 2a was identical to Experiment 1. Experiment 2b was identical to Experiment 2a except that a 30 s delay was interposed between the sample and choice runs of each trial of rewarded alternation (see [21]). In Experiment 2a, the amount of alternation following PBS infusion into the dorsal hippocampus was similar and comparable to uninfused performance (uninfused = 90%, PBS = 83%). In contrast, there was a substantial drop in choice accuracy following the infusion of either muscimol (58%) or AP5 (67%). A one-way repeated-measures ANOVA [model: drug condition2 × S9], found a main effect of drug condition (F2,16 = 6.3; p < 0.01) and post hoc pairwise comparisons (Student's Newman–Keuls) revealed significant differences between the vehicle and both drug conditions (p < 0.05) but not between the two drug conditions (p > 0.2). The introduction of the 30 s delay in Experiment 2b did not lead to a further drop of performance in either infusion condition (PBS = 83%; AP5 = 72%). Although not strictly appropriate (since the data were not derived in a counterbalanced manner), a repeated measures ANOVA was performed on the combined data of Experiments 2a and 2b (see Fig. 3). The ANOVA revealed a main effect of drug condition (F1,7 = 11.75; p < 0.05) but no effect of delay or drug condition × delay interaction (both F < 1; NS). Finally, performance accuracy (90%) during drug-free testing carried out after the last infusion confirmed that there was no lasting damage due to cannulae implantation or the microinjection process. 4 Discussion Experiment 1 showed that selective inactivation of the dorsal hippocampus results in significant impairment of spatial working memory performance whereas equivalent inactivation of ventral hippocampus does not. This result is consistent with previous lesions studies [7,18], but advances on these earlier findings by demonstrating that such dorsal/ventral differences are not due to indirect effects such as demyelination or compensatory changes that result from permanent brain lesions. Also, the impairment resulting from infusion of AP5 directly into the dorsal subregion (Experiment 2) identifies the importance of N-methyl-d-aspartate receptor (NMDAR) function in the dorsal hippocampus for spatial working memory performance. This data, and that obtained from previous cytotoxic lesions studies, are consistent with findings from a variety of different approaches which suggest a functional specialization along the septotemporal (dorsal/ventral) axis of the hippocampus. Evidence from electrophysiological single unit recording studies in both rats [24,25] and primates [26], from c-fos activation studies [27], and from structural magnetic resonance imaging studies [28], are all consistent with a functional dissociation between dorsal (posterior in primates) and ventral (anterior in primates) hippocampus, and with a preferential role for dorsal hippocampus in spatial learning and memory. To our knowledge, this experiment constitutes the first demonstration of differential effects of dorsal and ventral hippocampal inactivation on spatial working memory performance using T-maze rewarded alternation. It is worth noting, however, that the effects of selective inactivation of dorsal and ventral hippocampus on delayed alternation tasks have been examined previously using operant paradigms, but with mixed results. For example, Mao and Robinson [29] trained rats to press a lever on the left or right-hand side of the front wall of the operant chamber as indicated by the presence of a light stimulus (equivalent to the sample phase in the present study), then press a lever on the rear wall before being given a free choice of either front wall lever (equivalent to the choice phase in our study). The rats were required to alternate lever presses on the front wall in order to obtain food rewards. Although this task has some similarity to our T-maze task (both require alternation behaviour), bilateral infusions of muscimol into the dorsal hippocampus did not affect the proportion of correct responses. It is worth pointing out, however, that the doses used by Mao and Robinson were lower than the doses used in the present study (0.003–0.01 μg compared to 0.0375–0.3 μg in the present study). It is also worth noting that in their study dorsal infusions of higher doses of muscimol (0.03–0.2 μg) interfered with the rats’ ability to perform various non-spatial aspects of task performance such as correct lever presses at the sample stage and the rear lever press between the choice and sample phases. In a separate study, Maruki et al. [30], trained rats to alternate between pressing the left and right lever on each trial. To prevent mediating strategies, the rat was required to make a touch response to the central food-well during the ITI. Using this continuous alternation paradigm, the authors found that infusions of muscimol (0.07 μg/side) into the dorsal hippocampus reduced correct responding when the ITI was 20 s but not when it was 3 s. In a second experiment, they reported no effects of ventral infusions of muscimol relative to saline vehicle infusions on the same task. However, inspection of the data across the two experiments shows that the levels of performance in rats receiving muscimol infusions into dorsal hippocampus (70.9% correct) and in rats receiving muscimol into ventral hippocampus (72.3% correct) were virtually indistinguishable. The present study thus provides the first clear demonstration that spatial working memory performance is sensitive to dorsal but not ventral hippocampal inactivation. As noted by Mao and Robinson [29] direct intra-hippocampal infusion carries the risk of non-specific effects on performance. For example, it is possible that unequal quantities of muscimol entering each hemisphere could lead to a turning bias. First, it is worth pointing out that in our study both hemispheres were infused simultaneously. Second, in Experiment 1, rats receiving dorsal HPC infusions exhibited side preferences (i.e. they exclusively entered the same goal arm on the choice phase of each trial of a session) in only 13 of the 40 sessions (10 rats × 4 muscimol doses). It is also worth pointing out that such side preferences are commonly observed in rats with permanent HPC lesions and in our experience these preferences are not dependent on the size of the lesion in one particular hemisphere. They are generally considered to be a consequence, rather than a cause, of the memory impairment. Furthermore, the fact that in Experiment 1, errors were made to both goal arms in almost 70% of sessions argues against a performance account based solely on side-preferences elicited by unequal quantities of muscimol entering each hemisphere. An alternative account of the septotemporal differences found in Experiment 1 is that the spread of the drug within the hippocampus is simply greater when infused into dorsal than ventral HPC, effectively creating a larger dorsal than ventral HPC inactivation. Without autoradiography to determine the extent of muscimol diffusion we cannot rule out this possibility completely. However, when the data from the present study are considered along with the previous cytotoxic lesion studies, such an account seems unlikely. Evidence from permanent lesion studies suggests that lesion size itself is not the critical determinant of degree of dysfunction. For example, Moser and colleagues [6] found that rats with lesions encompassing 20–40% of HPC tissue starting at the ventral pole showed normal spatial learning in the watermaze, whereas rats with lesions of 20–40% of HPC tissue starting at the dorsal pole were profoundly impaired. Furthermore, we have previously tested rats with dorsal and ventral cytotoxic lesions of very similar sizes (50–55%) on the same T-maze rewarded alternation task as used in the present study, and have found that whereas dorsal lesioned animals resemble animals with complete hippocampal lesions, exhibiting chance levels of performance, ventral lesioned animals perform as well as sham operated controls [9]. These data strongly suggest that lesion location, and not lesion size, is the critical factor. In the present study, although no dose of muscimol into the ventral HPC significantly impaired performance compared to vehicle, there was some suggestion of a dose related decline in performance (see Fig. 2). This could reflect a role, albeit more limited, for the ventral hippocampus in spatial information processing. However, it is also possible that this slight drop in performance may reflect the spread of muscimol into mid-hippocampal and/or dorsal regions. In this regard, it is worth re-iterating that ventral hippocampal lesions, removing approximately 50% of the structure starting from the temporal pole, had absolutely no effect on performance on this very same spatial working memory, rewarded alternation task [7,9] Therefore, the evidence suggests that spatial working memory tasks such as rewarded alternation on the T-maze, like spatial reference memory tasks such as the standard fixed location version of the watermaze, are sensitive to dorsal but not equivalently sized ventral HPC lesions or inactivations. Nevertheless, it may be unwise to completely exclude a role for the ventral HPC in spatial information processing. Single-unit studies have shown that cells selective for specific spatial locations (“place cells”) do exist in ventral HPC, albeit they are fewer in number and their place fields are larger than those in dorsal HPC. Although the electrophysiological data are limited, McNaughton and colleagues have suggested a septotemporal gradient in spatial selectivity with higher resolution spatial discriminations processed in dorsal HPC [24,25]. In this way, cells in dorsal HPC might code for specific locations within a particular environment whereas ventral cells might code for different environments or contexts, although this may be just a subset of the contextual information that can be processed by the ventral subregion [5]. It remains to be seen if tasks requiring discrimination between different spatial environments or contexts are more sensitive to lesions of the ventral HPC. The results of Experiment 2 show that NMDARs in dorsal hippocampus contribute to performance on spatial working memory tasks. Previous work in this laboratory showed that i.c.v. infusion of AP5 impaired performance on the same rewarded alternation task [21]. The present results now suggest that this effect is, at least in part, due to the blockade of NMDARs in dorsal hippocampus. Similar spatial working memory impairments have also been observed following AP5 infusions into dorsal hippocampus on a spatial, matching-to-position version of the watermaze task [19]. Both of these results are consistent with the hypothesis that NMDAR-mediated forms of synaptic plasticity, similar to experimentally induced long-term potentiation [LTP; 31, 32], may underlie a flexible, rapidly acquired form of spatial memory, as typified by these spatial working memory tasks [20,33]. The observation that introducing a further 20 s delay failed to exacerbate the spatial working memory deficit might be considered surprising. In contrast to the present results, Tonkiss and Rawlins [21] showed that increasing the delay by 20 s did in fact increase the impairment in rats infused with AP5 i.c.v., on the same T-maze spatial working memory task. However, it is worth pointing out that in both cases, the effect of increasing delay was not investigated as part of a fully counterbalanced design, and therefore some caution is required when interpreting these results. Steele and Morris [19] did report a delay-dependent impairment in their matching to position watermaze task with a fully counterbalanced design, although it is worth noting that performance at the shortest delay was not consistently spared in their study. We have also recently tested genetically modified mice in which the NR1 subunit of the NMDAR has been selectively deleted, specifically from the dentate gyrus subfield of the hippocampus [34]. These mice exhibit deficits in LTP in both the medial and lateral aspects of the perforant path, whereas LTP in the CA1 subfield is normal. We assessed spatial memory on the radial maze in these mice, and found that they exhibited a very specific spatial working memory deficit. As with the present study, the animals displayed a spatial working memory deficit at the shortest-possible delays that we were able to test. It is of course possible that at even shorter delays performance may be spared in the AP5 rats and in the dentate gyrus NR1 knockout mice. Equally, it could be that by blocking NMDAR-dependent synaptic plasticity in the hippocampus, we have disrupted an existing spatial representation of the environment, or prevented the expression of memory in some way, for example, by impairing the ability to select the appropriate spatial response on the basis of information retrieved from memory (both of which might be expected to result in a delay-independent deficit). Against this, the dentate gyrus-specific NR1 knockout mice displayed perfectly normal acquisition and performance on the reference memory component of the radial maze task (i.e. learning which 3 arms were baited and which three arms were never baited). It may therefore be that disrupting hippocampal synaptic plasticity prevents the animals from rapidly forming a short-term spatial representation of the maze arm visited on the sample run. This ability to record or represent the recent experience of a particular spatial location or stimuli (i.e. the maze arm) is likely to be of crucial importance for efficient spatial working memory performance [35]. In conclusion, the present research has confirmed and extended previous findings from lesion studies, namely that the dorsal hippocampus has a greater involvement in spatial working memory than the ventral hippocampus. Furthermore, NMDAR activation within the dorsal hippocampus makes an essential contribution to this aspect of hippocampal information processing.

Osteoporos_Int-3-1-1820755

Balance training program is highly effective in improving functional status and reducing the risk of falls in elderly women with osteoporosis: a randomized controlled trial

Introduction The purpose of this study was to investigate the effect of a 12-month Balance Training Program on balance, mobility and falling frequency in women with osteoporosis. Introduction Osteoporosis is a debilitating, widespread disease, which affects approximately 55% of the population above 50 years old in the USA [1]. Falls among the elderly, especially for those with osteoporosis, are associated with high morbidity and mortality and can involve high-cost medical intervention [2]. In fact, falls are responsible for 90% of the growing increase in hip fractures [3] and are the sixth cause of death among patients aged over 65 [4]. Falls are multifactorial, and their causes are categorized as intrinsic (personal) and extrinsic (environmental) factors [5, 6]. Some examples of intrinsic factors include: altered balance, neurological diseases, sensory deterioration, musculoskeletal diseases, postural hypertension and the use of medication [7]. Research shows that altered balance is the greatest collaborator towards falls in the elderly [6, 8–12], with a high correlation between balance deficit and the incidence of falls [13, 14]. For this reason, studies regarding the risk of falling in osteoporosis are of high priority in clinical intervention. Diminishing the incidence of falls is a health priority, which reflects on both the quality of life and health costs [2]. Moreover, evidence suggests that exercise reduces the risk of fractures, showing an effect on the maintenance of bone mass and, more importantly, improving postural stability, mobility and, consequently, diminishing the risk of falls [15]. Indeed, improving balance should be an objective in the prevention of falls [16]. However, knowledge regarding balance deficit and the probability of falls is limited and controversial [17]. The literature shows that exercise may or may not be efficient in the control of falls [18], and the impact of prevention programs with balance training in diminishing falling frequency has yet to be established, principally in women with osteoporosis. Balance training has been investigated in healthy elderly individuals [3, 16, 19–21]; however, only one study regarding balance training in women with osteoporosis exists. Since patients with osteoporosis are at greater risk of fractures resulting from falls, further research in this group should be prioritized [22]. The purpose of the present study was to investigate the effect of a 12-month balance training program on functional and static balance, mobility and falling frequency in women with osteoporosis. Patients and methods Patient sample Sixty-six elderly women aged over 65 years old were consecutively selected from patients of the Osteometabolic Disease Outpatient Clinic of the Rheumatology Division, University of São Paulo. Only patients with osteoporosis, classified according to the World Health Organization [23] were included; with a bone mineral density (BMD) T-score lower than −2.5 standard deviation (SD), in the lumbar spine, femoral neck or total femur region. The following women were excluded: those with secondary osteoporosis, visual deficiency, severe auditive deficiency, or vestibular alteration of important clinical status, such as women who used assisted walking devices or who were unable to walk independently more than 10 meters [24]; those who planned to be out of town for more than 4 weeks during the 12-month study; and women who presented absolute or relative contraindications for exercise training according to the American College of Sports Medicine [25]. The patients were randomized consecutively into two groups: the group submitted for the Balance Training Program (Intervention Group), consisting of 34 patients; and the Control group, consisting of 32 patients without intervention. The Control group only received treatment for osteoporosis and orientation to prevent falls and return regularly (3-monthly follow-ups) to the Osteometabolic Disease Outpatient Clinic. All patients read and signed a term of free informed consent that described the procedures which would be realized during the research. Measured variables: interview and medical chart records Personal, family and clinical data were evaluated through an interview and medical chart records, with emphasis on the history of fractures, number of falls in the preceding year, use of medication for osteoporosis, and use of medication that favored the risk of falling, such as hypnotics, hypotensors and antidepressants. Functional state evaluation Static and dynamic balance and mobility were evaluated in all patients, before and at the end of the trial, by a physiotherapist who was blinded to the distinct group (Intervention, Control). Functional balance The Berg Balance Scale (BBS) is based on 14 items common to daily life activities used to evaluate functional balance [26]. The maximum score that can be achieved is 56, and each item possesses an ordinal scale of five alternatives which varies from 0 to 4 points. The test is simple, easy to administer and accompanies the evolution of elderly patients. It only requires a ruler and a watch and takes approximately 15 minutes to execute [26]. A score lower than or equal to 45 is considered evidence of altered balance [27]. Static balance Static balance was evaluated by the Clinical Test of Sensory Interaction for Balance (CTSIB), which consists of six sensory conditions (1: eyes open and firm surface, 2: eyes closed and firm surface, 3: eyes open, visual conflict and firm surface, 4: eyes open and unstable surface, 5: eyes closed and unstable surface, and 6: eyes open, visual conflict and unstable surface). Static balance is considered to be altered when an individual cannot remain at least 30 seconds in each of the six conditions [28]. Improvement in the test was defined as the capacity to complete the test during the final evaluation when unable to complete the same in the initial evaluation. Functional mobility Functional mobility was evaluated by the Timed “Up & Go” Test (TUGT) [29], which registers the time an individual takes to get up out of a chair, walk 3 meters, turn around, walk back and sit down again. Elderly individuals without balance deficit are capable of completing the test in less than 10 seconds. Falls The number of falls in the year prior to the study [30] was solicited and noted in the initial evaluation and at the end of the trial (final evaluation). During the year of the study, patients in both groups received a diary and were orientated to write down the days that they fell. At the end of the study, the difference in the number of falls/patient (final evaluation - initial evaluation) was compared between the Intervention Group and Control. Intervention The Balance Training Program consisted of 1 hour of exercises realized once a week, with a total of 40 classes, supervised by an experienced physiotherapist. This program was realized in a club (Associação Atlética Acadêmica Oswaldo Cruz - AAAOC) belonging to the Clinics Hospital, School of Medicine, University of São Paulo, located near to the Hospital. The balance exercises described by Tinetti and Suzuki [3, 11] were used. The type and mild to moderate intensity of the exercises used in the present study were chosen so that they could also be performed by elderly patients at home [3]. A list of weekly attendance controlled the absences of each patient. Basic warm-up and stretching exercises Prior to training, the patients participated in 15 min of warm-up and stretching exercises, consisting of head rotation, shoulder rotation and stretching of the upper and lower limbs. Walking was performed for 15 min with the supervision of a physiotherapist, who associated exercises for the upper limbs throughout the walk. Balance training Balance was realized in dynamic and static positions for a period of 30 min. This consisted of walking in the tandem position (one foot in front of the other), walking on the tips of the toes and on the heel, walking sideways, walking while raising the leg and the contra-lateral arm, standing on one leg, and standing in the tandem position, while gradually increasing the period of permanence in these last two static positions [3, 31]. Home-based exercises The patients were instructed and encouraged to continue the same exercises at home at least three times a week for 30 min. A manual with instructions and illustrations for each exercise was distributed. The frequency of participation in the home-based exercises was noted each week by the physiotherapist. Data analysis Sixty-six consecutive patients were randomized in the present study. Data analysis was realized on 60 patients (30 Intervention Group and 30 Control), as six patients desisted (four Intervention Group and two Control). In the Intervention Group the reasons for desistance were: physical limitations as a result of falls (n = 1), foot pain (n = 1), personal reasons (n = 2); while in the Control group the reasons were: physical limitations as a result of falls (n = 1), personal reasons (n = 1).   The data were expressed as the mean and standard deviation (SD) for each variable and differences between the Intervention and Control groups were tested by the Student’s t-test or Mann-Whitney test. The Chi-square test or Fisher’s exact test was used to compare the number of patients in both groups (Intervention Group vs Control) for hypnotic and diuretic drug use, fracture history, CTSIB conditions (1–6) and improvement in CTSIB conditions. P values <0.05 were considered significant. Results The basal characteristics of the patients of both groups were similar in relation to age, body mass index (BMI), fracture history, osteoporosis treatment, diuretics and hypnotics/antidepressants use and bone mineral density (BMD), with no statistically significant differences between the two groups (Table 1). Table 1Data at the onset of the study in relation to anthropometric parameters, fracture history, medication use and bone mineral density values (T-score) in the Intervention and Control groupsVariableIntervention (n = 30)Control (n = 30)p-valueAge, years74.57 ± 4.8273.40 ± 4.610.342*BMI, kg/m224.39 ± 4.4926.51 ± 5.320.100*Fracture history, n (%)13 (43.3)16 (53.3)0.438***Medication used for OP, n2.37 ± 1.502.30 ± 0.880.498**Hypnotics/Antidepressants, n (%)6 (20.0)7 (23.3)0.754***Diuretics, n (%)14 (46.7)16 (53.3)0.606***Lumbar spine, T-score−2.83 ± 1.07−2.62 ± 1.120.470**Femur neck, T-score−2.70 ± 0.75−2.75 ± 0.900.821**Total femur, T-score−2.10 ± 1.26−2.10 ± 1.090.990**Data expressed in means ± SD or percentageBMI: body mass index, OP: Osteoporosis*Student’s t-test**Mann-Whitney test***Chi-square test Similarly, in the first evaluation, no differences occurred when comparing the Intervention Group and the Control Group in reference to: Berg Balance Scale (BBS) score, the number of patients that could not complete the Clinical Test of Sensory Interaction for Balance (CTSIB) in the six sensory conditions, the Timed “Up & Go” Test (TUGT), and the number of falls/patients in the preceding year (p > 0.05) (Table 2). Table 2Data at the onset of the study for: Berg Balance Scale (BBS) score, number of patients that could not complete the Clinical Test of Sensory Interaction for Balance (CTSIB: condition 1: eyes open and firm surface; condition 2: eyes closed and firm surface; condition 3: eyes open, visual conflict and firm surface; condition 4: eyes open and unstable surface; condition 5: eyes closed and unstable surface; condition 6: eyes open, visual conflict and unstable surface), Timed “Up & Go” Test (TUGT), and number of falls/patient in the preceding year in Intervention Group and Control Intervention (n = 30)Control (n = 30)p valueBBS, score48.80 ± 4.1048.13 ± 5.360.900*CTSIB condition 1, n (%)0 (0.0)1 (3.3)1.000**CTSIB condition 2, n (%)2 (6.7)1 (3.3)1.000**CTSIB condition 3, n (%)2 (6.7)2 (6.7)1.000**CTSIB condition 4, n (%)4 (13.3)4 (13.3)1.000**CTSIB condition 5, n (%)15 (50.0)12 (40.0)0.604**CTSIB condition 6, n (%)12 (40.0)9 (30.0)0.589**TUGT, seconds14.31 ± 4.0313.86 ± 3.430.610*Falls/patient preceding year, n1.20 ± 1.880.87 ± 0.860.745*Data expressed in means ± SD or percentage.*Mann-Whitney test**Fisher’s exact test Adherence rate A high level of adherence was observed. Sixty percent of the patients participated in all of the exercise sessions at the club and absences occurred with the following justifications: doctor’s appointment, the realization of laboratory exams or for personal reasons. In relation to home-based exercise, 76.67% of the patients realized exercises at least once a week, 40% of the patients exercised every day and 36.67% from one to four times a week. Comparison between the Intervention Group and Control The difference in BBS score (final—initial evaluation) was greater in the group which suffered intervention (5.5 ± 5.67 vs − 0.5 ± 4.88, p < 0.001) (Table 3). Table 3Differences (final evaluation—initial evaluation) in: Balance Berg Scale (BBS) score, number of patients showing improvement in Clinical Test of Sensory Interaction for Balance (CTSIB condition 5: eyes closed and unstable surface; condition 6: eyes open, visual conflict and unstable surface), time of Timed “Up & Go” Test (TUGT), and number of falls/patient in the Intervention Group and Control Intervention (n = 30)Control (n = 30)p-valueDifference BBS, score 5.5 ± 5.67−0.5 ± 4.88<0.001*CTSIB condition 5, n (%)13 (43.3)1 (3.3)<0.001**CTSIB condition 6, n (%)12 (40.0)1 (3.3)0.001**Difference TUGT, seconds−3.65 ± 3.61+2.27 ± 7.18<0.001*Difference of falls/patient, n−0.77 ± 1.76+0.03 ± 0.960.018*Data expressed in means ± SD or percentage.*Mann-Whitney test**Chi-square test Similarly, the percentage of patients in the Intervention group whose static balance improved in two sensory conditions (CTSIB, condition 5: eyes closed, unstable surface; and condition 6: eyes open, visual conflict, unstable surface) was statistically significant when compared to Control (CTSIB condition 5: 13 patients vs 1, p  < 0.001; CTSIB condition 6: 12 patients vs 1, p < 0.001) (Table 3). Equally, a significant difference in the functional mobility, as measured by the TUGT (final—initial evaluation) was observed in the Intervention Group compared to Control (−3.65 ± 3.61 vs +2.27 ± 7.18, p < 0.001) (Table 3). Parallel to these improvements in functional and static evaluation, a reduction in the number of falls/patient (final-initial evaluation) was observed in the Intervention Group compared to Control (−0.77 ± 1.76 vs +0.03 ± 0.98, p = 0.018) (Table 3). Discussion Few studies have been developed regarding balance training in patients with osteoporosis. The present longitudinal prospective study demonstrated that a program of balance training realized over a period of 12 months was effective in improving the functional and static balance, mobility and diminishing the number of falls in elderly women with osteoporosis. In the present work, improvement in relation to functional balance was demonstrated by an increase in the BBS score in the final evaluation in the group submitted to the balance training program (Intervention Group). Similar results were shown in a study by Melzer et al. [32], where the patients who participated in the balance training obtained 64% improvement in 3 months. The authors showed that the group who underwent the balance training demonstrated better performance than the group submitted to muscular strength training. Programs that emphasize balance training are more effective at improving balance than those that consist primarily of aerobic, muscular strength or flexibility exercises [33]. Although it is a complex issue to evaluate the effectiveness of different types of exercises [34], balance training has shown beneficial results, with diminished risks of falls [35]. Another positive result of the present study was the improvement in the two difficult conditions of the Clinical Test of Sensory Interaction for Balance (condition 5: eyes closed, unstable surface, and condition 6: eyes open, visual conflict, unstable surface) in almost half the patients. Carter et al. [24] obtained an improvement in static balance using muscular strength training rather than balance training, though only in 6.3% of the patients. Thus, the present findings suggest that balance training leads to more evident positive results in static balance than does muscular strength training. In relation to functional mobility, improvement was demonstrated by a reduction in the TUGT time in the patients submitted to intervention. These results are relevant, since research shows that compromised mobility increases the risk of dependency three- to five-fold, in activities of daily life [36]. This is not surprising, considering that mobility is an important component of daily life activities, for example: going to the shopping mall, to the supermarket, to the doctor or the cinema. Increased dependency could lead to institutionalization and diminished quality of life. Good balance is considered fundamental for improving mobility and preventing falls [36]. In parallel to the improvement in functional evaluation, an important reduction in the frequency of falls was observed. Although the effect of exercise in the prevention of falls in the elderly is yet to be proved, some studies show that physical activity reduces the risk by 40% [37]. On the other hand, in a consensus on prevention of falls in the elderly, the only exercises recommended to prevent falls are those which specifically target balance training [38]. Many studies that show improvement in balance and mobility present similar characteristics in their exercises. This suggests that the content and intensity of the exercise program could be more important than other intervention variables [39]. Moreover, differences in the administration of the exercise program, the professional experience of those who apply the exercises, the location in which the sessions are held and whether the exercise is conducted in group or realized individually, are all fundamental parameters which influence the success of the exercise program[39]. An important factor for the success of the exercise program is adherence. Contrary to the study by Forcan et al. [40], who stated that adherence to exercise in the elderly is weak, in the present study good adherence was observed, with more than half the patients present at all sessions. The current findings are similar to other studies which showed adherence up to 97.5% [17]. The success in adherence in the present study is probably due to the location where the exercises were performed (a pleasant, natural environment), to social interaction, and to the supervision of a physiotherapist. A secure environment, session supervision and the opportunity for social interaction reduce the feeling of isolation. A social support system is considered important in group activities, and helps sustain adherence and the effectiveness of the weekly exercise sessions and also the adherence to home-based exercises [35]. Similarly to our study, Robitaille et al. showed that an exercise program performed in groups improves the balance of the elderly in the community [41]. Another relevant factor was the use of a manual of instructions and illustrations for the realization of home-based exercises, which contributed to the continuity and adherence of the exercises performed. Each exercise was appropriately prescribed and illustrated by a physiotherapist, giving the patient adequate support. Descriptive and illustrated pamphlets have been used in some studies, complemented by a home-based exercise program, with positive results [3, 42]. The present positive results could also be related to the state of health of these patients, which was good in our patients. Buchner et al. [43] showed that exercise can have beneficial effects on health and on the risk of falls in certain subgroups of the elderly. The physical and psychological benefits of the regularity of the sessions and the environment should never be ignored. Stimulating strategies and demonstrating ability in the transference from one exercise to another, consequently, maintaining enthusiasm during the exercises, can be practiced safely in groups [35]. Although our study did not use laboratory equipment [44] to substantiate the results obtained, we showed by reliable and reproducible scales and tests [26, 28, 29] that balance training performed once a week, supervised by a physiotherapist, and complemented by home-based exercises, is very effective in the improvement of functional and static balance and mobility, and in the reduction of falls in elderly women with osteoporosis.

Eur_Arch_Otorhinolaryngol-4-1-2279158

Voice quality after endoscopic laser surgery and radiotherapy for early glottic cancer: objective measurements emphasizing the Voice Handicap Index

We analyzed the functional outcome and self-evaluation of the voice of patients with T1 glottic carcinoma treated with endoscopic laser surgery and radiotherapy. We performed an objective voice evaluation, as well as a physical, emotional and functional well being assessment of 19 patients treated with laser surgery and 18 patients treated with radiotherapy. Voice quality is affected both by surgery and radiotherapy. Voice parameters only show differences in the maximum phonation time between both treatments. Results in the Voice Handicap Index show that radiotherapy has less effect on patient voice quality perception. There is a reduced impact on the patient’s perception of voice quality after radiotherapy, despite there being no significant differences in vocal quality between radiotherapy and laser cordectomy. Introduction Surgery and radiotherapy offer the same results in the treatment of early glottic carcinoma, with a cure rate of approximately 90% [1]. The introduction of endoscopic CO2 laser surgery in early glottic carcinoma (epidermoid carcinoma confined to the true vocal cords with normal mobility) has added controversy to the standard treatment choice. Laser surgery is quicker, which reduces cost considerably. However many institutions prefer to use radiotherapy in the belief that the voice will be better preserved after treatment [2]. Although the principal objective of oncology treatment is the complete eradication of the illness, normal voice preservation is another important consideration in the treatment choice of early glottic carcinoma. For this reason, post-treatment voice quality is a relevant factor to take into account when evaluating the results. Quantitative acoustic measurements are more regularly studied. These are obtained from tools that digitize and analyze the voice being investigated and quantify the characteristics that deviate from normality, understanding normality to mean when the voice is uniform in both amplitude and tone periodicity. The addition of noise to the voice signal is also a defect that affects voice quality, and this is the third acoustic characteristic that is studied using spectrography. Another of the factors to take into account is the auditive perception that is generated in the listener and is evaluated by ways of perceived voice quality, making use of semi-objective scales such as the GRBAS scale, as described by Hirano [3]. It is recommended that these measurements are made by two experts, although the parameters have shown sufficient reliability (inter- and intra-observer reproducibility) when used in a clinical setting [4]. Although all these measurements are important parameters in defining voice quality, they fail to provide information on the patient’s perceived voice quality. For this there is a method available that permits the patient to describe the sensations their voice gives: the Voice Handicap Index [5]. It is a questionnaire that reviews situations grouped into three areas (functional, physiological and emotional) and gives an idea of the subjective impact that a vocal problem produces in a specific individual. In this study, we present the objective and subjective analysis of voice quality following treatment of an early epidermoid glottic carcinoma. Results from the objective evaluation of the voice, along with the self-evaluation of voice quality quantified using the Voice Handicap Index of a group of patients treated with endoscopic laser surgery are compared with patients treated with radiotherapy. Materials and methods Voice analysis and quality of life studies of 19 patients treated with laser surgery and 18 with radiotherapy, suffering from early glottic carcinoma, were evaluated. Lesions were classified according to the American Joint Committee on Cancer [6]. In patients who received radiotherapy as the primary treatment, this was performed using a 6 mv linear accelerator to bilateral ports and one in front, with field sizes ranging from 5 × 5 to 6 × 6 cm. Wedges were used to establish dose homogeneity. In the case of patients treated with surgery, the treatment consisted of direct laryngoscopy for the complete resection (partial transmuscular cordectomy) of the lesion with CO2 laser. Objective voice analysis and voice quality studies, performed by both patients and examiners by means of an examination protocol including: perceptual analysis of dysphonia, acoustic analysis, aerodynamic efficiency and patient perception, were applied to both groups. In all patients, this protocol was undertaken at least 6 months after completing treatment. Perceptual analysis of dysphonia (GRBAS) Perceptual analysis of dysphonia was performed using the GRBAS scale [3]. Two experienced professionals evaluated the recorded voice samples simultaneously, classifying each sample from 0 to 3 (0 = normal, 1 = mild, 2 = moderate, 3 = severe). The severity of hoarseness is quantified under the parameter G (grade), which represents overall voice quality. B (breathiness): audible impression of turbulent air leakage through an insufficient glottic closure, which may include short aphonic moments (unvoiced segments). R (roughness or harshness): audible impression of irregular glottic pulses, abnormal fluctuations in F0, separately perceived acoustic impulses (as in vocal fry), includes diplophonia and register breaks. A (asthenicity): impression of weakness in the spontaneous phonation, hypokinetic or hypofunctional voice. S (strain, vocal tension): auditive impression of excessive force or tension associated with spontaneous phonation. Acoustic and spectrographic analysis The acoustic analysis was performed using Doctor Speech V3 software for Windows 95. The acoustic signal was recorded using the Voice Assessment application. The computer used was a Pentium II at 100 MHz and with 16 Mb of RAM and a Sound Blaster 16 sound card. The sampling frequency was 44,100 Hz and a high frequency range microphone was used. The microphone was located 10 cm from the patient’s mouth while they emitted the “e” sound at comfortable intensity and pitch levels in a soundproof chamber. The computer captured 3 s of sound. Once the signal was digitized, the computer calculated the following acoustic parameters: fundamental frequency (F0), jitter or frequency variation (%), shimmer or amplitude variation (%) and NNE or normalized noise energy, which measures the degree of noise produced by turbulent air escaping through the glottis during vocal emission. Using the same digitized voice sample, a narrow band spectrogram was generated (45 Hz) using the Speech Analysis application. The spectrograms obtained were grouped into four types according to the Yanagihara criteria [7]. Aerodynamic efficiency analysis This consisted in measuring the maximum phonation time (TMF) for the “a” vocal after instructing the patient to sustain this vocal for the longest time possible at a comfortable pitch and intensity. The patients were asked to repeat the test at least three times and the highest value was retained. Patient self-perception analysis (Voice Handicap Index) The patients completed the “Voice Handicap Index” via a self-evaluation form comprising 30 questions covering three domains [5]: functional, physical and emotional, translated from the original work in English by one of the authors. Each question was assigned a score of 0–4 (from least disability to most). In each item, the maximum score was 40 points and we classified them as mild disability (less than 20), moderate (21–30) and severe (more that 30). Adding the three scores together, the maximum possible was 120, and we classified the vocal disability as mild (less than 30), moderate (31–60), severe (61–90) and very severe (91–120). Statistical analysis The data obtained was gathered in the statistical database SPSS 12.0. The statistical analysis used was the Student’s t test in order to compare averages and the Chi-squared test for comparing proportions. The statistical differences were considered significant when P was lower than 0.05. Results In the patient group treated with CO2 laser, the ages were in the range 44–86 years, with an average age of 64 and a mean follow-up of 30 months (12–48). In the patient group treated with radiotherapy, the ages were in the range 55–81 years, with an average age of 67 and a mean follow-up of 43 months (6–81). All patients in both groups were males. In both groups, all lesions corresponded to stage I of the TNM classification. In the group of 19 patients treated with surgery, all the lesions were qualified as T1a and in the case of the 18 radiotherapy patients, 13 were classified as T1a and 5 as T1b. No metastases in the neck, or distant, were detected. At the time of the study, all patients were free from illness. In the group of patients treated with radiotherapy, the nominal total dose was 6,525 cGy, with 225 cGy daily doses for a total of 29 days. Of the PTV volume, 100% received minimum doses of approximately 5,700 cGy, a maximum dose of approximately 6,720 cGy and an average dose of 6,520 cGy. Aerodynamic efficiency and spectrographic analysis In Table 1, the maximum phonation times are presented for the “a” vocal along with the quantitative voice analysis of the vocal “e” obtained in the groups treated with laser and with radiotherapy. There was no significant difference between the parameters: fundamental frequency, (F0), jitter, shimmer and NNE on comparing both groups. However, there were significant differences in the maximum phonation time. Table 1MPT and acoustic analysis of the vocal /e/ after treatment: laser (n = 19) or radiotherapy (n = 18)VariableLaserRadiotherapyMPT11.83 ± 5.288.63 ± 3.23P < 0.05F0 (Hz)173.39 ± 47.41199.04 ± 51.46NSJitter (%)0.44 ± 0.240.72 ± 0.91NSShimmer (%)5.08 ± 4.724.07 ± 4.04NSNNE (dB)−5.82 ± 2.98−5.02 ± 4.44NSMPT maximum phonation time, F0 (Hz) fundamental frequency, NNE normalized noise energy In spectrographic terms, the 19 voice samples of patients treated with laser were classified as Grade I 4 (21%), Grade II 5 (25%), Grade III 7 (37%) and Grade IV 3 (15%). In the group of patients treated with radiotherapy, the 18 voice samples were classified as Grade I 1 (5%), Grade II 7 (39%), Grade III 5 (28%) and Grade IV 5 (28%). On comparing the spectrograms obtained in both groups, there were no statistically significant differences (P = 0.401). Perceptual dysphonia analysis (GRBAS) Table 2 shows the results obtained using GRBAS scale on patients treated with CO2 laser surgery. Mild dysphonia was found in 31.5% of the cases, moderate dysphonia in 37% and severe dysphonias in the remaining 31.5%. Table 3 shows the GRABS results of the patients treated with radiotherapy. Normal voices were observed in 11%, mild dysphonias in 44.4%, moderate dysphonia in 27.8% and severe dysphonia in 16.7% of the cases. On comparing the results obtained in the G domain of the GRABS scale, which corresponds with the degree of dysphonia, no significant difference was found between each group (P = 0.309). Table 2Results obtained using GRBAS scale on the patients treated with CO2 laser surgery (n = 19)0123TotalG0%31.5% (6)37% (7)31.5 (6)100% (19)R10.5% (2)58% (11)26% (5)5.5% (1)100% (19)A21% (4)42% (8)21% (4)16% (3)100% (19)B84% (16)16% (3)0%0%100% (19)S31.5% (6)31.5% (6)21% (4)16% (3)100% (19)G grade, R roughness, A asthenicity, B breathiness, S strainTable 3GRABS results of patients treated with radiotherapy (n = 18)0123TotalG11.1% (2)44.4% (8)27.8% (5)16.7%(3)100% (18)R55.6% (10)33.3% (6)5.6% (1)5.6% (1)100% (18)A55.6% (10)33.3% (6)11.1% (2)0%100% (18)B44.4% (8)27.8% (5)22.2% (4)5.6% (1)100% (18)S77.8% (14)16.7% (3)5.6% (1)0%100% (18)G grade, R roughness, A asthenicity, B breathiness, S strain Patients self-perception analysis (Voice Handicap Index) Table 4 shows the averages obtained from the “Voice Handicap Index” questionnaire in the functional, physical and emotional scales, as well as the scores obtained in both groups. Upon completing the comparison between the two groups, the statistical difference is significant, in favor of the radiotherapy patients in the functional and emotional ratings as well as the global scores. No significant differences were found in the physical scales. Table 4Averages obtained from the “Voice Handicap Index” questionnaire in the functional, physical and emotional scales, as well as the scores obtained in both groupsLaser RadiotherapyFunctional11.47 (0–32)2.83 (0–17)P < 0.05Physical12.68 (0–31)6.22 (0–18)NSEmotional4.63 (0–20)0.61 (0–5)P < 0.05Global28.79 (0–77)9.67 (0–29)P < 0.05 Discussion Early glottic carcinoma can be treated using endoscopic surgery, radiotherapy or partial open surgery. Lesions limited to the vocal fold are normally treated with endoscopic surgery or partial open surgery, with local control results of between 80 and 90% [8]. In choosing one treatment or another, one should contemplate the cure rate, larynx preservation rate, post-treatment voice quality, morbidity and treatment cost [9]. In recent years, endoscopic CO2 laser surgery has made headway compared to radiotherapy, based on its good oncology results and reduced morbidity. CO2 laser treatment in early glottic carcinoma has greatly improved voice quality compared to that obtained following cordectomy via laryngofissure. as documented by some authors such as Keilmann et al. [10]. However in other published studies, such as Schindler [11], this improvement is not as evident. One of the advantages of laser surgery is its low cost when compared to radiotherapy, as well as the additional benefit of being able to opt for radiotherapy at a later stage to treat a relapse or second primary malignancy. In many institutions, these tumours are treated with external radiotherapy because of the supposedly better functional and quality of life results obtained compared to patients treated with surgery [12]. Various studies have been published comparing voice quality after both treatments [13–15]. In some studies, voice quality is similar, while other authors maintain that the voice is better after radiotherapy than after laser surgery [16–18]. However, there are fewer published works that include patient opinions with regard to the impact the illness and the treatment has had on their quality of life [19–21]. In our work, no significant differences in fundamental frequency (F0), jitter, shimmer and NNE were found between the two groups. There were, however, significant differences in maximum phonation time, favoring the patients treated with CO2 laser cordectomy. Other authors do not find significant differences in the maximum phonation time between the groups [22–24]. In the study by Tamura et al. [13] the fundamental frequency is higher for patients treated with CO2 laser surgery, which suggests that this surgery has a greater impact on the vocal fold function than radiotherapy. Krengli et al. [18] found statistically significant differences in the vocal acoustic parameters and fundamental frequency, favoring radiotherapy patients. In terms of the spectrograms obtained in both groups, no statistically significant differences were found. According to our results, 48% of patients treated with laser, and 44% of radiotherapy patients have an aesthetically acceptable voice (type I and II dysphonia). There were no significant differences found between the two groups on comparing the scores obtained using the G parameter of the GRABS scale, which corresponds to the degree of dysphonia. Other studies such as Loughran et al. [20] failed to find statistically significant differences on comparing GRABS between patients treated with each technique. In our work, there is an elevated percentage of patients treated with CO2 laser (84%) who obtained low scores in the breathy voice domain (B), which shows that apart from individual healing patterns, or treatment-induced secondary glottic defects, post-cordectomy phonetic compensating mechanisms are important in determining new voice quality. According to Sittel et al. [25], glottic phonation voice quality is better than that obtained by the non-glottic phonation. Although one could believe that these patients had benefited from voice therapy, there are contradictory opinions on this [26]. While acoustic, physiological and perceptual measurements are important parameters in assessing vocal function, they do not provide information about the patient’s perception of their own voice quality. Given that voice quality, because of its potential impact on quality of life, can be an important factor in the choice of treatment, it is important to include this information when evaluating results. Not only should dysphonia as a by-product of laryngeal physiology be considered, but also the effects on patient quality of life must be considered as well. In our study, in order to quantify patient opinion as to the impact caused by treatment, the Voice Handicap Index has been employed. This is an instrument developed to help the professional in deciding the therapy, taking into account the patient’s subjective sensations with respect to their problem. It is a post-treatment global result evaluation method, from the point of view of the patient’s perceived global well being (physical, mental and social). Although it is a subjective evaluation based on the patient’s own perception, it can provide valuable data as to the reasons why patients with similar dysphonia characteristics can have different handicap severity indices [27]. VHI allows investigators to obtain information regarding the patient’s subjective perception as well as providing the physician with important information pre and post treatment. Given that the preservation of adequate phonation is an important consideration in early glottic carcinoma treatment selection and given equal oncologic results between the two treatments, the VHI proves to be a very important tool in the therapeutic decision-making process. In our study, low scores were obtained for both groups in the three scales, functional, physical and emotional. This could imply reduced impact in the quality of life of patients treated either with laser surgery or radiotherapy. The statistically significant differences are in favor of radiotherapy patients in the functional and emotional scales, as well as the global scores. Although acoustic and perceptual voice analysis in both groups showed no significant differences, the radiotherapy group scored less in the VHI than the surgical group. Loughran et al. [20] performed a study where they compared the results between both therapies in terms of the Voice Handicap Index as well as two other questionnaires aimed at evaluating the patient’s subjective voice quality (Vocal Performance Questionnaire, VPQ, and Voice Symptom Score, VoiSS). In this work, no significant differences were discovered between radiotherapy and CO2 laser surgery patients in any of the questionnaires, except for the emotional sub-scale inVoiSS. However, in other studies the average global score in the Voice Handicap Index questionnaire is lower for patients treated with endoscopic laser resection than in patients treated with radiotherapy. Peeters et al. [21] compared the results of both the Voice Handicap Index and a quality of life questionnaire (COOP/WOONCA) following both treatments. In their results, they found statistically significant differences in favor of laser surgery for the VHI and none in the quality of life results. They found higher scores for global VHI results in 40% of patients treated using surgery, and 58% for those treated with radiotherapy. Their conclusions reveal that the treatment for T1 glottic carcinoma frequently generates vocal problems in day-to-day life, influencing negatively certain social activities. This data contradicts the results of our study where VHI scores of both groups were low and the differences found favored the radiotherapy patient group. Recently, Cohen et al. [28] published a meta-analysis in order to classify the quality of life related to voice in patients with TI glottic carcinomas treated with radiotherapy compared with CO2 laser resection. Six studies with a total of 208 patients treated with surgery and 91 with radiotherapy had similar scores for VHI, from which they concluded that both treatments result in comparable vocal handicap levels for patients with T1 glottic carcinomas [28]. In other works such as that by Stoeckli [19], which value long-term results with regard to quality of life (QOL) of early glottic carcinoma patients, no differences were found between the two treatments and they conclude that both therapies provide good results in terms of quality of life. They show in addition, differences in the relative scores for questions such as swallowing, xerostomy and dental problems, favoring patients treated surgically, and show no differences with regard to perceived voice quality. These results could be related to the fact that xerostomy, edema, mucositis and fibrosis increase the sensation of handicap to such an extent that these effects on patient quality of life could be greater than those coming from the dysphonia itself in laser surgery patients. Few are the patients that have a so-called “normal” voice upon completing radiotherapy, above all taking into account that the larynx of these patients has undergone a surgical procedure in order to get a biopsy, has been intensely exposed to tobacco smoke and belongs generally to aged patients. All these factors negatively affect voice quality [29]. Despite this, the results of our work reveal that radiotherapy treatment for T1 glottic carcinoma generates lower repercussions in the subjective perception of residual dysphonia, as much in functional as in emotional aspects, when compared with CO2 laser surgery. The scores obtained in the VHI were low in the three domains for both groups, from which we can infer a scarce impact on the quality of life for laser surgery and radiotherapy for this type of patient. Conclusions Both CO2 laser resection and external radiotherapy as treatment for T1 vocal carcinoma offer similar objective measurement results (acoustic and spectrographic analysis) and subjective measurement results (GRABS scoring). However, in our series, the self-evaluation of the quality of voice, quantified by the VHI, shows a lower impact for radiotherapy patients.

Psychopharmacologia-3-1-1820758

Dopamine efflux in the nucleus accumbens during within-session extinction, outcome-dependent, and habit-based instrumental responding for food reward

Rationale Dopamine (DA) activity in the nucleus accumbens (NAc) is related to the general motivational effects of rewarding stimuli. Dickinson and colleagues have shown that initial acquisition of instrumental responding reflects action–outcome relationships based on instrumental incentive learning, which establishes the value of an outcome. Given that the sensitivity of responding to outcome devaluation is not affected by NAc lesions, it is unlikely that incentive learning during the action–outcome phase is mediated by DA activity in the NAc. Introduction Despite more than 25 years of intensive research there is still a vigorous and ongoing debate concerning the specific aspects of complex and adaptive behaviors that are mediated by brain dopamine (DA) function (Ikemoto and Panksepp 1999; Redgrave et al. 1999; Salamone and Correa 2002; Schultz 2002; Berridge and Robinson 2003; Joseph et al. 2003; Wise 2004; Kelley et al. 2005; Salamone et al. 2005; Young et al. 2005). Much of the attention is focused on the nucleus accumbens (NAc) and a consensus has formed around the theory that DA innervation of this structure plays a key role in incentive motivation, a Pavlovian conditioned appetitive state that can influence the vigor of approach behavior (Fibiger and Phillips 1986; Robbins et al. 1989; Berridge and Robinson 1998; Cardinal et al. 2002; Parkinson et al. 2002). Consistent with the importance of conditional stimuli (CS+) in the initiation of approach behavior, visual and olfactory stimuli associated with natural rewards such as food or sexually receptive conspecifics can evoke significant increases in DA efflux in the NAc that precede similar changes observed during consummatory behavior (Fiorino et al. 1997; Ahn and Phillips 1999, 2002). Depletion or antagonism of DA function in the NAc eliminates or diminishes the influence of Pavlovian CSs on the level of instrumental responding, i.e., Pavlovian to instrumental transfer (Cardinal et al. 2002; Parkinson et al. 2002), and additionally, shifts response choice from that with higher work demand and a food reward of greater value to that with lower work cost and lower reward value (Salamone et al. 1994; Salamone and Correa 2002). Contemporary views of instrumental conditioning propose that performance of an instrumental task is controlled by two distinct processes. Based on carefully designed experiments, Dickinson et al. (1995) have demonstrated that during initial learning trials (i.e., after exposure to 120 outcomes), an animal’s performance is based on the knowledge (or expectation) that an instrumental action will lead to a specific biologically significant outcome. After many more trials (i.e., after exposure to 360 outcomes), responses gradually shift from being outcome-dependent to habit-based. It is only in the early action–outcome controlled stage that instrumental performance is sensitive to manipulations that alter the incentive value of the outcome, underscoring the remarkable ability of rats to acquire and encode information relating current incentive value to an action–outcome contingency in as little as 120 outcome trials. Using outcome devaluation and Pavlovian to instrumental transfer tests, lesions of the basolateral amygdala have been reported to impair the capacity of rats to encode the relation between a specific action and the value of an outcome (Corbit and Balleine 2005). Given the generally accepted role of DA in the NAc in Pavlovian-based incentive motivation, the question arises as to whether DA activity in this region is involved in action–outcome and/or habit-based based stages of instrumental responding. A role for either the core or shell region of the NAc in the formation of action–outcome associations has not been confirmed (Balleine and Killcross 1994; Corbit et al. 2001; de Borchgrave et al. 2002), but deficits in the acquisition of instrumental responding have been reported after blockade of the NMDA class of glutamate receptors in the NAc core (Kelley et al. 1997). In a recent study, Yin et al. (2006) examined DA function during early action–outcome as distinct from later habit-based stages of instrumental responding. Mice, with a knockdown of the DA transporter and chronically elevated levels of DA, showed no deficits in acquisition of an instrumental task and responding early in training was still sensitive to tests of outcome devaluation (Yin et al. 2006). However, in rats with bilateral 6-OHDA lesions of the nigrostriatal DA system, responding on instrumental tasks remained sensitive to reward devaluation despite extensive training sessions (Faure et al. 2005). Thus, the nigrostriatal DA system, and more specifically, its innervation of the posterior lateral striatum, appears to be necessary for transition of instrumental conditioning from an action–outcome stage to a habit-based stage. Important insights into the role of DA transmission at different stages of instrumental behavior may be gained by examining in vivo changes in DA levels in major areas of DA innervation. Therefore, we conducted microdialysis experiments in the NAc and mediodorsal (MD) striatum at early (5th day) and later (16th day) stages of instrumental learning employing a protocol used successfully to demonstrate that responding after 120 but not 360 reinforced responses is sensitive to outcome devaluation and is therefore action–outcome as distinct from habit-based (Dickinson et al. 1995). Given the finding that quinolinic acid or NMDA-induced cytotoxic lesions of the NAc failed to alter suppression of instrumental responding after outcome devaluation (de Borchgrave et al. 2002), it is unlikely that activity in the NAc is involved in instrumental incentive learning. Rather, the finding that lesions restricted only to the NAc completely abolished Pavlovian to instrumental transfer is consistent with the involvement of DA function in this nucleus in conditioned appetitive states. Therefore, we hypothesize that DA efflux in the NAc may be comparable during both early action–outcome and later habit-based stages of instrumental responding. We also examined changes in DA levels in the MD striatum, as a control site for generalized activity; hence, we predicted no significant changes in DA efflux associated with instrumental responding. The design of this study also incorporated a “within-trial” extinction phase to test the hypothesis that DA efflux in the NAc but not MD striatum would be increased significantly by presentation of a Pavlovian CS+ paired previously with food pellets during instrumental responding in extinction. The relationship between response rate and magnitude of DA efflux in either NAc or MD striatum was also of interest. Materials and methods Surgery Long–Evans male rats (Charles River, Canada) weighing 280–310 g were implanted bilaterally with stainless steel guide cannulae (19 gauge, 15 mm) under anesthesia induced by xylazine (7 mg/kg) and ketamine hydrochloride (100 mg/kg) delivered intraperitoneally. Cannulae were implanted 1 mm below dura immediately above the NAc [in millimeter: +1.7 anteroposteriorly (AP) and ±1.1 mediolaterally (ML) from bregma] or MD striatum (+1.2 AP, 2.5 ML) and secured with dental acrylic and four stainless steel screws. Stylets maintained patency of the cannulae until probe implantation. An additional sham-cannula was embedded within the back half of the acrylic head cap for purposes of habituation (see Behavioral apparatus and training). Immediately after surgery, rats were moved to a reverse light-cycle (lights on 7 a.m.to 7 p.m.) colony room maintained at 20°C and housed individually in plastic bins lined with corncob bedding. Novel objects (e.g., egg cartons and paper rolls) were placed weekly in the cages to promote exploratory and play behavior. Rats were handled and weighed by the experimenter on a daily basis. Four days after surgery, rats were placed on a food restriction schedule for the duration of the experiment, maintaining their body weight at ∼85% of their free-feeding weight. The daily ration of food (20–25 g Purina Rat Chow) was given to the rats in their home cages following each day’s operant responding session. Water was available at all times except during the operant training and testing sessions. Behavioral apparatus and training Training and experiment sessions were conducted in a Plexiglas chamber (30×23×23 cm) with wire-mesh flooring. It was equipped with a retractable lever, a dispenser that delivered 45 mg Noyes pellets, and a light source (1 W, 12 V) located on the wall opposite the location of the lever. Pellets were dispensed into a photocell-equipped magazine located just left of the lever. The chamber was enclosed in a sound-attenuated ventilated box (Colbourn Instruments; Allentown, PA, USA) which had a small hole in the ceiling to allow passage of dialysis lines. Training began 8 days into the food-deprivation schedule. Before a rat was placed in a testing chamber, a stainless steel coil was attached to the sham-cannula. This allowed the rat to habituate to the weight and feel of being tethered to the stainless steel coil which normally sheathes microdialysis lines during experiments. Each session began with rats being placed in operant chambers under baseline conditions (coil attached, lever retracted, and light off), the length of which was varied from day to day. Illumination of the light and insertion of the lever signaled the availability of pellets contingent on lever presses. A computer was used to control the equipment and record the number of lever presses and nose poke entries into the magazine. At the start of the study, rats were trained to approach the magazine to retrieve 30 pellets delivered on a random-time 60-s schedule. Instrumental training sessions began the next day (day 1) on a RI 2-s schedule of pellet delivery. The schedule was changed to RI 15-s on day 2 and then to RI 30-s on day 3, and remained so for the remainder of the training and microdialysis sessions. Each instrumental training session was terminated when rats had responded for 30 pellets, except on days 4 and 15 when rats were allowed to press for 60 pellets (see Fig. 2). To minimize the effect of stress before microdialysis tests, rats remained overnight in the chambers under baseline conditions with water, after the training session on day 2. Microdialysis probes and high pressure liquid chromatography system Microdialysis probes were constructed as previously described (Ahn and Phillips 1999; Fiorino et al. 1997). They were concentric in design with a 2-mm semipermeable membrane (340 μm outer diameter, 65 kD MW cut-off, Filtral 12, Hospal; Neurnberg, Germany) with PE50 inlet and silica outlet tubing. A probe assembled in this manner typically had, at 21°C, in vitro recoveries of ∼22% DA. Once a probe was implanted in the brain through the guide cannula (see “Microdialysis experiments” section below for implant coordinates), a cylindrical brass collar secured the probe in place. The inlet tubing was connected to a liquid swivel (Instech 375s; Plymouth Meeting, PA, USA) which was mounted on top of the Colbourn box. Both inlet and outlet tubing were encased in a protective stainless steel coil which extended from the liquid swivel to the brass collar. A 2.5-ml gas-tight syringe (Unimetrics) and syringe pump (Model 22, Harvard Apparatus; South Natick, MA, USA) were used to perfuse a modified Ringer’s solution (in millimolar: 10 sodium phosphate buffer, 1.3 CaCl2, 3.0 KCl, 1.0 MgCl2, 147 NaCl, pH 7.4) through the probe at 1 μl/min. DA content in microdialysis samples were separated by high pressure liquid chromatography (HPLC) and quantified by electrochemical detection. The HPLC system consisted of, in sequence of flow, a Bio-Rad pump (Hercules, CA, USA), an SSI pulse damper (State College, PA, USA) a Valco Instruments two-position auto-Injector (EC10W; Houston, TX, USA), a Beckman reverse-phase column (Ultrasphere, ODS 5 μm, 15 cm, 4.6 mm internal diameter; Fullerton, CA, USA), an ESA guard cell (Model 5020; Chemlsford, MA, USA), an ESA analytical cell (Model 5011), an ESA Coulochem II Electrochemical detector, and a dual channel chart recorder. The electrochemical detection parameters were: +450 mV for the oxidation channel, −300 mV for the reduction channel, and −450 mV for the guard cell output. The mobile phase (in millimolar: 83 sodium acetate buffer, 27 EDTA, and 1.30 sodium octyl sulfate at pH 3.5, 10% methanol) flowed through the system at 1 ml/min. Microdialysis experiments In all experiments, each rat was tested with microdialysis on days 5 and 16. The side of probe implantation (i.e., left or right brain structure) was counterbalanced for each experiment day. The two microdialysis sessions were conducted using an identical protocol. The day before an experiment, rats were implanted unilaterally with probes in the NAc (exposed membrane spanned −6.0 to −8.0 mm DV from dura) or MD striatum (−4.0 to −6.0 mm DV from dura) after the day’s training session and kept overnight (∼16–18 h) in the test chamber with water under baseline conditions. The next morning, microdialysis samples were collected at 10-min intervals (10 μl volume) and analyzed immediately with HPLC. Baseline conditions were continued until four consecutive samples showed stable DA levels (i.e., <10% fluctuation between samples; times 1–4). In “Experiment 1”, the baseline period was followed by the concurrent illumination of the chamber and insertion of the lever. Rats were then allowed a 30-min period (times 5–7) during which they could lever press for food on a RI 30-s schedule. The session concluded with the retraction of the lever and a 40-min time-out period with the lights off (times 8–11). In “Experiment 2”, a 30-min extinction phase was incorporated (times 5–7) before a food reward phase. All aspects of the extinction phase were identical to previous rewarded training sessions except that lever presses did not lead to food delivery. A 20-min time-out period under baseline conditions (times 8–9) preceded a 10-min priming period when the light was on and five pellets were delivered randomly and noncontingently (time 10). The start of a food-rewarded phase (times 11–13) was marked by the insertion of the lever into the already illuminated chamber. During this period, rats were allowed to lever press for pellets on a RI 30-s schedule for 30 min. An experiment concluded with an additional 40-min postsession baseline period (times 14–17). Histology Rats were deeply anesthetized with chloral hydrate and intracardially perfused first with 0.9% NaCl and then phosphate-buffered formalin (3.7% formaldehyde). The removed brains were stored in 15% (w/v) sucrose in formalin for at least 24 h before being prepared as 50 μm coronal sections on 2% gel-coated slides. Cresyl violet staining was used to help verify placement of probe tracts. Only data from those rats with tracts in the shell/core region of the NAc (16 of 18 rats) and MD striatum (six of seven rats) of both hemispheres were included in the statistical analyses. Data analyses Neurochemical data were transformed into percentage of change from baseline (i.e., 0% representing the average concentration of the three samples preceding the final 4th baseline sample). Neurochemical data were analyzed using either a one-way (time) or two-way (day × time) repeated measures ANOVA followed by the Dunnett method of multiple comparisons, using the final baseline sample (time 4) as the control sample. The Huynh–Feldt correction for nonsphericity was applied to the degrees of freedom for all within-subject analyses. Comparisons between two means were assessed using paired t tests. A coefficient of determination (R2) was computed based on a linear regression of a scatter plot between lever presses/10 min (y-axis) and corresponding percent changes in DA efflux (x-axis), for data obtained on test days 5 and 16. Statistical analyses were performed using Systat or SPSS statistical packages. Results Experiment 1 Changes in extracellular DA levels in the NAc were compared after limited and extended training sessions, while rats lever pressed for food reward on a RI 30-s schedule. As shown in Fig. 1, the rates of responding approximately doubled from days 5 to 16, even though rats obtained and consumed a similar number of rewards during the two sessions [43.3 pellets (∼2.0 g) on day 5 and ∼46.2 pellets (∼2.1 g) on day 16]. The number of magazine entries also did not differ between test days. Separate one-way ANOVAs revealed a significant main effect of time on DA efflux on day 5 (F7,49=12.038, p<0.001) and day 16 (F7,49=5.325, p<0.044). Further analyses indicated that on both days, there was a significant increase in DA efflux above their respective baselines that remained elevated for the 30-min duration of instrumental responding for food pellets (Dunnett’s, p<0.05). Despite the doubling of response rates from days 5 to 16, the pattern and magnitudes of DA efflux on the two test days were not statistically different (maximal increase of 69±16% on day 5 and 71±28% on day 16), as a two-way repeated measures ANOVA failed to show a significant interaction of day × time on DA efflux (F7,98=0.598, p=0.528). However, a paired t test showed that during the first 10 min (time 5), DA efflux was significantly higher on day 16 than day 5 (68±24 vs 30±12%, respectively; p<0.05). Basal values of DA in the NAc (uncorrected for recovery) were 2.72±0.61 and 2.19±0.29 nM for days 5 and 16, respectively, and were not statistically different from each other. Fig. 1Change in DA efflux in the NAc (line graph) during instrumental conditioning on day 5 (left panel) and day 16 (right panel) in the same group of rats (n=8). Dotted lines highlight the Reward phase in which food pellets were delivered on a RI 30-s schedule (times 5–7). Lever presses (gray bars) and magazine entries (black bars) are shown per 10 min bin. Data are represented as mean±SEM. * indicates significant difference from the final baseline value (time 4) according to Dunnett’s method of multiple comparisons, p<0.05. † indicates significant difference from the corresponding data point on day 5 according to a paired t test, p<0.05 Experiment 2 Instrumental behavior As shown in Fig. 2, all rats in the NAc and MD striatal microdialysis groups learned to lever press for food pellets on a RI 30-s schedule and to retrieve the pellets, as indicated by the number of magazine entries. Training data for one rat in the MD striatal group was lost and not included in the following analyses. Instrumental behavior became more efficient through the initial days of training, and during the third and fourth training sessions, rats made significantly more lever presses than magazine entries. This increase in ratio of lever presses to magazine entries, from limited to extended training experience, may be explained by proposing that rats learned to use the auditory click made by the dispenser with the delivery of a pellet. Thus, rats learned to enter the magazine only when they heard the click. On days (4 and 15) before microdialysis tests, rats were allowed to press for 60 pellets, rather than the normal 30 pellets available on other days, and accordingly increased the rate of lever presses on these days. The mean number of lever press responses per training session across the RI 30-s schedule tended to be higher in the NAc group (from 327 to 835) than in the MD striatal group (from 367 to 436). However, an ANOVA test revealed no main effect of group (F1,11=2.414, p=0.149). ANOVA of magazine entry data similarly indicated that counts were comparable between the NAc and MD striatal groups (F1,11=0.366, p=0.557). Fig. 2Instrumental performance of rats in the NAc or MD striatal group over 16 training sessions. Rats were trained to lever press for food pellets on a RI 2-s schedule the first day, RI 5-s the next day, and then RI 30-s for the remainder of the study. Each training session terminated upon delivery of 30 pellets (exception: *60 pellets delivered on days before test). Test days (day 5 and 16) were composed of two phases, Extinction and Reward, which lasted 30 min each. Lever presses (gray and striped bars) and magazine entries (black bars) are shown per training session. Data are represented as mean+ SEM On test days, rats were tested for instrumental responding in extinction and then with food reward. During the extinction phase, the pellet dispenser was disconnected from the magazine, but still produced an auditory click according to the RI 30-s schedule. During the rewarded phase, all rats consumed every pellet delivered during the lever press sessions. During the rewarded component of the experiment, rats consumed ∼45.7 pellets (∼2.1 g) on day 5 and ∼46.2 pellets (∼2.1 g) on day 16. The similarity in food consumption and difference in magnitude of DA efflux (see below), across days 5 and 16, again supports the view that DA response is not a function of food reward. In both the NAc and MD striatal groups (Figs. 3 and 4), rate of instrumental performance approximately doubled from day 5 to day 16, during both the extinction and rewarded phases of the sessions. Over the 30-min extinction phase, rats displayed a typical decline in rate of lever presses, whereas over the 30-min reward phase, there was a slight increase in rate of responding. In the NAc group, the number of lever presses was significantly higher on day 16 than on day 5 during the first 10 min of the extinction (t test, p=0.003) and rewarded (t test, p=0.002) phases. In the MD striatal group, the lever press rates during the first 10 min of the extinction phase were comparable on days 5 and 16, but differed significantly between the 2 days during the first 10 min of the rewarded phase (p=0.038). In both the NAc and MD striatal groups, an ANOVA indicated that the number of magazine entries did not differ significantly between days 5 and 16. Fig. 3Change in DA efflux in the NAc (line graph) during instrumental conditioning on day 5 (left panel) and day 16 (right panel) in the same group of rats (n=8). Dotted lines highlight the Extinction phase during which an auditory CS+ was activated on an RI 30-s schedule in the absence of food pellet rewards (times 5–7) and a Reward phase in which food pellets were delivered on a RI 30-s schedule (times 11–13). P represents the period during which five priming pellets were delivered noncontingently. Lever presses (gray bars) and magazine entries (black bars) are shown per 10 min bin. Data are represented as mean±SEM. * Indicates significant difference from the final baseline value (time 4) according to Dunnett’s method of multiple comparisons, p<0.05. † Indicates significant difference from the corresponding data point on day 5 according to a paired ttest, p<0.05. § Indicates significant difference from the final baseline value (time 4; ttest, p<0.05)Fig. 4Change in DA efflux in the MD striatum (line graph) during instrumental conditioning on day 5 (left panel) and day 16 (right panel) in the same group of rats (n=6). See Fig. 3 for explanation of symbols. No statistically significant results were observed for neurochemical data set. † Indicates significant difference from the corresponding behavioral score on day 5 according to a paired ttest, p<0.05 DA efflux Basal values of DA in the NAc (uncorrected for recovery) were 2.58±0.32 and 2.16±0.16 nM for days 5 and 16, respectively, and were not statistically different from each other. Basal values of DA in the MD striatum (uncorrected for recovery) were 2.71±0.23 and 2.95±0.34 nM for days 5 and 16, respectively, and also did not differ statistically. In the NAc, the overall pattern of DA efflux across the different phases of the microdialysis test on day 5 appeared comparable to that observed on day 16 (Fig. 3), but statistical analyses revealed several key differences. Accordingly, an ANOVA identified a significant day × time interaction on DA efflux (F13,182=2.760, p=0.022), with a significant simple main effect of Time on day 5 (F13,91=8.690, p<0.001) and day 16 (F13,91=14.649, p<0.001). On both days, there was an increase in NAc DA levels during the initial 10 min of responding in extinction (10±7% above baseline on day 5 and 19±6% on day 16), but this increase was only significant after extended training on day 16 (paired samples t test, p=0.005). DA efflux then returned to baseline for the remainder of the extinction phase and time-out period. During the 10-min period preceding the rewarded responding phase, five pellets were noncontingently dispensed into the magazine; the purpose of this was to prime the rats to lever press again for food reward. During this period, DA levels did not differ from baseline values on day 5 (4±9%) but by day 16, were increased significantly above baseline (27±7%; Dunnett’s test, p<0.05). The reward phase of the session was accompanied by significant elevation of DA efflux on both days (maximal increase of 37±12% on day 5 and +83±15% on day 16; Dunnett’s test, p<0.05) that remained elevated after retraction of the lever and cessation of instrumental responding. DA levels then gradually declined towards baseline values over the remaining 60 min of the test session. During the initial 10 min of the reward phase (time 11), the magnitude of change on day 16 (83±15%) was significantly greater than the change in efflux observed on day 5 (37±12%; t test, p=0.013). In the MD striatum, there were no statistically significant changes in DA levels during the entire instrumental responding session on both days 5 and 16 (Fig. 4). Despite performing lever presses and magazine entries at rates comparable to the NAc group, DA levels in this group showed only slight fluctuations around baseline. Correlation between response rate and DA efflux Based on a linear regression of a scatter plot between lever presses/10 min and percent change in DA efflux, R2 values of 0.0014 for data from day 5 and 0.0065 on day 16 indicated that <1% of the variation in lever presses could be explained by a linear correlation between lever presses and DA levels, on both test days (Fig. 5). Fig. 5Correlation between instrumental response rates and NAc DA efflux. Scatter plot of lever presses/10 min (y-axis) and corresponding percent change in DA efflux (x-axis) for data obtained on day 5 (left panel) and day 6 (right panel). Shown on each graph is the best fit linear regression line and R2 value Histology The locations of all microdialysis probes are presented in Fig. 6. The 2-mm lengths of the dialysis membrane were in the NAc (shell-core boundary) or medial aspect of the MD striatum (just dorsal and lateral of the anterior commissure). Fig. 6Location of microdialysis probes in the NAc and MD striatum. Black bars represent 2 mm length of dialysis membranes. Numbers beside each plate correspond to millimeter from bregma. Coronal drawings were modified from Paxinos and Watson (1997) Discussion The present study examined the role of DA activity in the NAc in behaviors maintained by instrumental and Pavlovian incentive learning. DA efflux in the NAc was increased significantly during both early and later training stages of an instrumental response for food on a RI-30-s schedule of reinforcement (Figs. 1 and 3). It is important to note that this pattern of results was observed whether a period of extinction preceded a 30-min period of instrumental responding reinforced by food pellets. As such, the present findings confirmed previous reports of increased DA release in the NAc during lever pressing for food, employing fixed interval or ratio schedules of reinforcement (Salamone et al. 1994; Richardson and Gratton 1996; Cousins et al. 1999). Response actions during the early phase of training on interval schedules (i.e., in rats having received as few as 120 outcomes) have been characterized as goal- or outcome-directed (Adams and Dickinson 1981; Balleine and Dickinson 1992; Dickinson et al. 1995) and accordingly may represent instrumental incentives, as distinct from Pavlovian incentive processes related to incentive motivation (Parkinson et al. 2002). Continued performance of these instrumental actions leads to habitual responding which, unlike action–outcome learning, is impervious to outcome devaluation or contingency degradation (Dickinson et al. 1995). We failed to observe a selective increase in DA efflux when rats had limited as compared to extended training experience, as might be expected if dopaminergic activity in the NAc is related to instrumental incentive learning. Indeed, the magnitude of DA efflux was significantly greater after extended training when behavior is said to be no longer controlled by incentive learning and is based instead on habit. These increases in DA efflux were site-specific, as no significant changes in medial MD striatal DA efflux were observed throughout the different phases of this experiment (Fig. 4). Performance on instrumental tasks is often conducted under nonrewarded or extinction conditions to evaluate the control of behavior by Pavlovian incentive stimuli, unconfounded by unconditioned reward stimuli. In the present study, a significant increase in DA efflux in the NAc was observed only during the initial 10-min sample of responding in extinction on training day 16, but not on day 5 (Fig. 3). The specific CS+ present in this experiment was a distinct auditory “click” of the pellet dispenser which occurred on a RI 30-s schedule, by itself during extinction or accompanied by delivery of food pellets into the magazine during rewarded responding. As such, these data are consistent with previous reports of increased DA efflux in the NAc elicited by a CS+ (Phillips et al. 1993; Datla et al. 2002). In a Pavlovian to instrumental transfer protocol, a CS+ previously paired noncontingently to food reward can facilitate the acquisition of an instrumental response (Parkinson et al. 2002). Systemic administration of DA receptor antagonists during Pavlovian pairings of a CS+ with food reward blocks Pavlovian to instrumental transfer (Beninger and Phillips 1981; Dickinson et al. 2000). Damage to the shell or core of the NAc spares the acquisition of Pavlovian to instrumental transfer, but disrupts the potentiation by intra-NAc amphetamine on responding for the CS+ (Parkinson et al. 2002). Together, these findings suggest that a phasic increase of DA in the NAc, shown to occur after treatment with amphetamine (Taepavarapruk and Phillips 2003; Brebner et al. 2005), may mediate the facilitatory effects of a Pavlovian CS+ on instrumental responding. It is also of interest to note that the inclusion of an extinction session before a reinforced phase of instrumental responding attenuated the magnitude of DA efflux in the NAc observed when food reward was available on day 5, but not on day 16. This finding may be attributed to attenuation in the secondary reinforcement property of the CS+ associated with the delivery of food reward or possibly, an influence of frustrative nonreward engendered by extinction. In either case, it is apparent that these effects of extinction are restricted to the early phase of instrumental training. In earlier studies, Salamone et al. (1994) proposed that an important aspect of dopaminergic activity in the NAc is related to behavioral activation, exertion of effort, and possibly cost benefit analyses relating effort to value of reward stimuli (Salamone et al. 2003, 2005). In support of this hypothesis, consumption of large quantities of freely available food pellets or lab chow was not accompanied by increased DA efflux. It must be noted in passing that consumption to satiety of a large meal of a palatable food such as fruit loops, onion rings (Ahn and Phillips 1999), or sucrose (Hajnal and Norgren 2002), is accompanied by a significant increase in DA efflux in both the NAc and medial prefrontal cortex. Salamone et al. (1994) also observed a significant relationship between response rates of individual rats and the magnitude of DA efflux in the NAc. Specifically, an increase in DA release was only observed in rats that responded at medium to high rates of responding, whereas in rats with low response output, this measure did not differ from controls. Our data are also relevant to the relationship between response output and DA activity. In both Experiments 1 and 2, there was no evidence of a simple relationship between response rates and magnitude of DA efflux. As shown in Fig. 1, although the rate of lever presses were twice as high on day 16 compared to day 5, the magnitude of DA efflux did not different significantly between the 2 days. In Fig. 3, on day 5, initial response rates during the extinction and reward phases of the test were comparable during the first 10 min (times 5 and 11), yet the corresponding magnitude of DA efflux during the reward phase was three times greater than the extinction phase. A similar pattern was observed on day 16. Thus, different rates of responding were associated with similar magnitudes of DA efflux, and similar rates of responding were associated with different magnitudes of DA activity. Accordingly, no positive correlations between rate of lever press responding and magnitude of DA efflux in the Nac were observed after limited and extended training (Fig. 5). Finally, with respect to the appealing hypothesis that dopaminergic activity in the NAc is related to behavioral activation (Salamone et al. 2003, 2005), it must be emphasized that although the present data challenge this hypothesis, it cannot be refuted simply on the basis of the lack of a correlation between magnitude of DA efflux and intensity or degree of behavioral activation. The failure to observe a significant increase in DA efflux in the MD striatum during either action–outcome or habit-based instrumental responding provides a clear indication that DA transmission in this region of the striatum is not involved in instrumental conditioning or stimulus–response habit formation. These data are consistent with the finding that neither excitotoxic lesions nor reversible inactivation of the anterior MD striatum had any effect on acquisition or expression of action–outcome associations in instrumental conditioning (Yin et al. 2005). In contrast, lesions or inactivation of the MD striatum, posterior to the probe placements in the present study, impaired instrumental performance based on outcome–expectancy (Yin et al. 2004). Blockade of NMDA receptors in the dorsomedial striatum also disrupted action–outcome learning consistent with a role for glutamate-mediated synaptic plasticity in the encoding of action–outcome associations (Yin et al. 2005). The MD (“associative”) striatum receives inputs from association cortices (e.g., prelimbic region of the prefrontal cortex and premotor areas), as well as the basolateral amygdala, which appears to mediate the assignment of incentive value to the consequences of instrumental actions (Corbit and Balleine 2005). Integrity of the dorsolateral striatum has been shown to be required for habit formation in instrumental learning, and furthermore, rats with damage to this region of the striatum reverted to a state in which instrumental actions were goal-directed (Yin et al. 2005). This finding implies that the system involving the dorsolateral striatum responsible for habit formation can inhibit the circuit that mediates action–outcome or goal-directed instrumental actions. This in turn raises the possibility that the increase in NAc DA efflux observed in the present study after extended training provides a representation of instrumental incentive learning that is held in check by activity in the dorsolateral striatum. In conclusion, the present findings provide neurochemical evidence in support of previous data questioning the role of DA in the NAc in coupling incentive value to representations of instrumental outcomes (de Borchgrave et al. 2002). The data showing elevated DA efflux in the NAc during extinction in the presence of a Pavlovian CS+, in turn are consistent with a role for the NAc in incentive motivation (Fibiger and Phillips 1986; Robbins et al. 1989; Phillips et al. 1993; Balleine and Killcross 1994; Berridge and Robinson 1998). Rats received an apportionment of ∼60 food pellets during microdialysis test on days 5 and 16, yet the magnitude of DA efflux was significantly greater after extended training sessions. These data refute the hypothesis that dopaminergic activity in the NAc is a reflection of either reward value or reinforcement of instrumental responses (Wise 2004). The present “within subject” design revealed a hitherto unappreciated effect of extended training of instrumental responding with an interval schedule on the magnitude of DA efflux in the NAc. For reasons discussed above, this does not appear to be related to motor responding per se. Rather, we speculate that this effect may reflect the specific condition of a random or variable interval schedule of outcome presentation, in which extended training is necessary to appreciate that the probability of receiving a beneficial outcome at any particular time in the 30-min test session is always unpredictable. This degree of uncertainty may be highly compatible with the optimal conditions for activating midbrain DA neurons (Fiorillo et al. 2003), which in turn would result in a sustained increase in DA release in the NAc throughout a period of random reinforcement. This pattern of DA release could play an important role in maintaining a high level of motivation at the service of a variety of response strategies available to ensure access to objects essential for survival.

Mol_Immunol-1-5-1995235

The mouse complement regulator CD59b is significantly expressed only in testis and plays roles in sperm acrosome activation and motility

In mouse, genes encoding complement regulators CD55 and CD59 have been duplicated. The first described form of CD59, CD59a, is broadly distributed in mouse tissues, while the later identified CD59b was originally described as testis specific. Subsequent studies have been contradictory, some reporting widespread and abundant expression of CD59b. Resolution of the distribution patterns of the CD59 isoforms is important for interpretation of disease studies utilising CD59 knockout mice. Here we have performed a comprehensive distribution study of the CD59 isoforms at the mRNA and protein levels. These data confirm that expression of CD59b is essentially restricted to adult testis; trace expression in other tissues is a consequence of contamination with blood cells, shown previously to express CD59b at low level. In testis, onset of expression of CD59b coincided with puberty and was predominant on the spermatozoal acrosome. Ligation of CD59b, but not CD59a, markedly reduced spermatozoal motility, suggesting a specific role in reproductive function. 1 Introduction CD59, a broadly distributed glycosyl phosphatidylinositol (GPI)-anchored protein, is the principal regulator of complement membrane attack complex (MAC) assembly on cell membranes (Meri et al., 1990). A decade ago, we identified the mouse analogue of CD59 and showed that it was broadly distributed on cells and tissues (Powell et al., 1997). Deletion of the gene encoding mouse CD59 caused minimal disturbance in unchallenged animals but markedly enhanced susceptibility to complement-driven pathologies (Holt et al., 2001; Turnberg et al., 2003, 2004; Lin et al., 2004; Mead et al., 2004; Williams et al., 2004). Others demonstrated that the gene encoding CD59 is duplicated in the mouse; the protein products of these two genes were then termed CD59a for the first described, and CD59b for the product of the new gene (Qian et al., 2000). In this first report, it was stated that CD59b message was significantly expressed only in testis. We developed monoclonal antibodies specific for CD59a and CD59b, enabling us to confirm the broad distribution of CD59a and testis restricted expression of CD59b at the protein level; we concluded that CD59a was the principle regulator of MAC assembly in mouse tissues (Harris et al., 2003). This conclusion was challenged in a re-analysis of the pattern of distribution of mRNA encoding the two forms of CD59; these studies suggested that both CD59a and CD59b were broadly expressed in the mouse and contended that the latter was the major regulator of MAC in the mouse (Qin et al., 2003). These contentions were bolstered by the demonstration that deleting the gene encoding CD59b in mice caused a severe, spontaneous phenotype with anaemia, platelet dysfunction and fertility problems. In light of these data we reassessed the distribution patterns at protein and mRNA levels using a combination of specific monoclonal antibodies and shared primers for PCR in high sensitivity assays (Baalasubramanian et al., 2004). These analyses confirmed our earlier work showing that CD59a was broadly expressed while significant expression of CD59b was restricted to testis. Trace amounts of CD59b message and protein were detected in erythrocytes but quantitative assays of protein expression showed that CD59b expression on erythrocytes was less that 5% that of CD59a. Further, we confirmed that CD59a was dominant in protection of erythrocytes from MAC lysis. The findings of these painstaking studies have recently been challenged yet again (Qin et al., 2006). The appropriateness of the techniques used and specificity of probes and primers have all been questioned. These authors concluded that CD59b was broadly expressed and an important regulator of MAC assembly on erythrocytes and in tissues. We, and others, are using CD59a knockout mice in disease models to explore roles of MAC based on our evidence that CD59a is the principal regulator of the MAC in most tissues. If CD59b is indeed widely distributed then the value of studies in CD59a knockouts is in question. It is therefore essential that we test the evidence. Here using multiple methods we revisit these published studies and undertake new analyses to explore the expression patterns of CD59a and CD59b at the mRNA and protein level. We conclude that expression of CD59b at the mRNA and protein level is essentially absent in all tissues other than testis. Low level expression on blood cells was confirmed and trace detection of mRNA in tissues was shown to be likely due to blood contamination. We further analysed expression of CD59b in testis and showed that expression coincided with onset of puberty and was restricted to spermatozoa and their immediate precursors. Ligation of CD59b on spermatozoa with monoclonal antibody markedly inhibited sperm motility, suggesting a specific role in reproductive function. 2 Materials and methods 2.1 Mice Adult (8–16 weeks) and infant (1–5 weeks; pre-puberty) male mice, and embryos (day 5, 10 and 15) from C57BL/6(H-2b) background mice were used in our investigation. All experimental procedures were performed in compliance with Home Office and local ethics committee regulations. cd59a−/− mice generated as described previously (Holt et al., 2001), and back-crossed 10 generation onto the same background were used as controls. 2.2 Antibodies and reagents Rat anti-mouse CD59a (CD59a.1; IgG1) was generated in house (Harris et al., 2003). Mouse anti-mouse CD59b (CD59b.2; IgG1) was also made and characterized in house (Baalasubramanian et al., 2004). Separate aliquots of CD59a.1 and CD59b.2 were labelled with NHS-biotin (Sigma–Aldrich, Gillingham, Dorset, UK) and FITC-NHS (Perbio Science UK Ltd., Cramlington, UK) according to manufacturer's protocols. As negative controls, rat IgG1 and mouse IgG1 (purified in-house) were labelled in the same manner. FITC-labelled streptavidin was purchased from DAKO (Ely, Cambridgeshire, UK). HRPO-labeled donkey anti-rat IgG and HRPO-labeled donkey anti-mouse IgG were purchased from Jackson ImmunoResearch Europe (Newmarket, Suffolk, UK). 2.3 Semi-quantitative RT-PCR Total RNA was purified from all investigated mouse tissues using GenElute kit (Sigma–Aldrich) and controlled for DNA contamination by RT-PCR without using reverse transcriptase and employing a β-actin specific primer pair (Table 1). Only samples that did not show amplification were used in all analyses. Aliquots of these RNAs (1 μg each) were reverse transcribed using random hexamers and multiscribe reverse transcriptase according to the manufacturer's instructions (Applied Biosystems, Warrington, UK). Primer pairs specific for either CD59a or CD59b were as described by Qin et al. (2006), and a new primer pair common for both CD59a and b and amplifying a sequence in exon 4 comprising 199 bp for CD59a and 162 bp for CD59b, were used in the subsequent amplifying reactions for detection of CD59a and CD59b mRNAs (Table 1). Either 25 or 35 amplification cycles were used, except where attempting simultaneous detection of both mRNAs when 40 cycles were performed. Amplified products were separated either in 1% agarose or in 5% poly-acrylamide gels (PAAG). To obtain semi-quantitative data for expression of the two genes, we used as templates either 2 μl of the original cDNA, or a series of dilutions as indicated in figures. Gels were scanned and band intensity was measured by densitometry using Quantity One 4.3.0 software (BioRad, Hemel Hempstead, Hertfordshire, UK). The quantifications were carried out in triplicate and means and errors calculated. 2.4 Quantitative real-time PCR analysis The cDNAs from testis and liver prepared as described for the semi-quantitative RT-PCR, were subject to a Taqman assay as described previously (Baalasubramanian et al., 2004). One of the primer pairs was as described previously (Baalasubramanian et al., 2004), a perfect match for the sequence of CD59a mRNA and with a single internal nucleotide mismatch to CD59b mRNA; the second set was a perfect match for the same sequence in CD59b and with a single internal nucleotide mismatch to CD59a (Table 1). To measure the relative number of mRNA copies for CD59b in liver, perfused liver and testis we used the primer pair reported by Qin et al. (2006) to specifically amplify CD59b in a quantitative PCR (QPCR). Because these primers were not designed specifically for this assay, they did not meet the optimal annealing temperature requirements. To correct for this anomaly, the standard amplification programme was modified to include a pre-annealing step at 56 °C for 20 s prior to the extension phase. Primers were designed to β-actin (Table 1) as an internal control for normalization of starting cDNA levels. Quantitative PCR was performed on ABI PRISM 7000 using either TaqMan Universal PCR Master Mix or SYBR Green PCR Master Mix according to the manufacturer's instructions (Applied Biosystems) with 50 cycles of amplification. 2.5 Preparation of tissue lysates To obtain tissue lysates, freshly harvested mouse organs were immediately chilled and homogenized with ice-cold lysis buffer (PBS containing 2% NP40, 1 mM phenylmethylsulfonyl fluoride, 10 mM EDTA, 1 μg/ml leupeptin, and 1 μg/ml pepstatin; 1 g of tissue/1.5 ml of buffer) and incubated for 60 min on ice. Insoluble debris was removed by centrifugation (5000 × g, 15 min at 4 °C) and the supernatants stored in aliquots at −80 °C until use. In some experiments the mouse was perfused with saline at the time of sacrifice to reduce blood contamination in harvested organs. 2.6 SDS-PAGE and Western blot analysis Lysates were mixed 1:1 with sample buffer for SDS-PAGE and separated under non-reducing conditions in 15% gels. Separated proteins were transferred onto nitrocellulose membranes (Schleicher & Schuell, London, UK), and membranes blocked with 5% (w/v) non-fat milk in PBS (PBS-M). Membranes were then probed with the primary mAb diluted in PBS-M, washed in PBS containing 0.1% Tween-20 (PBS-T), then probed with HRPO-conjugated donkey anti-rat Ig in PBS-M to detect the rat anti-CD59a or HRPO-conjugated donkey anti-mouse Ig (Jackson) to detect the mouse anti-CD59b. After further washing in PBS-T, bands were developed using ECL (Perbio Science UK Ltd.) and captured on autoradiographic film (Kodak Ltd., Hemel Hempstead, Hertfordshire, UK). 2.7 Spermatozoa preparation and analysis Motile cauda epididymal spermatozoa were obtained by a ‘swim-up’ technique as previously described (Mizuno et al., 2004, 2005). Briefly, two cauda epididymi from an adult C57BL/6 mouse were roughly minced in 1 ml of DMEM (Invitrogen, Paisley, UK). This suspension was carefully overlayered with 1 ml DMEM and incubated at room temperature for 15 min. The upper layer was removed and spermatozoa pelleted by centrifugation (1000 × g) for 5 min at room temperature. Cells were washed twice by gentle centrifugation as above. For immunofluorescence studies, the re-suspended cells were smeared onto glass slides, air-dried immediately, fixed in acetone at room temperature for 1 min and stored at −20 °C until use. To prepare lysate, the swim-up spermatozoa from 4 cauda epididymi were pelleted and incubated with mixing in 100 μl of lysis buffer for 30 min on ice. Insoluble debris was removed by centrifugation (15,000 × g, 15 min at 4 °C) and the supernatant stored in aliquots at −80 °C. To compare CD59b expression in unactivated and acrosome-reacted spermatozoa, the acrosome reaction was induced essentially as described (Mizuno et al., 2004). Briefly, swim-up spermatozoa (106 cells/ml of DMEM) were incubated for 1 h at 37 °C with the calcium ionophore A23187 (Sigma–Aldrich) at 1 μM to induce the acrosome reaction. Control cells were incubated without ionophore. Acrosome-reacted and control spermatozoa were smeared on glass slides and immediately air-dried. To observe CD59b distribution, the smears were incubated with FITC-labeled CD59b.2. Nuclei were counterstained with DAPI (4′-6-diamino-2-phenylindole-2 HCl; 100 ng/ml final concentration; Sigma–Aldrich). On each slide, at least 100 cells were counted and the assay was carried out in triplicate. 2.8 Functional inhibition assay of CD59a and CD59b in mouse sperm Spermatozoa harvested by swim-up from two epididymi, either from wild type or cd59a−/− mice, were washed and suspended in 2 ml DMEM. Paired aliquots (200 μl) were incubated at 37 °C with mAbs CD59a.1, CD59b.2, or isotype-matched control mAb, each at 10 μg/ml. After 4 h of incubation, the sample was immediately placed on a glass haemocytometer slide and the total number of sperm and percentage remaining motile were counted under light microscopy. This experiment was performed in triplicate. 2.9 Statistical analysis All values are expressed as mean ± standard error (S.E.M.). The statistical analysis was performed by one-way ANOVA. When significant differences were observed, statistical analysis was further carried out using unpaired t-test between two groups. Significance between two groups was claimed when P < 0.05. 3 Results 3.1 CD59b mRNA is highly expressed only in testis We have previously demonstrated that CD59a is the primary regulator of MAC assembly in mouse (Baalasubramanian et al., 2004). However, in a recent work (Qin et al., 2006), abundant expression of CD59b mRNA in a number of tissues was reported. In an attempt to clarify this issue, which is of key importance for complement studies in mice, we repeated some of the experiments carried out by Qin and co-authors using the primer pairs designed by them. Under routine conditions (25 cycles of amplification), CD59a mRNA was detected in all tissues tested while CD59b mRNA was found only in testis (Fig. 1A). Increasing the number of amplification cycles to 35 (Fig. 1B) revealed weak expression of mRNA for CD59b in liver, blood cells, heart and lung. Amplified sequence was confirmed to be CD59b by sequencing. We next re-visited the primers used in our previous quantitative PCR analysis to assess expression of the two CD59 isoforms in different mouse tissues (Baalasubramanian et al., 2004). We had designed a primer pair that annealed to both isoforms and Taqman probes specific for either CD59a or CD59b to enable accurate quantitation. Qin et al. (2006) criticised these experiments on the basis that, while both primers used in our assay matched perfectly CD59a, each had a single internal nucleotide mismatch compared to the CD59b sequence (Table 1). They suggested, without evidence, that this mismatch favoured amplification of CD59a and caused our inability to detect CD59b in tissues other than testis and bone marrow. There is a large primer design literature that shows clearly that internal mismatches, unlike those at the 3′ end, do not significantly influence amplification (Sommer and Tautz, 1989; Kwok et al., 1990; Christopherson et al., 1997; Löffert et al., 1998). Nevertheless, we addressed further this issue by repeating our previous QPCR investigation but including a second primer pair from the same sequence matching perfectly CD59b mRNA but with a single mismatch to CD59a mRNA in the same position as in the original primer set (Table 1). Using the original primer pair that matched perfectly CD59a we obtained very similar results to those we previously published (Baalasubramanian et al., 2004). When we used the pair matching CD59b, there was a small increase in threshold cycle (Ct) for both CD59a and CD59b, likely a result of altered primer annealing temperature, but there was no difference in the calculated relative amounts for CD59a and CD59b mRNA in testis compared to the original primer set (Table 2). Of note, QPCR with either primer set did not detect any CD59b mRNA in liver, suggesting that the high cycle number PCR described above was detecting very small amounts of mRNA in these tissues. In summary, the data shows an 8-fold greater expression of CD59a mRNA in liver compared to testis, a 5-fold greater expression of CD59a mRNA compared to CD59b in testis and undetectable expression of CD59b in liver, regardless of primer set used. In light of these results we developed a semi-quantitative PCR to quantify the low levels of CD59b mRNA detected in tissues. In order to be able directly to compare the expression of both isoforms, we designed a new primer pair within exon 4 (Table 1), which is 100% homologous to both mRNAs. Amplification of DNA-free mRNA with these primers will result in bands of 199 bp for CD59a and 162 bp for CD59b. In testis we detected both isoforms as expected (Fig. 2A). Densitometric comparison of band intensities at higher dilutions of template, when the amplification reaction is in the linear range, showed approximately 5-fold higher expression of CD59a mRNA as compared to CD59b mRNA in testis, a result compatible with the QPCR data (Table 2) and our published results (Baalasubramanian et al., 2004). However, in liver we failed to detect presence of CD59b even after 40 cycles of PCR (Fig. 2B). We reasoned that the large excess of CD59a mRNA in liver out-competes trace amounts of CD59b mRNA for limited reagents in the initial cycles of amplification, thereby reducing the chance for amplification of the low abundance CD59b mRNA. To test this reasoning we performed semi-quantitative PCR in separate tubes, using the primers specific for either CD59a or CD59b mRNA (Qin et al., 2006). Firstly, to optimise the annealing and amplification efficiency for both primer pairs, we performed this reaction for testis mRNA (Fig. 3A). Similar band intensities for CD59a and CD59b were obtained for 5 × 105-fold and 105-fold template dilution respectively (Fig. 3A and B). This indicated an approximate 5-fold higher expression of CD59a mRNA compared to CD59b mRNA, supporting our results described above (Table 2) and published (Baalasubramanian et al., 2004), and confirming similar amplification efficiency for the specific primer pairs. Amplification of liver mRNA using these primers, and comparison of band intensities in dilutions of templates showed that expression of CD59b mRNA in liver was approximately 105-fold less compared to that of CD59a mRNA (Fig. 3C and D similar band intensities for CD59a and CD59b at 5 × 105 and 5-fold dilution respectively). In these latter experiments we used liver perfused with saline at the time of sacrifice to remove the bulk of entrapped blood; nevertheless, the trace mRNA detected might be from residual blood cells, which clearly express CD59b protein at low level (Baalasubramanian et al., 2004; Qin et al., 2006). To clarify this issue, we compared mRNA from perfused and non-perfused liver by QPCR using CD59b-specific primers and found the amount of CD59b mRNA in liver was reduced 12-fold by perfusion (Fig. 3E). For comparison, expression of CD59b in testis was 2200-fold higher than in unperfused liver and approximately 25,000-fold higher than in perfused liver. Taken together these results strongly suggest that the detectable traces of CD59b mRNA in liver and likely other organs are a consequence of contamination with blood cells. 3.2 Expression of CD59b protein is testis restricted We have previously reported, using immuofluorescence staining, that CD59b protein was highly expressed in testis but absent from all other organs tested (including liver, lungs, spleen, kidney and heart) (Baalasubramanian et al., 2004). We here extend these studies by Western blotting of testis, liver, lungs, plus aorta, chosen because of the suggested role of CD59 in vascular disease (Qin et al., 2004) (Fig. 4). Each organ was perfused with saline prior to preparation of protein lysates. CD59a was strongly detected in all the tissues; however, CD59b was detected only in testis lysates. These data confirm our published contention that organ expression of CD59b protein is restricted to testis. 3.3 Expression of CD59b mRNA in testis coincides with puberty and plays a role in spermatozoal motility We previously reported that CD59b expression in testis was restricted to developing and mature spermatozoa (Baalasubramanian et al., 2004). To confirm this germ cell restricted pattern we examined expression of CD59b mRNA in testis from pre-pubertal mice. CD59b mRNA was absent in testes harvested from mice at days 10 and 20 post-partum and appeared only from day 30 on, coincident with puberty (Fig. 5A). In contrast, CD59a mRNA was present in testes at all timepoints. RNA extracted from embryos at days 5, 10 and 17 were all positive for CD59a but negative for CD59b mRNA (Fig. 5A). These observations were confirmed by Western blotting (Fig. 5B). Expression of CD59b protein was highest in spermatozoa, intermediate in adult testis and absent from infant testis. CD59a was present in all the lysates, albeit at low level in spermatozoa and with a reduced apparent molecular mass compared with the testis protein, suggesting modification during sperm maturation. We previously reported that CD59b on spermatozoa was focussed on the head region in a highly granular pattern (Baalasubramanian et al., 2004). Here we have explored the expression pattern of CD59b in more detail. In freshly isolated spermatozoa, more than 80% showed this head/granular staining pattern, a minority showing a more diffuse staining on the head region (Fig. 6). CD59b was weakly expressed on spermatozoal tails and, in about two thirds, strongly in the mid-piece. Initiation of the acrosome reaction (with A23187) caused a precipitous loss of CD59b expression on sperm heads, more than 60% of cells being negative by 30 min and 90% by 180 min post-initiation, indicating that CD59b was shed with the outer acrosomal membrane (Fig. 6). These data suggested that CD59b might play a role in acrosome function, an essential component of sperm capacitation for fertilisation. Therefore, we next investigated whether ligation of CD59b with antibody influenced spermatozoal mobility, a surrogate marker for fertilisation capacity (Fig. 7). Ligation of CD59b caused a significant suppression of motility of spermatozoa harvested from both wild type and cd59a−/− mice. As a control, CD59a was similarly ligated but did not alter spermatozoal motility compared with the effect of a control antibody. The data suggest that CD59b but not CD59a has a role in regulating spermatozoal motility. These findings are of particular relevance in that a major feature of the CD59b knockout mouse was spermatozoal dysfunction that included diminished motility and infertility (Qin et al., 2003). 4 Discussion We have recently examined the distribution patterns of the two isoforms of CD59 in the mouse and concluded, based upon its broad distribution, that CD59a is the primary regulator of MAC assembly in mouse (Baalasubramanian et al., 2004). We were unable to detect the expression of CD59b, either at mRNA or protein level, in brain, lungs, heart, liver, spleen and kidney. These data have recently been questioned by Qin et al. (2006) who first used a BLAST search of the mouse EST database and found ESTs matching the CD59b sequence from several tissues and organs. These in silico data were supported by RT-PCR analyses using primer sets specific for CD59a and CD59b, respectively that showed abundant expression of CD59b mRNA in all tissues tested, indeed, expression in testis was lower than in other organs. The apparent absence of CD59b mRNA in the CD59a knockout mouse in these published data was unexplained. Here we designed a number of quantitative and semi-quantitative assays to comprehensively address this controversial issue. Our data unambiguously demonstrated that expression of CD59b is essentially restricted to testis (Figs. 3 and 4). A comparative quantitative analysis of expression of CD59b mRNA in perfused and unperfused liver (Fig. 3) showed a 12-fold decrease in expression of CD59b following perfusion, strongly suggesting that the trace detection of this mRNA was a consequence of contamination with blood cells. This would explain why Qin et al. (2001) were able to detect CD59b in multiple mouse tissues using Northern analysis in which they loaded 10 μg of mRNA in each lane, a huge excess for this sensitive procedure. The contamination likely also explains the presence in multiple tissues of CD59b-specific EST sequences. We further demonstrated that the testis expression of CD59b coincides with puberty, supporting our findings of expression only on spermatozoa and their immediate precursors (Fig. 5). We found that CD59b was released from spermatozoa heads upon acrosome activation (Fig. 6), an observation that strongly support a role for this protein in functioning of acrosome. Ligation experiments with antibodies showed that CD59b but not CD59a was involved in spermatozoa motility (Fig. 7), providing support for an earlier report describing decreased motility and viability of sperm in cd59b KO mice (Qin et al., 2005). 5 Concluding remarks The data presented here, obtained using a broad panel of reagents and methods designed to give unbiased and unequivocal results, show that CD59b expression is limited. CD59b mRNA is abundantly expressed only on male germ cells and present in trace amounts in bone marrow and blood cells, but is absent from other organs where trace detection of mRNA is likely due to blood contamination. CD59b protein is abundant only on developing and mature spermatozoa. Erythrocytes express CD59b at low levels that we have previously quantified as less than 200 molecules per cell, irrelevant for protection from complement when CD59a is present at 2500 molecules per cell (Baalasubramanian et al., 2004). We show a unique distribution pattern of CD59b protein on spermatozoa, the precipitous loss of this protein with outer acrosomal membranes and effects of CD59b ligation on sperm motility. We conclude that CD59a is indeed the principal regulator of MAC expressed in the mouse and that the CD59a knockout is an appropriate model for studying the roles of MAC and its regulation in disease models. The CD59b knockout mouse might prove of value for studies of fertility; however, it should be noted that the mouse described by Halperin and co-workers is, for unexplained reasons, also markedly deficient in CD59a (Qin et al., 2006), limiting its utility for studying specific roles of CD59b in vivo.

Fam_Cancer-3-1-1914241

Family history is neglected in the work-up of patients with colorectal cancer: a quality assessment using cancer registry data

In the diagnostic work-up of hereditary non-polyposis colorectal cancer (HNPCC, Lynch syndrome), high-risk patients can be identified using information from the family history on cancer (‘Amsterdam criteria’ and ‘Bethesda guidelines’). To investigate to what extent the medical specialists apply these criteria to patients with colorectal carcinoma and a suspicion of HNPCC, we collected information on diagnostic work-up of 224 patients of seven hospitals in the region of the Comprehensive Cancer Centre West in Leiden, The Netherlands. These patients were diagnosed with colorectal cancer between 1999 and 2001 and satisfied at least one of the Bethesda guidelines. A complete family history was recorded for 38 of the 244 patients (16%). Patients with a complete family history were more likely to be referred to the Clinical Genetic Centre than those with an incomplete or absent family history (53% vs. 13% and 4%, respectively; P < 0.0001), and more likely to be analyzed for microsatellite instability (MSI), which is a characteristic of HNPCC (34% vs. 6% and 1%, respectively; P < 0.0001). We conclude that the family history is neglected in the majority of patients with colorectal cancer and MSI-analysis is only performed in a small proportion of the patients that meet the guidelines for this analysis. Introduction Colorectal cancer (CRC) is one of the most common forms of cancer in Western society. Every year 9,500 patients in the Netherlands are diagnosed with the disease and almost half of them die from it (Dutch Cancer Registry, 2002). It is estimated that in around 20% of the patients with a colorectal tumour genetic factors play a role in the aetiology [1]. About 1–5% of the patients with CRC are thought to have hereditary non-polyposis colorectal cancer (HNPCC, Lynch Syndrome); a dominant hereditary disease, which is caused by a defect in one of the DNA-Mismatch Repair (MMR) genes [2]. The most important clinical characteristics of CRC associated with HNPCC, are the relatively young age at which patients are diagnosed with the disease (average <45 years old) and the proximal localisation of the tumour in the colon. Besides an increased risk of developing a tumour in the colon, there is an increased risk of developing a tumour elsewhere in the body, especially in the endometrium (lifetime risk: 50%), the small intestines, the ovaries, the brain, the urinary tract, the biliary tract and the development of a keratoacanthoma or a carcinoma of the sebaceous glands of the skin [3]. The identification of patients with hereditary colorectal carcinoma is of great importance for the patient, because the treatment and follow-up of the tumour differ from those with non-hereditary colorectal carcinoma [4]. Furthermore, the identification of these patients is important, because it offers efficient manners for the prevention of colorectal carcinoma and other forms of cancer for the patient himself as well as his family [5]. It has been shown that a colonoscopy every three years can lead to a decline in mortality of at least 65% [6]. The genetic defect in HNPCC can be detected by immunohistochemic staining of the MMR proteins in tumour cells [7]. Another method to select families for mutation analysis of the MMR genes is analysis of errors in repetitive DNA-sequences, i.e., Micro Satellite Instability (MSI) [8]. MSI is found in around 15% of the non-selected CRC and in more than 95% of the colorectal tumours associated with HNPCC [9]. In 1996, guidelines were formulated for patients whose tumours should be analyzed for MSI [10]. These so-called Bethesda-guidelines describe practically all situations, where there is a suspicion of HNPCC. Recently these guidelines have been revised (Table 1) [11]. Table 1Guidelines for the performance of MSI-analysis of colorectal tumour [11]Revised Bethesda-guidelinesA person with colorectal carcinoma diagnosed at age ≤50A person with colorectal carcinoma and MSI-associated pathologya<60 yearsA person with colorectal carcinoma and a HNPCC associated tumourbA person with colorectal carcinoma and a first degree relative with a colorectal or HNPCC associated tumour; at least one of the tumours is diagnosed before the age of 50Three relatives diagnosed with colorectal carcinoma or a HNPCC associated tumour, diagnosed at any age; one patient needs to be a first degree relative of the other twoaThe presence of tumor-infiltrating lymphocytes, so called, "Crohn's like lymfocyte reaction", mucinous or signet ring cell carcinoma differentiation or medullary growth patternbCarcinoma of the endometrial tissue, stomach, small intestines, pancreatic gland, biliary tract, urinary tract, ovaries, brain, keratoacanthoma and carcinoma of the sebaceous glands Before the discovery of the MMR-genes, the most common approach in the diagnostic work-up for HNPCC was to use the ‘Amsterdam criteria’. These criteria are met if there are, within one family, three individuals with a colorectal (or another HNPCC-associated kind of) tumour, of whom one person is a first degree family member of the other two and at least one carcinoma is diagnosed before the age of fifty [12]. To evaluate the Amsterdam criteria in patients with CRC, a complete history on cancer in the patient’s family has to be obtained. Until now it is not known whether an adequate family history is taken of all patients with a colorectal carcinoma. We also do not know to what extent medical specialists use the above mentioned clinical Bethesda guidelines and if the tumours of all the patients who match the criteria are tested for MSI. The objective of the present study was to answer these questions using data of the Cancer Registry of the Comprehensive Cancer Centre West (CCCW) in Leiden, The Netherlands. Patients and methods We selected patients who were diagnosed with a primary and invasive colorectal tumour in the period 1999–2001 from the Cancer Registry (CR) of the Comprehensive Cancer Centre West (CCCW) in The Netherlands. The patients had to satisfy one of the following two Bethesda guidelines: the patient had to have more than one tumour, i.e., one colorectal carcinoma and a second one (colorectal cancer or another HNPCC-associated kind of tumour), or the patient had to be fifty years or younger at diagnosis. The selected patients were considered to have an indication for the performance of MSI-analysis and/or referral to the Clinical Genetic Centre (CGC). Patients with a carcinoma in situ or a carcinoïd of the appendix were not included in the analysis. Between 1999 and 2001, 434 patients who complied with the above mentioned criteria were diagnosed with CRC in one of the twelve hospitals in the CCCW-region. Seven hospitals gave permission for the collection of information concerning family history, MSI-analysis and referral to the CGC. We extracted this information from the various (electronic) medical reports. The family history was considered complete if the medical records reported on cancer in the family, and if so, information about the age at the time of the diagnosis, the type of cancer and the occurrence of cancer within first-degree and second-degree family members. Data were collected of 244 patients. Of these patients, 120 patients had multiple tumours, 109 patients were fifty years or younger at the time of diagnosis, and 15 patients had both characteristics. For comparisons between patients with multiple tumours and patients who were young at diagnosis, those with both characteristics were allocated to the “multiple tumours” group. The data were analyzed using SPSS statistical software (version 12.0.1). Univariate comparisons of proportions between patient groups were performed by Chi-squared test. Multivariate logistic regression analysis was used to study whether the presence of a complete family history or referral to a CGC could be explained by age, sex, inclusion criterion (multiple tumours or young age at diagnosis), hospital or type of medical specialist. Results The study group consisted of 244 persons, who complied with one of the Bethesda guidelines and therefore were considered to be referred for MSI-analysis and/or genetic counselling. The male:female ratio was 49:51 and did not differ between the groups selected on the basis of multiple tumours or age ≤50 years at diagnosis. A complete family history was recorded in the medical records of 38 (16%) of the 244 patients. For 136 patients (55%) limited information on the family history was available, and for 70 (29%) patients no information on the family history was found in the medical records. In the seven participating hospitals, a family history was reported for 38–91% of the patients. Of the 38 patients with a complete family history, 20 (53%) were referred to the CGC. This percentage was higher than that of patients with an incomplete family history (13%) and that of patients without any information on family history (4%) (P < 0.0001, Table 2). MSI-analysis was performed more often in the patients with a complete family history: 34% of patients with a complete family history compared to 6% of patients with an incomplete family history and 1% of patients without any family history (P < 0.0001) (Table 2). Presence of a complete family history and the performance of MSI-analysis were not associated with age, sex, inclusion criterion (multiple tumours or young age at diagnosis), hospital or type of medical specialist (multivariate logistic regression analysis; data not shown). Table 2Diagnostic work-up for HNPCC in 244 patients with colorectal cancer, by completeness of the family history as reported in the medical recordsDiagnostic workupFamily history complete (n = 38)Family history incomplete (n = 136)Family history absent (n = 70)Referred to CGC20 (53%)17 (13%)3 (4%)MSI-analysis performed13 (34%)8 (6%)1 (1%)Results of MSI-analysis3 MSI, 10 stable7 stable, 1 unknown1 stableDiagnosis of HNPCC6 (16%)3 (2%)1 (1%) Discussion We used the Bethesda-guidelines to select a group of patients with a suspicion of HNPCC. These patients were diagnosed with colorectal cancer between 1999 and 2001, a period during which MSI-analysis and the Bethesda guidelines were already available. Therefore we expected that for these patients, physicians would have examined and reported their patients’ family history and that MSI-analysis would have been performed. In our study group, however, the family history of the patients diagnosed with colorectal carcinoma was not sufficiently examined and reported in the medical records. For this reason, we believe the Bethesda-guidelines were not sufficiently applied by the physicians. As a consequence, MSI-analysis was performed on a small proportion of the tumours. More patients with a complete family history in their medical records were referred by their physicians to the CGC than patients without such a family history. MSI-analysis was also performed more often in this group. We expect that in a low-risk population, i.e., patients with colorectal cancer who do not meet the Bethesda guidelines, these results would be even more dramatic. On the one hand, our results may appear to be better than they actually are. We collected our data using medical records from various medical specialties, while the treating physician will not have this overview in practice. On the other hand, it is possible that when a physician examined a family history and none of the family members was diagnosed with cancer, he did not report it in the medical records. In this case, the family history was considered as absent, although it in fact was examined. Nevertheless we expect that if MSI-analysis was performed or the patient was referred to the CGC, this would have certainly been reported. We found that the attention for HNPCC in the diagnostic workup for CRC differed widely. For the seven participating hospitals, the proportion of patients with a reported family history on cancer ranged from 38% to 91%. Furthermore, only half of the approached hospitals were willing to cooperate. For these reasons, we cannot generalise our results for the whole CCCW-region. Nevertheless, we conclude that the family history appears to be neglected in the majority of patients with colorectal cancer in our study period, and that MSI-analysis was only performed in a small proportion of the patients that meet the guidelines for this analysis. Possibly, the attention for identification of patients with HNPCC has increased in more recent years. Our findings underscore the importance of implementation of family history and Bethesda guidelines in the physician education.

Intensive_Care_Med-3-1-1915596

Elimination of daily routine chest radiographs in a mixed medical–surgical intensive care unit

Objective To determine the impact of elimination of daily routine chest radiographs (CXRs) in a mixed medical–surgical intensive care unit (ICU) on utility of on demand CXRs, length of stay (LOS) in ICU, readmission rate, and mortality rate. Introduction Chest radiographs (CXRs) are frequently obtained as a complement to physical examination of critically ill patients [1, 2]. There are two different schools of thought regarding the utility of CXRs in the intensive care unit (ICU): The CXRs should be made on indication only, specifically when there is a sound reason to obtain a film (so-called on demand CXRs); or CXRs should be obtained routinely every day, that is, without any specific reason (so-called daily routine CXRs). Argument for the latter strategy is the high prevalence of findings on CXRs of ICU patients [3]; however, interpretation of studies on the usefulness of daily routine CXRs is hampered because of major differences in methodology [4]. Importantly, most studies did not attempt to discriminate between clinically relevant and irrelevant findings. We recently demonstrated that daily routine CXRs hardly ever reveal potentially important abnormalities and seldom result in a change in therapy [5]. While it can be recommended to discontinue a daily routine CXR practice in ICU patients, elimination of these CXRs may have several disadvantages. Firstly, eliminating daily routine CXRs bears the risk that the number of on demand CXRs increases. In addition, elimination of daily routine CXRs might result in on demand CXRs being obtained more frequently during off-time hours, which may cause an inverse rise of costs. Secondly, length of stay (LOS) in ICU, readmission rate and mortality rate might be negatively influenced by this change in CXR practice. To evaluate the impact of elimination of daily routine CXRs we determined the change in on demand CXR practice in our multidisciplinary ICU, where a daily routine CXR strategy was applied until performance of this study. In addition, we evaluated the diagnostic and therapeutic value of on demand CXRs before and after this intervention. Finally, LOS in ICU, readmission rate, and mortality rate during a daily routine CXR strategy were compared with those during an on demand CXR strategy. Materials and Methods Subjects A prospective, nonrandomized, controlled design with an intervention was used for this study. Of all patients, all CXRs taken in the adult ICU department of the Academic Medical Center in Amsterdam, Netherlands, from 1 March 2004 to 31 July 2004 and from 1 September 2004 to 31 January 2005 were studied. This department is a closed-format tertiary care, referral, 28-bed multidisciplinary ICU. The patient population consists of cardiothoracic surgery patients, medical patients (including cardiology patients and pulmonary disease patients), and surgery patients (including trauma patients and neurosurgery patients). Patients who were admitted during the period in between phases 1 and 2, as well as patients that were readmitted, were not analyzed. The study protocol was approved by the local ethics committee. Protocol The study period was divided into two parts: phase 1, a 5-month phase before the intervention during which the daily routine CXR strategy was practiced; and phase 2, a 5-month phase which began 1 month after the intervention. The intervention consisted of a change in the ordering practice of CXRs: no standing orders for daily routine CXRs; each (on demand) CXR required a clinical indication, such as admittance to the ICU, insertion of central venous lines, intra-aortic balloon pump or tracheal and chest tubes, an increase in oxygen requirement, or a change in pulmonary secretions with or without fever (see Table E1). For phases 1 and 2, CXR volume data were collected prospectively. Type of, and reason for, admission was registered for all patients. Severity of illness was scored by means of acute physiology and chronic health evaluation (APACHE) II for all patients. Data on LOS in ICU, readmission to ICU as well as ICU, and hospital mortality rate, were collected from the National Intensive Care Evaluation (NICE) database [6]. The LOS was calculated from day and time of arrival at, and discharge from, ICU. The total number of hours in ICU were divided by 24 to determine the exact LOS in ICU in days. Diagnostic and therapeutic value of on demand CXR Diagnostic and therapeutic value of on demand CXRs was determined as described previously for daily routine CXRs [5]. In short, the attending physician completed a specially developed data sheet on radiological abnormalities which was printed on the back of the normal CXR request form. It was to be ticked whether a certain finding was expected, and whether it was “old” (i.e., already present on preceding CXR) or “new” (i.e., not present on preceding CXR). All CXRs were interpreted by an independent radiologist on the day the on demand CXR was performed. Similar to the ICU physicians, the radiologist structurally interpreted these on demand CXRs for each patient (i.e., the radiologist ticked whether radiological abnormalities were absent or present and, if an abnormality was present, whether it was judged to be an “old” or “new” finding). If a predefined finding was unexpectedly found, then we determined whether any action was taken because of the new unexpected finding. To do this, two of us (M.G. and M.J.S.) and two independent observers carefully read the medical records, checked the patient data management system (Metavision, iMDsoft, Sassenheim, The Netherlands) and searched the hospital information system for the following: orders for sputum cultures or performance of a bronchoalveolar lavage for culture, or start of, or a change in, antimicrobial therapy in case of unexpected infiltrates on the CXR; repositioning of tubes in case of malposition of orotracheal tubes (ignoring planned extubations); ultrasound of the thorax in case of pleural effusion on the CXR, start or change in medication (diuretics); insertion of a pleural drain; and repositioning of devices in the case of malposition of medical devices other than orotracheal tubes (ignoring planned changes such as removal of intravenous lines). The observers were not involved in the daily care of the patients, and ICU physicians were not aware of this part of the observation. As a consequence, the clinical relevance of the predefined abnormalities could not be evaluated in some cases, specifically in cases of large atelectasis and severe pulmonary congestion, since start of physiotherapy, changes in levels of positive end-expiratory pressure, and the use of diuretics might have been triggered by other (clinical) findings. Statistical analysis All data are expressed as means (± SD), or medians (interquartile ranges). A Mann-Whitney U-test was used for analyzing continuous variables. A chi-square test was used to compare the groups in phase 1 and phase 2. The incidences of expected and unexpected findings, and clinically important abnormalities, were compared by chi-square test. A p-value < 0.05 was considered to be statistically significant. All calculations were performed using SPSS version 12.0.1 software (SPSS, Chicago, Ill.). Results Study population We evaluated 1376 patients over the two periods. Patient profiles on entering this study are summarized in Table 1. A total of 3894 CXRs were obtained from 754 patients in phase 1; these included 2457 daily routine CXRs and 1437 on demand CXRs. A total of 1267 CXRs were obtained from 622 patients in phase 2. These CXRs were, by definition, all on demand CXRs. Table 1Demographic data. APACHE-II Acute Physiology and Chronic Health Evaluation II, CXRs chest radiographs, CI confidence intervalPhase 1Phase 2Significance (p)No. of patients 754 622Age (years; mean, SD) 60 (16) 62 (16)0.02Gender (male; n) 475 (63%) 398 (64%)0.70CXRs while patients being mechanically ventilated (n)a3194 (82%)1115 (88%)< 0.001APACHE-II score 16.4 ± 6.9 16.4 ± 7.21.00Patient subgroups Cardiac surgery (n) 317 (42%) 306 (49%)0.01 Medical (n) 197 (26%) 119 (19%) Surgical (n) 144 (19%) 131 (21%) Neurosurgical/neurology (n) 69 (9%) 46 (7%) Other (n) 27 (4%) 20 (3%)Length of stay in ICU (days; median IQR) 1.9 (1.0–4.6) 1.9 (0.9–4.6)0.95Mortality ICU (n) 94 (12%) 62 (10%)0.49 Hospital (n) 132 (18%) 104 (17%)0.70 Predicted hospital mortality (%) 181 (24%) 155 (25%)0.69 Observed/predicted ratio (95% CI) 0.73 (0.59–0.90) 0.67 (0.53–0.83)aAll patients were mechanically ventilated at any time during stay in ICU. Expressed is the percentage of CXRs during which patients were on the ventilator while the CXR was performed Utility of CXRs The number of CXRs per day for the whole ICU declined from 22.6 ± 4.9 to 8.2 ± 3.2 (p < 0.05; Fig. 1). Adjusting for patient volume, the ratio of CXRs per patient day decreased from 1.1 ± 0.3 to 0.6 ± 0.4 after the intervention (p < 0.05). The median number of CXRs per patient for the complete stay in ICU declined from 3 (range 2–5) during phase 1, to 1 (range 1–2) after the intervention. The number of on demand CXRs increased minimally after the intervention, and the distribution over 24 h, did not change (see ESM, Fig. E1). Fig. 1Number of CXRs/day during the study. Phase 1: daily routine CXR strategy, i.e., a daily routine CXR was made every morning, from March to July; phase 2: on demand CXR strategy, i.e., each CXR required a clinical indication, from September to January. Open bars: mean number (± SD) of on demand CXRs/day; closed symbols: mean number (± SD) of all CXRs/day Diagnostic and therapeutic value of on demand CXRs The diagnostic and therapeutic value of on demand CXRs increased with elimination of daily routine CXRs (Tables 2, 3). Before intervention, 38 expected predefined abnormalities were found (2.6% of all on demand CXRs in 4.9% of all patients), and after the intervention 64 expected predefined abnormalities were found (5.0%; p < 0.05) in 9.5% of cases (p < 0.05). All these findings led to a change in therapy. Before intervention, 147 unexpected predefined abnormalities were found (10.2% of all on demand CXRs in 15.9% of all patients), of which 57 (4.0 in 6.4%) led to a change in therapy. After intervention 156 unexpected predefined abnormalities were found (11.6% of all on demand CXRs in 19.1% of all patients), of which 64 (4.8 in 9.5%; p < 0.05) led to a change in therapy. Subgroup analysis revealed no differences between phases 1 and 2, except for medical patients, in which there was a significant rise in the number of on demand CXRs that showed an unexpected predefined major abnormality (p < 0.05 vs phase 1; see ESM, Table E2). Table 2Expected and unexpected findings on on demand chest radiographsPhase 1 (n = 1437)Phase 2 (n = 1267)AbnormalitiesExpectedExpected+foundUnexpected+foundExpectedExpected+foundUnexpected+foundLarge atelectasis 37 (2.6) 2 (0.1) 13 (0.9) 49 (3.9) 3 (0.2) 15 (1.2)Large infiltrates 57 (4.0) 3 (0.2) 21 (1.5) 69 (5.4) 5 (0.4) 27 (2.1)Pulmonary congestion 98 (6.8) 8 (0.6) 25 (1.7)104 (8.2)14 (1.1) 22 (1.7)Pleural effusion 41 (2.9) 3 (0.2) 17 (1.2) 43 (3.4) 4 (0.3) 27 (2.1)Pneumothorax or pneumomediastinum 68 (4.7) 4 (0.3) 17 (1.2) 39 (3.1)c 3 (0.2) 12 (0.9)Malposition of invasive devices350 (24.4)18 (1.3) 54 (3.8)392 (30.9)c35 (2.7)c 52 (4.1)Total no. of abnormalities6513814769664c155Total no. of CXRs with abnormalitiesa641 (44.6)38 (2.6)133 (9.2)384 (30.3)c63 (5.0)c147 (11.6)cTotal no. of patients with CXRs with abnormalitiesb580 (76.9)37 (4.9)120 (15.9)223 (35.9)c58 (9.5)c119 (19.1)Numbers in parentheses are percentagesa Absolute number of chest radiographs (CXRs; percentage of all daily routine CXRs)b Absolute number of patients (percentage of all patients with on demand CXRs)c p < 0.05 vs phase 1Table 3Unexpected findings on on demand chest radiographs resulting in a chance in therapy. ND not definedPhase 1 (n = 1437)Phase 2 (n = 1267)AbnormalitiesResulting in a change in therapyResulting in a change in therapyLarge atelectasisNDNDLarge infiltrates10 (0.7%)14 (1.1%)Pulmonary congestionNDNDPleural effusion11 (0.8%)12 (0.9%)Pneumothorax or pneumomediastinum11 (0.8%) 9 (0.7%)Malposition of invasive devices25 (1.7%)29 (2.3%)Total no. of abnormalities5764Total no. of CXRs with abnormalitiesa56 (3.9%)61 (4.8%)Total no. of patients with CXRs with abnormalitiesb48 (6.4%)59 (9.5%)ca Absolute number of chest radiographs (CXRs; percentage of all daily routine CXRs)b Absolute number of patients percentage of all patients with on demand CXRs)c p < 0.05 vs phase 1 LOS in ICU, readmission rate and mortality rate The LOS in ICU was not different in phase 1 as compared with phase 2 (Table 1). Total readmission rate was similar (8.4% in phase 1 vs 7.6% and phase 2, risk difference 0.8% (95% CI: 2.1–3.7%, P = 0.6), and did not change with the intervention for the different subgroups. There were no statistically significant differences in ICU and hospital mortality rates before and after the intervention (Table 1). Discussion The present study demonstrates the impact of elimination of daily routine CXRs in a mixed medical–surgical ICU. We found a sharp decline in the total number of CXRs, while only a minimal increase in the number of on demand CXRs was observed. In addition, the number of CXRs in off-hours was similar between the two periods. Elimination of daily routine CXRs did neither affect LOS in ICU and readmission rate nor ICU and hospital mortality rate. Although the diagnostic and therapeutic value of on demand CXRs was significantly higher after the intervention, we considered this difference clinically irrelevant. When one considers the increase in diagnostic and therapeutic value of on demand CXRs after elimination of daily routine CXRs indirect proof of the “value” of daily routine CXRs, one must also recognize its futility regarding the therapeutic value. Indeed, the percentage of CXRs with unexpected findings that truly led to a change in therapy was similar in the two study phases. Since readmission rate and mortality rate remained unchanged after the intervention, we conclude that the true value of daily routine CXRs in our multidisciplinary ICU is very low. Interestingly, only in medical patients did the number of CXRs that showed an unexpected predefined major abnormality increase after elimination of daily routine CXRs. The reason for this finding remains unexplained. The distribution of abnormalities encountered on CXRs of these patients was similar in the two study phases; however, neither readmission rate nor differences in raw or risk-adjusted ICU and hospital mortality rates of medical patients was affected by the change in CXR practice. One interesting finding was the decrease in abnormalities presumed to be present on CXRs. Indeed, a 30% reduction in expected predefined findings was observed in phase 2. This finding remains unexplained and we can only speculate on its cause. Firstly, it may be that physicians learned from experience that many of their expectations proved to be untrue during the actual carrying out of the study. This may have caused them to be more reluctant in scoring for expected findings. Alternatively, physicians may have become less enthusiastic about the study, which might have resulted in failure to comply with study rules at some moments (i.e., they did not fill in the back of the formal CXR request form); however, there was no change in expectations of physicians regarding abnormalities that truly led to a change in therapy. More importantly, if the backside of the formal forms were not filled out, as a rule the CXR was simply not obtained. Indeed, collection of data was complete regarding this issue, there were no on demand CXRs without a completed form. Our study has, at least partially, overlap with two other studies [7, 8]. Price et al. performed a nonrandomized controlled study on the financial impact of elimination of daily routine CXRs [7]. They showed that elimination of daily routine CXRs in a pediatric ICU resulted in decreased variability in ordering practice, fewer CXRs per patient, and an accompanying cost savings, while not influencing LOS. In addition, cost reduction with the change in radiology policy was significant in their study. This is in line with our results, since we found a substantial decline in radiology costs (see ESM). Besides the fact that this study was performed in a pediatric ICU, making generalization of study results difficult, their study did not include all patient categories. Indeed, postoperative cardiovascular surgery patients continued to receive daily routine CXR. We specifically included this patient group in our study because cardiovascular patients form one of the largest categories in many adult ICU. Krivopal et al. performed a randomized controlled trial to determine whether there is any difference in diagnostic, therapeutic, and outcome efficacy between protocols utilizing daily routine CXRs and those utilizing on demand CXRs in mechanically ventilated patients [8]. In their study a daily routine CXR strategy compared with an on demand CXR strategy was not associated with a negative effect on LOS or mortality; however, this study was small, including not more than 94 patients. We did not collect information on less evident findings on CXRs. Less evident findings (such as atelectasis less than two lobes, infiltrates less than one lobe, or small pleural effusions [5]), however, might still influence daily management of ICU patients. Since LOS in ICU was not altered for the whole group, readmission rate and mortality rate remained unchanged after the intervention, we suggest that changes of less evident CXR findings are not at all important, at least in our ICU. In other ICUs, such as open-format ICUs, less evident findings might be of more clinical importance, however; therefore, our results must be interpreted with caution, it might be that our results are not easily translated to other types of ICU. Several important drawbacks of our study must be mentioned. Firstly, our study did not include a strict method for tracking complications as a result of elimination of daily routine CXRs. Indeed, several abnormalities might have been missed (or discovered too late) which might (or do) have impact on clinical outcome. Examples of these types of abnormalities include pneumothorax causing weaning problems, the malposition of devices such as central venous lines, causing extravasation of fluid, or orotracheal tubes, potentially causing injury to the vocal cords. Considering these examples, such a strict method may mandate a daily check of all invasive devices. Although possible complications of elimination of daily routine CXRs could be discussed in daily bedside rounds, daily radiology conferences and daily multidisciplinary meetings during the performance of our study, no clinically important complications were reported as the result of elimination of daily routine CXRs; thus, although we assume that the elimination of daily routine CXRs does not cause any complications, we cannot be certain that this was truly the case. Secondly, as mentioned previously, it is of importance to realize that results that come from one center may simply not be similar for other centers: differences in staffing; especially during off-hours, and differences in case mix may be of great influence on outcome when abandoning daily routine CXRs. Thirdly, as mentioned previously, we found a reduction in expected predefined finding in phase 2. We assumed that the cause of this reduction might be that the physicians became less enthusiastic about the study, which might be seen as a limitation of the study. Conclusion In conclusion, in our mixed medical–surgical ICU elimination of daily routine CXRs leads to a sharp decline in the total number of CXRs, while only minimally increasing the number of on demand CXRs. Although we cannot be certain whether we missed important findings by abandoning daily routine CXRs, its elimination did neither affect LOS in ICU, nor readmission rate and ICU and hospital mortality rates. Electronic supplementary material Electronic Supplementary Material (DOC 165K)

Anal_Bioanal_Chem-4-1-2413088

Disposable electrochemical flow cells for catalytic adsorptive stripping voltammetry (CAdSV) at a bismuth film electrode (BiFE)

Catalytic adsorptive stripping voltammetry (CAdSV) has been demonstrated at a bismuth film electrode (BiFE) in an injection-moulded electrochemical micro-flow cell. The polystyrene three-electrode flow cell was fabricated with electrodes moulded from a conducting grade of polystyrene containing 40% carbon fibre, one of which was precoated with Ag to enable its use as an on-chip Ag/AgCl reference electrode. CAdSV of Co(II) and Ni(II) in the presence of dimethylglyoxime (DMG) with nitrite employed as the catalyst was performed in order to assess the performance of the flow cell with an in-line plated BiFE. The injection-moulded electrodes were found to be suitable substrates for the formation of BiFEs. Key parameters such as the plating solution matrix, plating flow rate, analysis flow rate, solution composition and square-wave parameters have been characterised and optimal conditions selected for successful and rapid analysis of Co(II) and Ni(II) at the ppb level. The analytical response was linear over the range 1 to 20 ppb and deoxygenation of the sample solution was not required. The successful coupling of a microfluidic flow cell with a BiFE, thereby forming a “mercury-free” AdSV flow analysis sensor, shows promise for industrial and in-the-field applications where inexpensive, compact, and robust instrumentation capable of low-volume analysis is required. Introduction Mercury electrodes, normally in the form of mercury films for flow systems, have been widely used for performing stripping voltammetry as a result of their high sensitivity and reproducibility, but, due to increasing concern and legislation regarding the toxicity of mercury, alternative working electrode (WE) materials have been investigated. Metals such as Au [1, 2], Ag [2, 3], Ir [4] and even W [5] as well as bare and coated carbon electrodes [6, 7] have all been investigated for stripping analysis of metals. In 2000, bismuth-film electrodes [8] were mooted as a replacement for mercury-film electrodes, as bismuth exhibits negligible toxicity and is more environmentally friendly in comparison. Mercury use is increasingly regulated, motivating the search for mercury-free methods. For example, Sweden banned the sale of switches, apparatuses and instruments containing mercury in 1993 (Ordinance SFS 1991:1290) and instructed the Swedish Chemicals Inspectorate (KemI) to look into a total ban on the handling of mercury in Sweden, including chemicals used for analysis. The Inspectorate proposed that mercury for chemical analysis should be exempted from the general ban until 31st December 2008 in order to enable the development and implementation of mercury-free analysis methods [9]. In 2005 the EU commission proposed a ban on mercury exports by 2011 as part of a strategy to reduce mercury emissions and protect against exposure [10], and recently MEPs voted to bring this date forward to 2010 [11]. A variety of substrates have been used for bismuth films. While platinum [12–14] and gold [13] have both been investigated, the majority of films have been deposited on carbon substrates. Most studies have used glassy carbon [8, 13, 15–32], though wax-impregnated graphite [22, 24], pencil-lead graphite [33], carbon paste [13, 34–36], screen-printed carbon inks [21, 37, 38], carbon fibres [8, 13, 14] and boron-doped diamond [39] have all been employed as substrates. The use of Bi2O3 containing pastes [34, 40] and a bismuth bulk electrode [41] have also been reported. The use of BiFE has recently been reviewed [42]. Bismuth-film electrodes have mainly been applied to anodic stripping voltammetry (ASV) and adsorptive stripping voltammetry (AdSV) techniques for metal ion analysis, although cathodic detection of nitrophenols [17, 27, 41] and amperometric detection of glucose [12] have also been reported. Cd [28], Pb [18, 37], Zn [31, 32] and mixtures of all three ions [8, 20, 22, 23, 33, 36, 41] have been the most extensively studied by ASV. Tl, Cu [16], In [16, 43], Mn [39] and Sn [44] have also been analysed. Fewer reports have been made employing AdSV, though Ni [15] and Co [28–30] and Ni with Co [24, 26] have been examined using dimethylglyoxime (DMG). Cr [21, 25, 45], U [46, 47], Al [48], V [49] and Mo [50] analysis by AdSV on bismuth-film electrodes has also been performed. Stirred solutions have normally been used, though Economou et al. have reported the use of a rotating disc electrode [22–25] and a Nafion-coated BiFE in a thin layer flow cell (0.5 mm spacer) for sequential injection-ASV of metals [51, 52]. Hutton et al. generated an in-situ BiFE in a thin layer (0.3 mm spacer) flow cell (Bioanalytical Systems Inc., West Lafayette, IN, USA) for flow injection analysis of nitrophenols with cathodic amperometric detection [27]. Adsorptive stripping voltammetry of DMG-complexed Co and Ni has found widespread use in trace metal analysis. The enhancement of the Co-DMG response using nitrite, i.e. catalytic adsorptive stripping voltammetry (CAdSV), has also been the subject of several publications, for example van den Berg et al. [53, 54] and the review of catalytic systems by Bobrowski and Zarębski [55]. CAdSV of Co on a BiFE was reported by Krolicka et al. [29, 30] using an ex situ plated film on glassy carbon. Most BiFEs have been plated from acetic buffers, although Krolicka et al. used a plating solution of Bi(NO3)3 (0.02 M), LiBr (0.5 M), and HCl (1 M) with a plating potential of −0.25 V for 45 s [addition of bromide and strong acid enabled a highly concentrated Bi(III) plating solution to be produced]. Stripping was typically carried out in 0.1 M ammonia buffer (pH 9.2) containing DMG (1 × 10−4 M) and NaNO2 (0.5 M). Voltammograms were recorded in the differential pulse mode and a 15-fold enhancement of the Co-DMG stripping signal was reported. Recently the authors reported the use of 40% carbon fibre-filled nylon 6/6 injection-moulded electrodes as working electrodes for the determination of Cu(II) by CV and ASV techniques [56]. In this work, the electrodes were moulded from a 40% carbon fibre-filled polystyrene, which was also found to be suitable for performing electrochemical detection. This paper presents the first report of the use of CAdSV at an in-line plated BiFE in an integrated three-electrode injection-moulded plastic micro flow cell. Experimental Instrumentation Voltammetric measurements were performed using an Autolab electrochemical system (PSTAT 10, Eco Chemie B.V., Utrecht, The Netherlands) controlled with GPES software (v4.6, Eco Chemie) running on a PC. Rotating disc electrode experiments The rotating disc electrode (RDE) was a glassy carbon electrode (6.1204.000, Metrohm, Buckingham, UK) 3 mm in diameter controlled with a variable speed (500–3000 rpm) electrode rotator (Part No. 628-10, Metrohm). The disc electrode was polished with an alumina slurry (0.3 μm Al2O3, Metrohm) before use. A standard (12 mm in diameter) glass double junction Ag/AgCl/KCl (3M) reference electrode was used (CR4/DJ/AG, Thermo Electron, Auchtermuchty, Fife, UK) with a Pt wire auxiliary 0.5 mm in diameter (99.9%, Aldrich, Gillingham, UK). The electrodes were mounted in a small-volume 50-ml beaker used as an electrochemical cell. Flow cell experiments A gravity-fed flow system was used comprising a reservoir formed from a disposable syringe body (20 ml), and narrow-bore PTFE tubing (0.032 in. i.d., Lee Products, Gerrards Cross, UK). The flow was controlled with a manual two-way valve (Part No. 2420, Omnifit, Cambridge, UK). Barbed (1/16″) polypropylene female Luer fittings (Cole-Parmer, Hanwell, London, UK) enabled connection of the flow system to the injection-moulded flow cell. Electrical connection to the Autolab electrochemical system was made using crocodile clips. Chip fabrication The injection-moulded flow cells were produced in-house. Figure 1 depicts the fabricated device showing the three integrated electrodes (WE, reference and auxiliary). Solid models created using AutoCAD (Mechanical Desktop 2004, Autodesk, San Jose, CA, USA) were converted into macro commands for a CNC milling machine (Datron CAT3D M6, Datron Technology, Milton Keynes, UK) to produce the milled aluminium moulds for the injection-moulding machines (Babyplast 6/6 or 6/10, Cronoplast SL, Barcelona, Spain). A four-stage process was required to manufacture a ready-to-use device. The polymer electrodes were injection moulded (moulding temperature of 220 °C) from a conducting polymer (40% carbon-fibre-filled high-impact polystyrene (HIPS), RTP 487, RTP Company (UK) Plastics Ltd., Bury, UK). The reference electrodes were coated with Ag (Ag evaporation slug, 99.99%, Aldrich) using an e-beam evaporator (Auto500 with EB3 e-beam unit, Edwards High Vacuum International, Crawley, West Sussex, UK) to a thickness of 125 nm. The WE, reference and auxiliary electrodes were then incorporated into the flow cell by an overmoulding procedure. The mould cavity for the flow cell design comprises a base plate into which the preformed electrodes are inserted in the appropriate recesses and a top plate with a relief pattern that forms the flow channel and integrated fluidic connectors. The flow cell was moulded (moulding temperature of 220 °C) from crystal polystyrene (Northern Industrial Plastics Ltd. Chadderton, UK). Using a 500-W ultrasonicwelder (Mini delta 9500, FFR Ultrasonics Ltd., Queniborough, Leicestershire, UK), with a weld time of 1 s and a hold time of 2 s, the top and base plates were sealed together to form a complete and ready-to-use device. The flow channel dimensions were 1 mm wide, 1 mm deep and 30 mm long. The working and auxiliary electrodes were 5 mm wide, the reference was 2 mm wide and the spacing between them was 2 mm. Fig. 1The injection-moulded electrochemical micro flow cell. Here (A) is the Ag-coated electrode, which forms the reference (Ag/AgCl) element; (B) is the working electrode, on which the bismuth film electrode is formed; (C) is the auxiliary electrode; (D) is the device inlet and (E) is the device outlet. The flow channel is 1 mm × 1 mm and 30 mm long Reagents and solutions All aqueous solutions were prepared using >18MΩ water (Elga Maxima Ultra Pure, Vivendi, High Wycombe, UK). The acetate buffer was prepared from acetic acid (glacial 99.8% AnalaR grade, BDH, Poole, UK) and sodium acetate (99+% ACS grade, Aldrich, Gillingham, UK). The ammonium buffer was prepared from ammonium chloride (99.8% AnalaR grade, BDH) and ammonium hydroxide (28–30% NH3 ACS grade, Aldrich). Bismuth plating solutions were prepared with bismuth(III) nitrate pentahydrate (99.999%, Aldrich,) either in acetate buffer or with lithium bromide (99+%, Aldrich) in hydrochloric acid (1 M volumetric standard, Riedel-de Haën, Gillingham, UK), as described in the “Procedure” section. The mercury plating solution was prepared in nitric acid from a 1000 ppm mercury(II) chloride (98+%, Aldrich) stock solution, as described in the “Procedure” section. Sample solutions were made from cobalt (995 μg/l, Aldrich) and nickel (1 g/l, Fluka, Gillingham, UK) atomic absorption standards with dimethylglyoxime (99+%, Acros, Loughborough, UK) and sodium nitrite (97+%, Aldrich). The saturated AgCl solution was prepared from silver nitrate (99.9% AnalaR grade, BDH) in potassium chloride (99.5% AnalaR grade, BDH). Nitric acid (2 M volumetric standard, Riedel-de Haën) was used for electrode cleaning, as described in the “Procedure” section. Procedure Formation of the Ag/AgCl miniaturised reference electrode Using an external auxiliary platinum wire located in the inlet of the microdevice, the Ag electrode was coated in AgCl (in saturated KCl solution that was also saturated with AgCl through the addition of a small quantity of AgNO3) at 2 V for 3 s. Ex situ electroplating of the BiFE on the rotating disc electrode (RDE) Two electroplating solutions were investigated, (A) and (B). Acetate buffer Bi(III) plating solution. 100 ppm Bi(III) in 1 M acetate buffer (pH 4.5). Plated for 5 mins at −1.0 V in a quiescent solution.LiBr/ HCl Bi(III) plating solution. 0.02 M Bi(III) in 0.5 M LiBr and 1 M HCl. Plated for 60 s at −0.28 V in a quiescent solution. The RDE was then carefully rinsed before transferring it to the analysis solution. In-line electroplating of the BiFE on the flow cell working electrode Two electroplating solutions were investigated, (A) and (B). Acetate buffer Bi(III) plating solution. 100 ppm Bi in 0.1 M acetate buffer (pH 4.5). Plated for 5 mins at −1.0 V at a flow rate of 37.1 μl/s.LiBr/ HCl Bi(III) plating solution. 0.02 M Bi(III) in 0.5 M LiBr and 1 M HCl. Plated for 120 s at −0.28 V at a flow rate of 37.1 μl/s. After formation of the BiFE, the plating flow stream was flushed with water and switched to the analysis stream. Bismuth film cleaning procedure The Bi(III) film was cleaned of the remaining adsorbed complexes by holding the electrode at a potential of −1.3 V for 30 s in 0.1 M HCl. Bismuth film removal Bi(III) films were removed by applying a conditioning potential at +0.3 V, −0.5 V and 0 V for 2 × 50 s in a flowing solution of 0.1 M HNO3. In-line plating of MFE on the flow cell working electrode The Hg plating solution was 200 ppm in 0.1 M HNO3. Plated for 120 s at −1 V at a flow rate of 9 μl/s. After film formation the cell was flushed with water and switched to the analysis stream. Mercury film cleaning procedure The film was cleaned at a potential of −1.4 V for 10 s in 0.1 M HNO3. Mercury film removal Films were removed at +0.8 V in a flowing solution of 0.1 M HNO3 for 60 s. Results and discussion The in-line prepared BiFE was assessed for its suitability in performing CAdSV of Co(II) and Ni(II). Various parameters, such as flow rates, solution composition, plating parameters and square-wave conditions, were studied to investigate the electrochemical behaviour of the Bi(III) films prepared on the injection-moulded carbon fibre electrodes. Investigation of the effect of flow rate during the plating and accumulation stages Using the acetate buffer-based plating solution, i.e. the 100 ppm Bi(III) in 0.1 M acetate buffer, the effect of varying the flow rate during BiFE plating procedure was examined at 20, 25 and 37 μl/s by varying the height of the reservoir in the gravity-fed flow system (with sample conditions as given in Fig. 2). The maximum response was obtained for the stripping peaks at a flow rate of 37 μl/ s and so this rate was subsequently employed in this study. Fig. 2Influence of flow rate during the accumulation stage on the Ni(II) and Co(II) CAdSV peaks, where the flow rates are: (a) 5.8, (b) 15.7, (c) 25.6, (d) 37.1, (e) 42.9 and (f) 55.5 μl/s respectively. Carried out with a 10 ppb sample of Co(II) and Ni(II) in 0.1 M ammonium buffer (pH 9.2) with 0.1 mM DMG and 0.5 M NaNO2. SW conditions: Conditioning potential −1.3 V (10 s), deposition potential −0.8 V (120 s), equilibration time 5 s, pulse frequency 15 Hz, pulse amplitude 40 mV and step potential 4 mV. Scan: initial −0.8 V to end potential −1.3 V. Data were smoothed with a level-2 Savitzky–Golay filter The effect of varying the flow rate during the accumulation stage on the peak heights of Co(II) and Ni(II) was studied over the range of 5.8–55.5 μl/s; the results are depicted in Fig. 2. The response for Co(II) increased linearly with increasing flow rate with a slope of −0.036 μAs/ μl and an intercept of −1.26 μA with a correlation coefficient (R2 = 0.96), whereas that for Ni(II) did not show significant variation. A flow rate of 37 μl/s was selected, as this gave an acceptable response without adverse peak broadening and was compatible with the BiFE plating step flow rate, thereby simplifying the instrumentation. Effect of composition of the BiFE plating solution Reported methods for AdSV of Co(II) and Ni(II) have generally employed acetate buffer (pH 4.5) containing 100 mg/l Bi(III) [15, 24, 26, 28]. However, Królicka et al. [29, 30] suggested the use of a bromide-modified plating solution. Hence, both plating solutions were assessed using the RDE and flow cell setups. Figure 3 depicts the differing performances of the two plating solutions. The LiBr-containing plating solution gave the best stripping responses on both the RDE and the flow cell and was therefore used for subsequent studies. Fig. 3Comparison of different Bi(III) plating solutions. a Effect of (a) 100 ppm Bi(III) in acetate buffer (1 M, pH 4.5) and (b) 0.02 M Bi(III) in 0.5 M LiBr- and 1M HCl-based preplating solutions on RDE at 500 rpm. b Effect of (c) the acetate buffer (0.1 M, pH 4.5) and (d) LiBr/HCl preplating solutions on the flow cell. c Comparision of acetate buffer pH 4.5 Bi(III) preplating solution on (e) flow cell (0.1 M) and (f) RDE (1 M). d Comparison of LiBr preplating solution on (g) flow cell and (h) RDE. Carried out with a 10 ppb sample of Co(II) and Ni(II) in 0.1 M ammonium buffer (pH 9.2) with 0.1 mM DMG and 0.5 M NaNO2. SW conditions: Conditioning potential −1.3 V (10 s), deposition potential −0.8 V (120 s), equilibration time 10 s, pulse frequency 25 Hz, pulse amplitude 50 mV and step potential 5 mV (except trace (e) which was at 15 Hz, 40 and 4 mV respectively). Scan: initial −0.8 V to end potential −1.3 V Effect of solution parameters on the system behaviour Buffer concentration The effect of changing the ammonium buffer concentration over the range 0–0.5 M is shown in Fig. 4a. Increasing the buffer concentration over the range 0–0.1 M had a marked effect on the height of the Co(II) peak, but increasing the concentration further did not improve the peak height due to the increased background signal. As Co(II) and Ni(II) were sufficiently resolved at 0.1 M, this concentration was selected for all further experiments. Fig. 4Effect of solution parameters on the CAdSV behaviour of a 10 ppb Co(II) and Ni(II) sample: (a) ammonium buffer (pH 9.2) concentration over the range 0–0.5 M, (b) DMG concentration over the range 0–0.35 M and (c) nitrite concentration over the range 0–0.8 M. Supporting electrolyte conditions were: (b and c) 0.1 M ammonium buffer (pH 9.2), (a and c) 0.1 mM DMG and (a and b) 0.5M NaNO2. SW conditions were: conditioning potential −1.3 V (10 s), deposition potential −0.8 V (120 s), equilibration time 10 s, pulse frequency 25 Hz, pulse amplitude 40 mV (except c: 50 mV) and step potential 4 mV (except c: 5 mV). Scan: initial −0.8 V to end potential −1.3 V Effect of DMG concentration Figure 4b shows the effect of increasing DMG concentration, over the range 0.01–0.35 mM, on the peak current. The Co(II) signal increases almost linearly with increasing DMG concentrations, whereas the signal for Ni(II) steadily decreased to 0.2 mM whereupon it levelled off. The signal for the Co(II) peak shifted to a slightly more negative potential (−4 mV) than that for Ni(II) with increasing concentration. A concentration of 0.3 mM was employed in the subsequent studies. Effect of nitrite concentration Figure 4c shows the influence of nitrite concentration on the sample signal. The Co(II) signal shows a rapid increase in signal to 0.3 M and a slower increase thereafter, whereas the Ni(II) peak height remained level to a concentration of 0.6 M. Concentrations of greater than 0.6 M lead to increased background current and so a nitrite concentration of 0.5 M was subsequently employed. Figure 5 shows the results obtained from the addition of nitrite to the sample system. Addition of nitrite to separate samples of Ni(II) and Co(II) results in increases in response for both metals, Fig. 5a and b, whereas the addition of nitrite to a mixture of 10 ppb Co(II) and Ni(II) results in an almost unchanged Ni(II) peak height whilst that for Co(II) has increased elevenfold, Fig. 5c. Figure 5d shows the reduction (16.6%) of the Co(II) peak height upon the addition of 10 ppb Ni(II), while Fig. 5e displays the reduction (51.9%) of the Ni(II) peak height upon the addition of 10 ppb Co(II). Fig. 5a Effect of nitrite on stripping peak for 10 ppb Ni(II) when (a) no NaNO2 is present; (b) 0.5 M NaNO2 is present. b Effect of nitrite on stripping peak for 10 ppb Co(II) when (c) no NaNO2 is present; (d) 0.5 M NaNO2 is present. c Effect of nitrite on the stripping peaks for 10 ppb Co(II) and Ni(II) when (e) no NaNO2 is present; (f) 0.5 M NaNO2 is present. d Effect of addition of 10 ppb Ni(II) on the peak height for 10 ppb Co(II) in the presence of 0.5 M NaNO2 when (h) Co(II) only is present and (g) both Co(II) and Ni(II) are present. e Effect of addition of 10 ppb Co(II) on the peak height for 10 ppb Ni(II) in the presence of 0.5 M NaNO2 when (j) Ni(II) only is present and (k) Co(II) and Ni(II) are present. Carried out in 0.1 M ammonium buffer (pH 9.2) with 0.1 mM DMG. SW conditions: conditioning potential −1.3 V (10 s), deposition potential −0.8 V (120 s), equilibration time 5 s, pulse frequency 25 Hz, pulse amplitude 50 mV and step potential 5 mV. Scan: initial −0.8 V to end potential −1.3 V Effect of varying the square-wave (SW) stripping parameters Frequency The effect of frequency was studied in the range 12.5–100 Hz, Fig. 6a depicts the results obtained. The peak heights for both metals increased with increasing SW frequency (as expected from increasing the effective scan rate) and shifted towards more negative values. However, increasing the frequency leads to insufficient time for the background current to decay and leads to an increase in the contribution to the signal and hence a distortion in peak shape. A frequency of 25 Hz was generally the maximum employed. Fig. 6Effect of varying the SW stripping parameters on the CAdSV peaks of a 10 ppb Co(II) and Ni(II) sample. The variables are (a) frequency (12.5–100 Hz), (b) step potential (0.15–7 mV), (c) amplitude (5–70 mV), (d) deposition potential (−0.3 to −0.9 V), (e) deposition time (30–210 s). Supporting electrolyte: 0.1 M ammonium buffer (pH 9.2) with 0.1 mM DMG and 0.5 M NaNO2. SW conditions were (unless being investigated as above): conditioning potential −1.3 V (10 s), deposition potential −0.8 V (120 s), equilibration time 5 s, frequency 25 Hz, pulse amplitude 50 mV (except b and e: 40 mV) and step potential 5 mV (except e: 4 mV). Scan: initial −0.8 V to end potential −1.3 V Step potential Figure 6b shows the effect of the step potential on the peak heights. The response for Co(II) rose rapidly with rising step potentials (i.e. with an increase in effective scan rate) up to 3 mV and thereafter levelled off, whereas the response for Ni(II) increased slowly to 7 mV. Peak resolution remained the same across this step potential range, with both peaks shifting towards more negative potentials. Values of 4 or 5 mV were generally found to be ideal for adequate peak definition. Amplitude The effect of the SW pulse amplitude was studied over the range 5–80 mV. Both peaks shifted to more positive values, see Fig. 6c and they both initially increased rapidly in height (5–20 mV) whereupon the responses for Co(II) and Ni(II) levelled off as a result of the increase in contribution from the background current. A SW pulse amplitude of either 40 or 50 mV was employed. Deposition potential Figure 6d shows the influence of the deposition potential on the peak currents. At potentials less than −0.4 V the Ni(II) signal is completely suppressed and the Co(II) signal was markedly reduced due to the oxidation of the bismuth film (at around −0.46 V in alkaline solutions). Both peaks gradually increase with potential in the range of −0.4 V to a maximum at −0.8 V and then exhibit a sharp reduction in adsorption at a more negative potential of −0.9 V resulting from further reduction of the analyte complex. A deposition potential of −0.8 V was selected, as this gave the maximum response for both metals. Accumulation time The influence of the accumulation time on the stripping signal was examined. Figure 6e depicts the response in peak currents for 10 ppb each of Ni(II) and Co(II). For both Co(II) and Ni(II) the response increases with accumulation time up to 150 s for Co(II) and 180 s for Ni (II), after which the response levels off, indicating saturation of the surface. An accumulation time of 120 s was used for subsequent measurements as this gave the best compromise between sensitivity and analysis time. Analytical characterisation The simultaneous determination of Co(II) and Ni(II) over the concentration range of 1–20 ppb was investigated. The determination was carried out in 0.1 M ammonium buffer (pH 9.2) with 0.1 mM DMG and 0.5 M NaNO2. SW conditions: conditioning potential −1.3 V (10 s), deposition potential −0.8 V (120 s), equilibration time 5 s, pulse frequency 25 Hz, pulse amplitude 50 mV and step potential 5 mV. Scan: initial −0.8 V to end potential −1.3 V. The BiFE exhibited a well-defined response to changing concentration and the peak current response (y, μA) was linear over the concentration range (x, ppb) of 1–20 ppb, as expressed in the calibration plots for Co(II),y = −11.4x − 85.5 (R2 = 0.96), and Ni(II),y = −1.64x – 7.20 (R2 = 0.92), respectively. The stability of the BiFE formed on the injection-moulded substrates was investigated by performing replicate runs on the same film. Ten replicate runs gave an average peak height for Ni(II) of −1.05 ± 0.10 × 10−5 A (rsd: 9.6%) and for Co(II) −8.19 ± 0.82 × 10−5 A (rsd: 10.0%). To compare the working electrode behaviour, AdSV and CAdSV of Co(II) and Ni(II) were also performed at a MFE in the flow cell. The solutions needed to be deaerated before analysis on the MFE. Neither AdSV or CAdSV of Co(II) on a MFE was possible in these particular flow cells due to a significant shift in the stripping peak to an extreme cathodic potential (<−1.4 V vs Ag/AgCl), which led to analytical uncertainty at the edge of the supporting electrolyte potential window. However, AdSV of Ni(II) was possible, as illustrated in Fig. 7. It has been noted previously by this group [57, 58] that the main drawback with MFEs is the difficulty involved in regenerating the active mercury surface after use, necessitating the inclusion of cleaning and mercury film renewal procedures. In contrast to the MFEs, the BiFEs formed in these flow cells enabled the generation of reproducible, quantitative analytical data, due to the enhanced stability of the bismuth films during the cleaning cycles, and the reduced cathodic potential for the cobalt stripping peak. Fig. 7AdSV of Ni(II) on a mercury film electrode. Here decimal numerals indicate the film number and roman numerals indicate replicate analyses carried out on the same film. Carried out with a 200 ppb sample in 0.1 M ammonium buffer (pH 9.3) with 0.1 mM DMG (deaerated) at a flow rate of 16 μl/s. SW conditions: conditioning potential −1.4 V (10 s), deposition potential −0.5 V (200 s), equilibration time 5 s, pulse frequency 25 Hz, pulse amplitude 10 mV and step potential 4 mV. Scan: −0.5 V to end potential −1.4 V. Data were smoothed with a level-2 Savitzky–Golay filter Conclusions The application of a BiFE for the determination of Co(II) and Ni(II) by square-wave CAdSV under flow conditions in a non-deaerated solution has been demonstrated. The injection-moulded carbon-fibre-filled polystyrene electrodes have proven to be suitable supports for the formation of bismuth films. The LiBr-containing plating solution was found to offer superior performance for both the RDE and the flow cell under the alkaline conditions required for CAdSV. The application of a flow stable BiFE in an inexpensive sensor system permits the possibility of autonomous on-site industrial and environmental monitoring. The inherent disposability of the devices coupled with their mercury-free status should advance the scope of the sensor to point-of-care clinical applications.

Glycoconj_J-4-1-2234451

High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles

Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall glycans immobilized on nitrocellulose was assessed. Hierarchical clustering of microarray binding profiles from newly produced mAbs, together with the profiles for mAbs with previously defined specificities allowed the rapid assignments of mAb binding to antigen classes. mAb specificities were further investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls in plant materials. Introduction Carbohydrate microarrays provide a means of rapidly screening the interactions between glycans and other molecules [1–7]. Applications for this technology include the screening of protein–glycan interactions, characterization of carbohydrate-active enzymes and the analysis of the specificities of monoclonal antibodies (mAbs) and carbohydrate-binding modules [8, 9]. mAbs are powerful tools for investigating the biological roles of glycans but producing them is time consuming, labour-intensive and costly. Hybridoma-based mAb production involves the fusion of spleen cells from an immunized animal with myeloma cells. The resulting hybridoma cell lines are both immortal and secrete antibody into the cell supernatant [10]. This procedure is conventionally performed in a one-by-one fashion, such that each animal is immunized with a single antigen and the resulting antibodies are screened for desired specificities using enzyme-linked immunosorbent assays (ELISAs). However ELISA-based screening is low throughput because only a limited amount (∼100 μl) of hybridoma supernatant is available for screening during the initial stages of mAb production and this is typically only sufficient to test each mAb against just one or two antigens. An alternative approach involves ‘shotgun’ immunisation with a mixture of antigens [11]. This potentially results in the simultaneous generation of antibodies with a range of specificities, but the time limiting step then becomes the detailed retrospective screening of their specificities. However, microarrays offer a means of rapidly screening limited amounts of hybridoma supernatant against multiple antigens and therefore greatly increasing throughput in the identification of valuable cell lines. Here we report the use of shotgun immunisation followed by microarray-based screening of hybridoma supernatants in order to produce mAbs against plant cell wall glycans. Plant cell walls are fibre composites that contain some of the most complex glycans known [12, 13]. In addition to their biological roles, many cell wall components have important industrial applications including as functional food ingredients, pharmaceuticals, nutriceuticals, fibres and increasingly, bio-fuels [14–16]. Cell wall glycans can be broadly grouped into cellulose, hemicelluloses, pectins and glycoproteins [17]. Cellulose microfibrils are cross-linked by hemicelluloses such as xyloglucans, xylans and mixed linkage glucans forming a tough load-bearing matrix which is embedded in pectic polysaccharides [18]. Pectins are the most complex and heterogeneous family of cell wall glycans and are comprised of a series of galacturonic acid-rich polymers including homogalacturonan (HG), rhamnogalacturonan I (RGI), rhamnogalacturonan-II (RG-II) and xylogalacturonans (XG). In addition, structurally complex arabinan, galactan and arabinogalactan polymers may be present as side chains to the galacturonan-rich backbone domains [18]. The protein moieties of cell wall glycoproteins are often rich in hydroxyproline (Hyp) and these polymers, which are collectively referred to as Hyp-rich glycoproteins (HRGPs), include the extensins and arabinogalactan-proteins (AGPs) [19–21]. The fine structures and relative amounts of cell wall components vary greatly not only among plants, but also between organs, tissues, cells, and even between different micro-domains within a single cell wall. This complexity and heterogeneity presents a major barrier to detailed analysis and our understanding of many aspects of plant cell wall structure and function is far from complete. Several mAbs with specificities for diverse plant cell wall components have been developed and these are powerful tools for the analysis of cell walls [22]. However, the repertoire of mAbs currently available covers only a small proportion of the glycan structures that have been identified and there is a pressing need for a wider range of mAbs to facilitate the further characterization of cell walls. We have developed a method of rapidly screening mAb specificities using microarrays of cell wall glycans including pectins with different degrees and patterns of methyl-esterification, pectic side chains (e.g. arabinan and galactan), hemicelluloses (e.g. xylans, mannans, and xyloglucans), HRGPs (e.g. AGP-rich gums), β-linked glucans (e.g. (1→3)(1→4)-β-glucan and (1→3)-β-glucan) and celluloses (e.g. hydroxylethyl cellulose and carboxylmethyl cellulose). A further 14 samples were sequentially extracted from A. thaliana using CDTA and NaOH which are known to predominately solublize pectic polymers and hemicelluloses respectively [23]. Experimental procedures Immunisation and generation of monoclonal hybridomas Cell wall polymers were isolated from 6 week old wild type A. thaliana plants, ecotype Col-0, grown in soil at 22°C with cycles of 10 h of light/14 h of darkness. One g dry weight of a mixture of leaves, stems and roots was homogenized to a fine powder in liquid nitrogen. The homogenate was incubated with 20 ml 50 mM 1,2-Diaminocyclohexanetetraacetic acid (CDTA; pH 7.5) for 3 h at 18°C and centrifuged for 20 min at 4,400 rpm. The supernatant was collected and dialyzed extensively against deionized water (dH2O) in dialysis tubing (6–8,000 kDa molecular weight cut off) to remove low molecular weight molecules and freeze dried. The material was dissolved in phosphate-buffered saline (PBS) to generate the immunogen. Rats were used for antibody production so as to make subsequent comparisons with existing mAbs, most of which were produced in rats, as valid as possible. The immunization of rats, hybridoma preparation and cloning procedures were as described previously [24]. Briefly, two male Wistar rats were each injected subcutaneously with 250 μl of an emulsion of the isolated cell wall material at 1 mg/ml in PBS with an equal volume Freund’s complete adjuvant on day 0. On days 40 and 79 the injections were repeated using incomplete adjuvant. Tail bleeds were taken 10 days after injections to assess the immune response. On day 198 a pre-fusion boost was given to the selected rat and 3 days later, the spleen was removed and lymphocytes were isolated and fused with rat myeloma cell line IR983F [25] using standard polyethylene glycol fusion of lymphocytes and myeloma cells. Hybridoma lines were initially screened by ELISA with the immunogen coated onto microtitre plates (MaxiSorp, Nunc, Roskilde, Denmark) at 50 μg/ml. Previously described monoclonal antibodies Arrays were probed with 23 mAbs with previously described specificities and details of these are provided in Table 1. All were rat antibodies except mAbs CCRC-M1, BS-400-02, BS-400-03, and BS-400-04 which were produced in mice and PAM1 which was produced by phage display. Table 1Previously characterized monoclonal antibodies used to probe glycan arrays. HG, homogalacturonanSpecificityNameRef.Un-esterified HGPAM1[31]Un-esterified/Calcium ion cross-linked HG2F4[34]Partially methyl-esterified HGJIM5[33]Partially methyl-esterified HGJIM7[33](1→4)-β-galactanLM5[37](1→5)-α-arabinanLM6[24]Fucosylated xyloglucanCCRC-M1[38]Non-fucosylated xyloglucanLM15(1→4)-β-mannan/galacto-(1→4)-β-mannanBS-400-4[39](1→4)-β-xylanLM10[40](1→3)(1→4)-β-glucanBS 400-3[41](1→3)-β-glucanBS 400-2[32](1→4)-β-xylan/arabinoxylanLM11[40]Arabinogalactan-proteinJIM4[43]Arabinogalactan-proteinLM2[42]Arabinogalactan-proteinMAC207[43]Arabinogalactan-proteinJIM8[44]Arabinogalactan-proteinJIM13[43]Arabinogalactan-proteinJIM16[43]Arabinogalactan-proteinJIM14[43]ExtensinLM1[45]ExtensinJIM19[46]ExtensinJIM20[46] Glycan samples used on the array Details of the 50 defined glycans used are provided in Table 2. Most glycans were dissolved in dH2O. Arabinoxylan and glucuronoxylan were prepared by boiling in dH2O for 10 min. and then standing for 3 h at 18°C before use. Glucomannan was prepared by wetting with 95% ethanol followed by addition of dH2O. The mixture was heated to boiling point and stirred for 20 min until dissolved. Pachyman was prepared by dissolution in a minimal volume of 10% (w/v) sodium hydroxide followed by neutralization with acetic acid. 14 samples on the arrays were cell wall polymers extracted from A. thaliana organs listed in Table 2 using CDTA and 4 M NaOH. Fifty milligrams (fresh weight) of each organ collected from at least four separate plants were homogenized to a fine powder prior to adding 300 μl of 50 mM CDTA (pH 7.5). After incubating with rotation for 4 h at 20°C, the extracts were centrifuged at 4,400 rpm for 10 min and the supernatants (‘CDTA extracts’) removed. Pellets were resuspended in 300 μl of 4 M NaOH and samples were incubated with rotation for 4 h at 20°C prior to centrifugation at 4,400 rpm for 10 min. Supernatants were ‘NaOH extracts’. Table 2Samples included on the glycan arraysAlphanumerical codesSamplesA1Arabinan (sugar beet)B1Pectin (apple)C1Galactan (lupin)D1Homogalacturonan (sugar beet)E1Pectin (lime) B15F1Pectin (lime) B43G1Pectin (lime) B71H1Pectin (lime) 96A2Pectin (lime) F11B2Pectin (lime) F19C2Pectin (lime) F43D2Pectin (lime) F76E2Pectin (lime) P16F2Pectin (lime) P24G2Pectin (lime) P32H2Pectin (lime) P41A3Pectin (lime) P46B3Pectin (lime) P60C3Pectin (lime) P76D3RGI (soybean)E3RGII (A. thaliana)F3Xylogalacturonan (pea)G3MHR I (apple)H3MHR II (carrot)A4MHR III (potato)B4MHR HS1 (apple)C4MHR HS2 (apple)D4Xylogalacturonan (apple)E4AGP (P. patens)F4Seed mucilage (A. thaliana)G4Xyloglucan/mannan (tomato)H4Glucomannan (konjac)A5Gum (guar)B5Gum (locust bean)C5Gum arabic (acacia)D5Gum (karaya)E5Gum (tragacanth)F5AGP (larch)G5Arabinoxylan (wheat)H5β(1-3),(1-4)-glucan (lichenan)A6Mannan (ivory nut)B6Xyloglucan (tamarind)C6Glucuronoarabinoxylan (maize)D6Hydroxyethyl celluloseE6β(1-4)-glucan (avicel)F6Carboxymethyl celluloseG6Alginic acidH6β(1-3),(1-6)-glucan (laminarin)A7β(1-3)-glucan (pachyman)B7β(1-4),(1-6)-glucan (pullulan)C7CDTA extract (A. thaliana flowers)D7CDTA extract (A. thaliana siliques)E7CDTA extract (A. thaliana stem top)F7CDTA extract (A. thaliana stem middle)G7CDTA extract (A. thaliana stem base)H7CDTA extract (A. thaliana leaves)A8CDTA extract (A. thaliana roots)B8NaOH extract (A. thaliana flowers)C8NaOH extract (A. thaliana siliques)D8NaOH extract (A. thaliana stem top)E8NaOH extract (A. thaliana stem middle)F8NaOH extract (A. thaliana stem base)G8NaOH extract (A. thaliana leaves)H8NaOH extract (A. thaliana roots)Alphanumerical codes refer to the position of samples on arrays. Source organisms are in parenthesesRGI Rhamnogalcturonan I; RGII rhamnogalacturonan II; MHR modified hairy region; AGP arabinogalactan-protein Post-printing modification of glycans Glycan samples on selected arrays were modified in situ after printing by enzymatic digestion. For the data in Fig. 6 selected arrays were digested with endo-α(1-5)-L-arabinanase or endo-β(1-4-)-galactanase (both from Aspergillus niger, Megazyme (Bray, Ireland) used at 1 U/ml in 200 mM sodium acetate pH 4.0. Printing of arrays Glycans were applied to nitrocellulose membrane (0.45 μm pore size, Schleicher and Schuell, Dassel, Germany) at two concentrations (0.2 and 0.04 mg/ml) and in duplicate such that each sample was represented by four spots. CDTA and NaOH extracted A. thaliana material was printed as extracted and as a five fold dilution, also in duplicate. Printing was performed using a microarray robot (Microgrid II, Genomic Solutions, Ann Arbor, MI, USA) equipped with split pins (MicroSpot 2500, Genomic Solutions). Pins were washed twice in dH2O after deposition of each sample. Probing of arrays Arrays were blocked by incubation for 1 h in PBS (140 mM NaCl, 2.7 mM KCl,10 mM Na2HPO4, 1.7 mM KH2PO4, pH 7.5) containing 5% w/v low fat milk powder (5%MPBS). Arrays were then probed for 2 h with antibodies diluted in 5%MPBS. All antibodies were used as 1/10 dilutions except CCRC-M1 which was used at 1/50, and BS-400-2, BS-400-3 and BS-400-4 which were used at 1/200. After washing with PBS, arrays were incubated for 2 h in either anti-rat or anti-mouse secondary antibody conjugated to alkaline phosphatase (Sigma, Poole, UK) diluted 1/5000 in 5%MPBS. After washing in PBS, arrays were developed using a substrate containing 5-bromo,4-chloro,3-indolylphosphate (BCIP) and nitroblue tetrazolium (NBT) in BCIP/NBT buffer (100 mM NaCl, 5 mM MgCl2, 100 mM diethanolamine, pH 9.5). Scanning and analysis Arrays were scanned, converted to 16 bit grey-scale TIFFs, transformed to negative images and uploaded into ImaGene 6.0 microarray analysis software (BioDiscovery, El Segundo, CA, USA). Semi-automatic gridding was used to create an analysis area for each spot, and a 5-pixel zone around each spot was used for calculation of local background signals. Individual spot signals were defined as the mean pixel value within each spot area (red zone in Fig. 1d) minus the median pixel value in the surrounding local background area (green zone in Fig. 1d). The mean of the four individual spot signals for each sample was defined as the ‘mean sample value’ (MSV) and ‘total mean sample values’ (TMSVs) were the means of MSVs from three separate experiments. Selected data sets (TMSVs) were used to generate heatmaps (Figs. 2b and 5b) using online heatmapper software (http://bbc.botany.utoronto.ca/ntools/cgi-bin/ntools_heatmapper.cgi). For each antibody in the heatmap, the maximal TMSV was set to 100% and all other values within that data set adjusted accordingly. A cut off of 5% of the maximal TMSV was imposed. Hierarchical cluster analysis was performed on TMSVs obtained for 23 previously characterized antibodies and 7 newly produced ones using Epclust software (http://ep.ebi.ac.uk/EP/EPCLUST/). Clustering was based on correlation measure based distance and average linkage. Fig. 1Microarrays of plant cell wall glycans. a, b A library of 50 glycan polymers were spotted onto nitrocellulose membranes in an area 22 mm × 22 mm. Arrays included pectins, HRGPs, hemicelluloses and glucans (samples, A1–B7) and the position of these classes of polymer on the arrays are indicated by colour coding in (b). Fourteen samples extracted from Arabidopsis thaliana using either CDTA (samples C7–A8) or NaOH (samples B8–H8), were also included. Details of the samples are provided in Table 2. c Each sample was printed at the two concentrations indicated and in duplicate. d Microarray analysis software (ImaGene 6.0) was used to quantify spot signals and individual spot signals were defined as the mean pixel value within each spot area (red zone) minus the median pixel value in the surrounding local background area (green zone). e–g Two pairs of three arrays were printed and probed with the anti-homogalacturonan mAb JIM5 on three separate occasions. The mean sample values (MSVs) for each pair were quantified and plotted against MSVs of an array produced on a different occasion. These are presented as scatter plots, with the R2-values for the plots indicatedFig. 2Probing of cell wall glycan arrays with mAbs with known specificities. a Glycan arrays were probed with 23 mAbs with previously defined specificities and 5 representative examples are shown: PAM1 (anti-homogalacturonan); BS-400-2 (anti-(1→3)-β-glucan; LM5 (anti-(1→4)-β-galactan); JIM13 (anti-arabinogalactan-protein); JIM5 (anti-homogalacturonan). A control array was probed with secondary antibody, but no primary mAb. Arrays were scanned and converted into 16 bit greyscale TIFFs. b Heatmap showing the total mean sample values (TMSVs) for the binding of each mAb to each sample. The maximal TMSV for each mAb was set to 100 and all other values were adjusted accordingly. A cut off of 5% of the maximal TMSV was imposed and all values at, or below this are represented as black boxes Indirect immunofluorescence labeling of plant materials Resin embedded and fresh plant material was sectioned and labelled with antibodies as described previously [26, 27]. LM14 and LM13 hybridoma supernatants were used as 1/10 dilutions in 5%MPBS. Sections were counterstained for cellulose with Calcofluor white (fluorescent brightener 28, Sigma, Poole, UK) used as a 0.005% aqueous solution. Competitive-inhibition ELISA assays The ability of arabinose, galactose, rhamnose and oligoarabinosides to inhibit the binding of LM14 and LM13 to the immuogen in ELISAs was assessed as described previously [28]. Sugar composition analysis of modified pectic hairy regions (MHRs) Sugar composition was determined using methanolysis as described previously [29]. MHRs were treated with 2 N HCl in dry methanol for 16 h at 80°C, followed by 1 h of 2 M CF3CO2H (TFA) at 121°C. The released sugars were analysed using high-performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) as described previously [30]. Results Production of microarrays of plant cell wall polymers The layout of the arrays is shown in Fig. 1 and colour coding indicates the distribution on the arrays of different classes of cell wall polymer (Fig. 1a,b). A detailed list of the samples used is provided in Table 2. Each sample was represented on the arrays by four spots (two concentrations, printed in duplicate) (Fig. 1c) and microarray analysis software was used to quantify background-corrected spot signals (Fig. 1d). As shown by the arrays in Figs. 2a and 3, the consistency of the printing and probing replicates within arrays was high. However, reproducibility between arrays was also tested. A total of six arrays (3 × 2 pairs) were printed and probed with the anti-HG mAb JIM5 on three separate occasions. The MSVs were quantified and the values for each array were plotted against those from an array produced on a different occasion. The R2-values obtained for these plots were all >0.98, indicating a high degree of reproducibility between arrays (Fig. 1e–g). Fig. 3Probing of cell wall glycan arrays with newly produced mAbs. Glycan arrays were probed with mono- or multiclonal antibodies generated following shotgun immunisation with plant cell wall polymers. Four representative examples are shown. Arrays were scanned and converted into 16 bit greyscale TIFFs Probing of plant cell wall glycan arrays Arrays were probed with a range of mAbs with previously defined specificities for epitopes occurring on the major classes of cell wall polymers. Details of the mAbs used are provided in Table 1. A total of 23 mAbs were tested and 5 representative examples are shown in Fig. 2a. The TMSVs for the binding of these mAbs to each sample (derived from three independent experiments) are shown as a heatmap in Fig. 2b, where spot signals are correlated to colour intensity. mAb binding profiles were in good agreement with the published results for the antibodies tested. For example, mAb PAM1 has specificity for HG with a low degree of methyl-esterification (DE) [31] and consistent with this, PAM1 bound to lime pectin samples with DEs of 11–19% (samples E1, A2 and B2). PAM1 did not bind above background to other pectin samples with higher DEs, or to any other cell wall glycans. mAb BS-400-2 is specific for (1→3)-β-glucan [32] and, of the defined samples, only bound to this polymer (sample A7) on the arrays. (1→3)-β-glucan is known to be extractable from plant cell walls using NaOH and consistent with this, BS-400-2 also bound to weakly to NaOH-solubilized extracts from A. thaliana (samples B8-H8). As expected, the epitopes recognized by some mAbs were detected on multiple samples and this was the case for mAbs LM5 (anti-(1→4)-β-galactan), JIM13 (anti-AGP) and JIM5. However, the binding profiles of these mAbs were also consistent with their known specificities. For example, JIM5 bound with greatest avidity to pectin samples that contained abundant HG domains (such as the lime pectins E1-C3), but not to pectic fragments lacking HG (such as the arabinan or galactan samples A1 and C1). To generate a new series of cell wall-directed mAbs, rats were immunized with an immunogen consisting of a crude extract of cell wall material solubilized from A. thaliana using CDTA. 13 cell lines were selected by an initial ELISA analysis against the immunogen and these were subsequently probed against the cell wall polymer arrays and four representative examples are shown in Fig. 3. Antibodies that bound with very low avidity, or appeared to have identical binding profiles to previously generated antibodies were not selected for further analysis whilst the data for the remaining seven were subjected to hierarchical cluster analysis. Hierarchical clustering of antibody binding profiles Cluster analysis was used to rapidly obtain information about the specificities of 7 new antibodies based on the similarly of their binding profiles on the arrays to 23 previously characterized mAbs (Fig. 4). Clustering indicated that most of the antibodies tested separated into 4 broad clusters: HG-binders; pectic side chain-binders; HRGP-binders and hemicellulose-binders. Three antibodies with specificity for (1→3)(1→4)-β-glucan, (1→3)-β-glucan and (1→4)-β-mannan did not cluster with any other mAbs within the 0.55 (distance measurement units) cut off imposed. These broad clusters were in agreement with the previously defined specificities of the mAbs tested, and this was also the case within sub-clusters. For example, within the HG-binders cluster, mAbs JIM5 and JIM7, which both bind to partially methyl-esterified HG [33], clustered closely, whilst mAbs 2F4 and PAM1, which both bind to non-methyl-esterified HG also clustered closely [31, 34]. Similarly, within the hemicellulose-binders, the anti-xylan mAbs LM10 and LM11 formed a sub-cluster, as did the two anti-xyloglucan mAbs CCRC-M1 and LM15. One of the new mAbs (1H12, designated LM13 when cloned) was grouped with the pectic side chain-binders LM5 (anti-(1→4)-β-galactan) and LM6 (anti-(1→5)-α-arabinan). However, most of the new antibodies tested were grouped in the HRGP-binders cluster and within this, clustered most closely with the anti-AGP mAbs MAC207, JIM4 and JIM16. One of the new mAbs within this group (1B3, designated LM14 when cloned) was, together with LM13 subject to more detailed epitope characterization in order to further explore the evidence gained from the clustering analysis. Fig. 4Cluster analysis of antibody binding profiles to arrays. 23 mAbs with previously defined specificities, and 7 newly produced antibodies (red diamonds in black boxes) were analyzed using Epclust software. Hierarchical clustering was performed using clustered correlation measure based distance and average linkage. The binding profiles of all the mAbs analyzed clustered into four broad groups: 1 homogalacturonan-binders; 2 pectic side chain-binders; 3 HRGP-binders; and 4 hemicellulose-binders. Three mAbs were non-clustering outliers (NCO). The specificities of the 23 previously defined mAbs are listed in Table 1 Detailed characterization of the epitopes recognized by LM14 and LM13 Cluster analysis of LM13 and LM14 suggested that the epitopes recognized by these mAbs may contain neutral sugars such as arabinose and galactose, that are abundant in the side chains of both pectins and HRGPs. The glycan arrays contained five 5 samples of branched (or ‘hairy’) regions of pectic polymers modified by enzymatic digestion (‘modified hairy regions’ or MHRs) that were rich in neutral sugars and for which monosaccharide composition was determined (Fig. 5). The binding of LM14 and LM13 to these samples was therefore of particular interest. LM14 bound only to an MHR from carrot (sample H3), whilst LM13 bound to MHRs from potato and apple (samples G3, A4 and B4). It was noteworthy that the two MHR samples to which LM13 did not bind (samples H3 and C4) had the lowest arabinose content, suggesting that the epitope recognized by this mAb may contain arabinose. In order to explore this possibility further, the sensitivity of the LM13 epitope to arabinanase digestion was investigated. To do this, cell wall glycan microarrays were digested with arabinanase prior to probing with mAbs, and the TMSVs obtained with and without digestion were compared. These data are presented as scatter plots (Fig. 6a–d) in which signals from enzyme treated arrays were plotted against signals from untreated arrays. As a positive control, arabinanase digested and untreated arrays were probed with the anti-(1→5)-α-arabinan mAb LM6 [24]. As expected, the binding of LM6 to all samples was reduced to some extent by arabinanase digestion (Fig. 6a). However, the effect of arabinanase digestion on LM13 binding was even more pronounced than for LM6 and LM13 binding to all samples on the arrays was essentially abolished (Fig. 6b). This suggested that the epitope recognized by LM13 either contained arabinan and was degraded by arabinanase, or that the epitope was attached to arabinan and was released from arrays by arabinanase. In contrast, LM14 binding to all samples was essentially unaffected by arabinanase digestion (Fig. 6c). The effect of galactanase digestion on mAb binding to arrayed samples was also assessed. LM13 binding was reduced to some extent by galactanase digestion, but the reduction was far less for than arabinanase digestion (Fig. 6d). LM14 binding to all samples was unaffected by galactanase digestion, and the ability of this enzyme to degrade galactan was indicated by the fact that the binding of the anti-(1→4)-β-galactan mAb LM5 was essentially abolished by galactanase digestion (data not shown). The epitope recognized by LM13 was further characterized using competitive inhibition ELISA (ciELISA) assays (Fig. 6e). The binding of LM13 to immobilized arabinan was inhibited to 50% of maximal binding by approximately 450 μg/ml of both arabino-α1,5-octaose and arabino-α1,5-pentaose, but oligoarabinosides of lower degrees of polymerisation and rhamnose, galactose and glucuronic acid failed to inhibit binding at the highest concentration tested of 1 mg/ml;. The binding of LM14 to immobilized antigen was not inhibited by any of the haptens tested at concentrations up to 1 mg/ml (data not shown). Fig. 5Binding of LM13 and LM14 to samples to pectic modified hairy regions (MHRs). a Monosaccharide composition of 5 pectin modified hairy regions (MHRs) from apple (samples G3, B4 and C4), carrot (sample H3), potato (sample A4). b TMSVs for the binding of mAbs LM14 and LM13 to these samples. The maximal TMSV for LM13 and LM14 (to all samples on the array) was set to 100 and all other values adjusted accordingly. A cut off of 5% of the maximal TMSV was imposed and all values at, or below this are represented as black boxesFig. 6The epitope recognized by LM13 is arabinanase sensitive. a–d Scatter plots showing the effect of galactanse and or arabinanase on the binding of LM13, LM14 and LM6 (anti-(1→5)-α-arabinan) to glycan arrays. Arrays were incubated in enzymes after blocking but before probing. TMSVs were determined and plotted against each other. Spots that lie on the dotted line indicate that enzyme digestion had no effect on mAb binding, whereas spots that lie below the dotted line indicate where enzyme digestion reduced mAb binding. e The binding of LM13 to oligoarabinosides was tested by competitive inhibition ELISA, in which arabinan was the immobilized polymer. Oligoarabinosides with degrees of polymerization less than five failed to inhibit LM13 binding, even when used at 1 mg/ml. Both arabino-α1,5-octaose and arabino-α1,5-pentaose inhibited the binding of LM13 by 50% of maximal binding when used at approximately 450 μg/ml Cluster analysis suggested that LM13 was most similar to that of mAbs with specificity for pectic side chains, whilst the binding of LM14 was most similar to that of anti-AGP mAbs. AGPs can be distinguished from pectic polymers using SDS-PAGE because whereas AGPs resolve as characteristic smears, pectic polymers typically do not enter gels [35]. Western blots were prepared using the same A. thaliana CDTA-extracted material that was used for immunisation and probed with LM13 or LM14 (Fig. 7). LM13 did not bind to material on these blots whereas LM14 bound to a high molecular weight smear (∼70–200 kDa) that is characteristic of AGPs. Fig. 7Immunoblot of monoclonal antibodies LM13 and LM14. The CDTA-soluble Arabidopsis thaliana fraction that was used as the immunogen was separated by SDS-PAGE with 30 μg protein loading per lane. A Western blot of this material was probed with LM13 and LM14. LM13 did not bind to the blot but LM14-probing produced a smear in the region of 70–200 kDa, which is characteristic of AGPs Immunolocalization of the LM14 and LM13 epitopes In order to further investigate the epitopes recognized by LM13 and LM14 in planta a range of plant materials were probed with these mAbs using indirect immunofluorescence labelling. In transverse resin-embedded sections of A. thaliana inflorescence stems, LM13 bound specifically to epidermal cells and most strongly to the tangential cell walls (Fig. 8a). Plant cell wall glycans may be altered during extraction and/or immobilization such that epitopes are changed from their native state in planta. In order to test if this was a factor in determining the arabinase sensitivity of the LM13 epitope, sections of A. thaliana stems were also digested with arabinanase prior to probing with LM13. As shown by comparison of Fig. 8a,b, the binding of LM13 to A. thaliana stem epidermal cell walls was abolished following arabinanase digestion indicating that the epitope-structure immobilised on arrays recognized by LM13 reflected that observed in planta. In contrast to LM13, LM14 bound to some extent to all cell walls in an equivalent section through an A. thaliana stem (Fig. 8d). Labelling of the surface of intact roots of A. thaliana seedlings indicated that LM13 bound very weakly whereas LM14 bound strongly to the surface of root hairs (Fig. 8e and f). In transverse sections through tobacco stems, the LM13 epitope was associated with cell walls adjacent to metaxylem cells of the vascular tissue—a distinctly different labelling pattern to that observed in A. thaliana stems (Fig. 8g). In contrast, LM14 did not label any cell walls in tobacco stems (Fig. 8h). Fig. 8Indirect immunofluorescence labelling of plant material with LM14 and LM13. LM13 bound strongly to inner and outer radial walls of epidermal cells in transverse sections of A. thaliana inflorescence stem sections. The section is counterstained with calcofluor (blue) that labels β-linked glucans. b LM13 binding to A. thaliana inflorescence stem sections when sections were treated with arabinanase. Arrowheads in a and b indicate the epidermal surface. c Control in which an equivalent stem to that in a was labelled with the absence of primary antibody. d, An equivalent section to that shown in a labelled with LM14 which bound to all cell walls. e LM13 bound very weakly to the intact surface of the roots of A. thaliana seedlings. f LM14 bound strongly to the surface of root hairs (arrowheads) of Arabidopsis seedling roots. g In transverse sections through tobacco stem LM13 bound strongly to cell walls adjacent to files of metaxylem cells (mx). h LM14 did not bind to equivalent sections through tobacco stems. Scale bars = 50 μm for a–d, g and h; 20 μm for g inset, 50 μm for e and f Discussion The work presented here demonstrates the potential of glycan microarrays for overcoming a major bottleneck in anti-glycan mAb production. Specifically, the use of microarrays enabled 50 μl of hybridoma supernatant to be screened rapidly and simultaneously against >60 potential epitope-bearing target molecules. A novel aspect of this work was the use of cluster analysis of array data to rapidly predict antibody specificities by comparison with previously defined mAbs. Subsequent detailed analyses of the specificities of the new mAbs LM13 and LM14 indicated they bound to pectic and AGP class of polymers respectively, as was predicted by the cluster analysis. These results indicate that if a relatively large set of probes with defined specificities are available to serve as references, this is an effective method for high-throughput initial mAb screening. One potential limitation of shotgun immunisation could be immuno-dominance, such that an immune response is elicited against a limited subset of the injected antigens. In this study two mAbs, LM13 and LM14 were selected with specificity for two different classes of molecule. However, of the seven mAbs selected for cluster analysis of binding profiles, six clustered with mAbs with specificity for AGPs and one with mAbs to pectic side chains, suggesting that AGPs were the immuno-dominant antigens in this case. It is therefore likely that such a multi-antigen approach towards cell wall polymers may be most effective to obtain a panel of mAbs with a range of specificities within a single class of polymer. The arrays we constructed were based on the non-covalent attachment of glycans to nitrocellulose. This approach has the advantage that molecules can be immobilized directly without the need to create functional groups, and nitrocellulose has been previously shown to be a effective substrate for the immobilization of diverse glyans [35, 36]. Non-covalent attachment has the potential drawback that variations in the effectiveness of immobilization may result from differences in the structural properties of the arrayed molecules [8]. All of the arrayed samples used in this work were recognized to some extent by at least one of the mAbs used (as shown in Fig. 4), indicating that all were immobilized to some degree. Nevertheless, the spot signals obtained can only provide semi-quantitative information about mAb binding. Two new probes, LM13 and LM14 were produced against cell wall polymers and both have distinctly different specificities to previously generated antibodies. Cluster analysis, ciELISA data immunoblotting and arabinanase sensitivity suggested that the epitope recognized by LM13 is an arabinan-containing structure that occurs as a side chain on pectic polymers. Pectic side chains typically consist of either galactan, arabinan or type I arabinogalactan chains in which arabinose usually occurs as a terminal sugar [18]. It was of note that LM13 binding to arrays was reduced to some extent by galactanase digestion. However, the galactanase used had very low arabinanase side activity (<0.03 U/ml specific activity with an arabinan substrate compared to 780 U/ml specific activity with a galactan substrate). It is possible therefore that galactanase digestion resulted in the indirect loss of the LM13 epitope by cleavage of galactan to which an arabinan-containing epitope is attached. The fact that LM14 bound to both pectin-derived MHR samples and to AGP-like material on a blot suggests that this mAb binds to an epitope of type II arabinogalactan that may occur on both pectins and AGPs. The novel patterns of recognition on A. thaliana and other plant materials indicates that these are useful new probes for the analysis of cell wall glycans polymers and complex cell wall architectures.

Photosynth_Res-3-1-1769345

The atypical iron-coordination geometry of cytochrome f remains unchanged upon binding to plastocyanin, as inferred by XAS

The transient complex between cytochrome f and plastocyanin from the cyanobacterium Nostoc sp. PCC 7119 has been analysed by X-ray Absorption Spectroscopy in solution, using both proteins in their oxidized and reduced states. Fe K-edge data mainly shows that the atypical metal coordination geometry of cytochrome f, in which the N-terminal amino acid acts as an axial ligand of the heme group, remains unaltered upon binding to its redox partner, plastocyanin. This fact suggests that cytochrome f provides a stable binding site for plastocyanin and minimizes the reorganization energy required in the transient complex formation, which could facilitate the electron transfer between the two redox partners. Introduction In oxygen-evolving photosynthetic organisms, the cytochrome b6–f complex couples proton translocation across the thylacoid membrane to the electron transport between photosystems I and II (Allen 2004). In this complex, cytochrome f (Cf) transfers electrons from the Rieske iron sulphur cluster to a soluble metalloprotein that acts as the immediate electron donor of P700 cofactor at Photosystem I. The Cf subunit consists on a 28.2 kDa N-terminal soluble domain anchored to the membrane by C-terminal helix (Gray 1992). Its soluble domain is an atypical c-type cytochrome because both its β-sheet secondary structure and the unusual heme axial coordination (Martinez et al. 1994) with the N-terminal amino acid Tyr-1 acting as an axial ligand. The most ubiquitous electron carrier between Cf and P700 is plastocyanin (Pc) (Sandman et al. 1983), which is a type I cupredoxin (Gough and Chotia 2004). Its structure consists on an anti-parallel β-barrel with a single copper atom (Coleman et al. 1978; Sykes 1985; Redinbo et al. 1994) coordinated by two nitrogen atoms and two sulphur ones from highly conserved residues (H39, C89, H92 and M97 in Nostoc cyanobacterium). The mechanism of the electron transfer (ET) reaction between Pc and its physiological partners has been studied extensively (Hope 2000; Hervás et al. 2003; Díaz-Quintana et al. 2003) highlighting the role of electrostatic and hydrophobic interactions on binding. Also, the solution structures of several Pc–Cf complexes have been reported (Ubbink 2004; Díaz-Moreno et al. 2005a, Lange et al. 2005; Musiani et al. 2005). In all the cases, these structures are consistent with a single conformation ensemble in which the hydrophobic patch surrounding Tyr-1 in Cf docks the hydrophobic patch of Pc. Despite the large amount of data concerning the nature of the interactions influencing the binding between the redox partners, little is known about the effects of complex formation on the metal cofactors, and how they can modulate the ET process. An analysis about whether the Fe coordination in Cf is altered upon binding to Pc would be interesting to address the above-mentioned effects, especially due to the fact that the N-terminal amino acid Tyr-1, which is involved in the Pc–Cf interface (Ubbink 2004; Díaz-Moreno et al. 2005a, Lange et al. 2005; Musiani et al. 2005), acts as axial ligand. To get a deep insight on these subjects we have studied the Fe K-edge of free and Pc-bound Cf by X-ray absorption spectroscopy (XAS) in solution. Materials and methods Protein samples Nostoc sp. PCC 7119 Pc was purified from E. coli cells transformed with the pEAP-WT plasmid (Molina-Heredia et al. 1998). Production and purification of the soluble domain of Nostoc sp. PCC 7119 Cf were as previously described (Albarrán et al. 2005). For the XAS experiments, the Pc and Cf samples were concentrated to the required volume by ultrafiltration methods, and exchanged into 10 mM sodium phosphate pH 6.0. After concentration, stock solutions of 10 mM Pc and 4.5 mM Cf were obtained. Protein concentration was determined by optical spectroscopy using an absorption coefficient of 4.5  mM−1 cm−1 at 598 nm for oxidized Pc (Molina-Heredia et al. 1998) and 31.5 mM−1 cm−1 at 556 nm for reduced Cf (Albarrán et al. 2005). Oxidized Cf (Cf III) was prepared by adding an equimolecular amount of potassium ferricyanide, followed by gel filtration in a Pharmacia Superdex G75 column to remove ferrocyanide. Reduced Cf (Cf II) was obtained upon addition of a tenfold excess of sodium ascorbate to a resulting Cf III sample. Oxidized Pc (PcII) was obtained by addition of either ferricyanide, as described for Cf. The oxidized complex (PcII–Cf III) was prepared by adding an aliquot of a 10 mM PcII onto a Cf III sample up to reach a final concentration of ca. 2 mM each. Under these conditions, the percentage of Pc bound to Cf was estimated to be ca. 87%, according to the binding constant calculated from NMR data (Díaz-Moreno et al., 2005a). The reduced complex (PcI–Cf II) was obtained upon addition of a tenfold excess of sodium ascorbate to a PcII–Cf III sample prepared as above. XAS measurements The X-ray absorption spectra were recorded at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France), which was operating with a ring current of 200 mA and energy of 6 GeV. The Fe K-edge (7112 eV) was measured at beam line BM29 using a double crystal monochromator fitted with a pair of flat Si (311) crystals detuned to 50% fwhm of the maximum transmission, for the suppression of high energy harmonics. All measurements were made at room temperature in fluorescence mode, using a 13-element Canberra solid-state germanium detector. The samples were contained in a PTFE cell equipped with two 12 μm Kapton foil windows. Energy calibration was achieved by measuring an iron foil for the Fe edge. The spectra of the foil were measured simultaneously, with the data in transmission mode, to be used as an internal reference. Ionization chambers filled with the appropriate mixture of gases were used as detectors. For all measurements, each data point was collected for 4 s, and several scans were averaged to achieve a good signal-to-noise ratio. In no case protein photoreduction or damage was observed, even in those samples in which the measurements were made over several hours. For the oxidized forms free and Pc-bound Cf, the edge remained at fix energy position from the first to the last scan, thus confirming the absence of protein photoreduction. The lack of radiation-induced changes in the samples was also confirmed by UV-vis measurements before and after XAS measurements. XAS data analysis The background subtraction required to obtain the extended X-ray absorption fine structure (EXAFS) functions χ(k) from the measured X-ray absorption spectra were performed using the AUTOBK code from the University of Washington. E0 was defined as the maximum of the first derivative of the absorption edge. To analyze the EXAFS spectra, the theoretical phases and amplitudes were calculated using the FEFF 6.0 code (Rehr et al. 1992; Newville et al. 1995). The fit to the experimental data was performed using the FEFFIT program (version 2.54) (Newville et al. 1995). Results and discussion XANES region Figure 1 (upper panel) shows the X-ray absorption near edge structure (XANES) spectra corresponding to free Cf in its reduced and oxidized states. In both spectra there is a pre-edge feature corresponding to the forbidden transition 1s−3d, which is sensitive to the electronic and geometric structure of the atom (Fe) site (Westre et al. 1997). In fact, the intensity of this transition agrees with the octahedral environment around the iron atom in Cf (Martinez et al. 1994). Fig. 1XANES region of the Fe K-edge XAS spectra of free and Pc-bound Cf. Upper, experimental data for oxidized and reduced forms of free proteins, Cf III (continuous line) and Cf II (dashed line). Middle, data for the oxidized species, Cf III (continuous line) and PcII–Cf III complex (dashed line). Lower, data for the reduced species, Cf II (continuous line) and PcI–Cf II complex (dashed line). The presence of a pre-edge signal at 7113 eV is marked by arrows On the other hand, the edge position, determined by the maximum in the first derivative of the absorption spectrum corresponding to free Cf II, appears ca. 1 eV below that of Cf III, as previously found for the reduced and oxidized states of two closely related proteins, as heart cytochrome c (Cheng et al. 1999) and Nostoc cytochrome c6 (Díaz-Moreno et al. 2006). In case of cytochrome c (Cheng et al. 1999), photoreduction of the oxidized species was observed only after the 16th spectrum of the series. In our case, however, there is no photoreduction as inferred from the fact that the edge position remains fixed for all the spectra along each series i.e. reduced or oxidized, free or bound. The reason not to observe any sample photo-reduction is due to the moderate brightness of the EXAFS spectrometer deliberately chosen for this investigation. In addition, the same shift has also been reported upon oxidation from Fe2+ to Fe3+ at pH 7 in the heme protein cytochrome c (Shulman et al. 1976). Moreover, there are some differences in both XANES spectra, particularly in the first feature after the edge. Figure 1 also shows the XANES regions of the absorption spectra of Cf bound to Pc in the oxidized and reduced state. The XANES region of the absorption spectra for PcII–Cf III and Cf III are identical (Fig. 1, middle panel), indicating that the electronic and geometrical configuration around the iron centre remains unchanged when bound to Pc. However, this is not the case for the reduced proteins (Fig. 1, lower panel). In fact, the spectrum of the PcI–Cf II complex exhibits some differences compared with that of free CfII. With regard to the pre-edge, a slight increase in the intensity of this feature is observed for CfII when bound to PcI, according to the decrease in electron density of the Fe atom. The shape of the first feature after the edge slightly changes too, thus suggesting certain modifications in the geometrical distribution of the ligands around the iron atom. It is worth noting that the spectrum of the reduced complex is very similar––except for the pre-edge signal and edge position––to that of the oxidized complex and free oxidized Cf, as shown in Fig. 3. This suggests that the iron geometry in reduced Cf is distorted upon association to Pc, approaching to that of the iron centre in the oxidized form, either free or bound to Pc. EXAFS Region The extracted EXAFS signals of Cf in the reduced and oxidized states are shown in Fig. 2 (left panels) as k-weighted spectra. The choice of a k-weighting instead of a k3-weighting is based on the following facts. First, we were interested in determining the local environment around the absorbing atom and, in particular, at the first coordination shell. This shell is formed by iron and light elements, such as nitrogen, and their backscattering amplitude drops quickly at increasing energy (k value). Therefore, they have a larger contribution when the transforms are made in k rather than in k3. Second, if a k3-weighting is used, the highest energy part of the spectrum is emphasized and, in this case, the EXAFS signal would be dominated by the spectral region where the signal-to-noise ratio is poorer. Fig. 2EXAFS data (panels a and b) and their FT modules (panels c and d) at the Fe K-edge of Cf, either free or bound to Pc. The EXAFS spectra of free proteins (Cf II and CfIII) are shown in panela by continuous line for Cf II and dashed line for Cf III. In panel b, the EXAFS spectra of PcI–Cf II and PcII–Cf III complexes are represented by continuous and dashed lines, respectively. The corresponding FT modules of the EXAFS spectra for free Cf (Cf II and CfIII) are represented in panel c by closed and open circles, respectively. Panel d shows the FT modules for Pc-bound Cf forms: PcI–Cf II by closed circles and PcII–Cf III by open circles. The best fits of the FT data for both reduced (Cf II and PcI–Cf II) and oxidized states (Cf III and PcII–Cf III) are represented by continuous and dashed lines, respectively We should note here that the XAS data were collected at room temperature in order to maintain consistency with previous room temperature NMR measurements made on the same systems (Díaz-Moreno et al. 2005a, b). Although a better signal to noise should be obtained at cryogenic temperatures, the transient complexes we study are not stable upon freezing, unless the partners are chemically cross-linked. Similar experimental conditions are required by EPR. Although EPR measurements on free and Pc-bound Cf would help to study the iron geometry, any comparison between results inferred from NMR or XAS, on one hand, and EPR, on the other, would be unreliable. As can be seen, the EXAFS function is not a simple wave but a combination of different waves corresponding to the contributions from the neighbouring atoms at different distances around the iron centre. Indeed, the EXAFS signals of Cf do not change upon binding to Pc, thereby indicating that the local coordination environment around the iron centre remains unchanged. The Fourier Transform (FT) of the corresponding spectra and the best fits, obtained with the parameters included in Table 1, are also shown in Fig. 2 (right panels). As a starting model, the values obtained from the X-ray diffraction (XRD) structure of Phormidium Cf (Carrel et al. 1999) were used. In all cases there is a main signal around 1.5 Å, which corresponds to the first coordination sphere formed by the four nitrogen atoms of the porphyrin ring (NA, NB, NC and ND), along with the nitrogen atoms of the two iron axial ligands Tyr-1 (N) and His-26 (Nε2). The intensities of the remaining signals are significantly lower than the first one because they correspond to contributions from lightweight elements lying at longer coordination distances. In order to fit the data, six simple scattering paths from the Fe atom to the six ligand N atoms were selected. Δr1 and Δr2 were used as two fitting parameters corresponding to changes in the distances of Fe to the two axial N atoms and to the four phorphyrin N atoms, respectively. The Debye–Waller parameter, σ2, related to system dynamic disorder was also fitted, with σ12 accounting for the bonds between the Fe atom and the two axial N atoms and σ22 accounting for the bonds between the Fe atom and the four porphyrin N atoms. The fifth adjustable parameter was the internal potential correction (ΔE0). Table 1Best-fit structural parameters resulted from the EXAFS analysis for free and Pc-bound Cf, in both oxidized and reduced stateSystemLigandΔE0 (eV)r(M–L) (Å)σ2(M–L) (Å2)CfIIIN (NC)−7 ± 41.96 ± 0.020.002 ± 0.001N (NA & ND)1.97 ± 0.02N (NB)1.98 ± 0.02N (Nε2)2.44 ± 0.060.010 ± 0.010N (N)2.49 ± 0.06CfIIN (NC)−6 ± 21.97 ± 0.010.002 ± 0.001N (NA & ND)1.99 ± 0.01N (NB)2.00 ± 0.01N (Nε2)2.43 ± 0.020.004 ± 0.003N (N)2.48 ± 0.02PcII–CfIIIN (NC)−7 ± 31.97 ± 0.010.002 ± 0.001N (NA & ND)1.98 ± 0.01N (NB)1.99 ± 0.01N (Nε2)2.40 ± 0.030.011 ± 0.009N (N)2.45 ± 0.03PcI–CfIIN (NC)−6 ± 31.97 ± 0.020.003 ± 0.001N (NA & ND)1.98 ± 0.02N (NB)1.99 ± 0.02N (Nε2)2.40 ± 0.030.010 ± 0.009N (N)2.45 ± 0.03S02, factor of amplitude of reduction; r(M–L), metal–ligand distanceS02 = 1.0, Δk = 3.5–11 (1/Å), ΔR = 1.1–2.1 (Å), R factor (free Cf III) = 0.044, R factor (free Cf II) = 0.004, R factor (PcII-bound Cf III) = 0.004, R factor (PcI-bound Cf II) = 0.003 Although there are no significant changes in the metal–ligand distances, the Fe K-edge X-ray absorption data suggests a small distortion of the Fe2+ metal centre geometry when Cf II binds to PcI. The resulting Fe2+ geometry resembles that of Cf III, either free or bound to PcII (Fig. 3). Although the Fe atom of Cf shows a well-defined position at the heme group, which provides a rigid frame, the small distortion on the Fe2+ metal centre when Cf II binds to PcI can be explained by the smaller size of Fe3+ compared to Fe2+: The Fe3+ atom fits well into the heme ring whereas the Fe2+ atom is somewhat shifted out of the plane (Schnackenberg et al. 1999); however, the Fe2+ atom could be driven into the ring upon complex formation, thus yielding a structure of PcI-bound Fe2+ similar to that of Fe3+ in free and PcII-bound Cf III. Fig. 3XANES region of the Fe K-edge XAS spectra of free CfIII and PcI–bound CfII. Experimental data for Cf III and PcI–Cf II are shown in continuous and dashed lines. The presence of a pre-edge signal at 7113 eV is marked by arrows This tiny perturbation is consistent with the intensity changes at the pre-edge region of CfII in presence of PcI, because they correspond to transitions to the iron dz2 and dx2y2 orbitals, which are pointing towards the axial and equatorial ligands, respectively. As the reducing electron is not located in these orbitals, such subtle changes may have little influence on the redox potential. This situation is clearly different to that previously reported for cytochrome c6, which shows a perturbation on the pre-edge region corresponding to its oxidized species, instead of the reduced ones, upon binding its reaction partner, photosystem I (Díaz-Moreno et al. 2006). In such case the pre-edge corresponds to transfer to a half-occupied non-bonding d orbital, which is that receiving the reducing electron, so the observed changes can be correlated with changes in the redox potential of the heme protein. Regarding to this point, it is important to note that the heme plane is normal to the interaction surface of cytochrome c6, but parallel in the case of Cf. Thus, our results indicate that the bond between iron and the N-terminus nitrogen is strong enough to prevent a distortion of the iron-coordination geometry upon binding of Cf to Pc, as well as to provide a stable binding site to the copper protein. Such a stiffness of the coordination sphere minimises the reorganisation energy concomitant to charge transfer, thus optimising the ET process.

Clin_Oral_Investig-3-1-2134843

Nonsurgical and surgical periodontal therapy in single-rooted teeth

The purpose of this study was to compare the effect of tooth related and patient related factors on the success of non-surgical and surgical periodontal therapy. In 41 patients (22 female) with untreated and/or recurrent periodontitis, no therapy, scaling and root planing (SRP), or access flap (AF) were assigned according to probing pocket depth (PPD). PPD and vertical relative attachment level (RAL-V) were obtained initially, 3 and 6 months after therapy. Baseline data were compared according to therapy, jaw, tooth type, and site. Factors influencing clinical parameters were identified using multilevel analyses. Baseline PPDs were deeper interproximally, in the maxilla and at premolars compared to buccal/oral sites, mandibular, and anterior teeth. At 6 months, PPD reduction and RAL-V gain were significantly greater at sites receiving SRP and AF as compared to untreated sites (p < 0.001). PPD reduction and RAL-V gain were significantly less (p < 0.005) in smokers as compared to nosmokers and at interproximal sites (p < 0.0001) as compared to buccal/oral sites. RAL-V gain was less in aggressive periodontitis, and PPD reduction was less in the maxilla (p < 0.001). In sites with greater bone loss and infrabony defects, a poorer response was observed regarding RAL-V gain or PPD reduction, respectively. The conclusions of the study are the following: (1) Nonsurgical and surgical periodontal therapies are effective in single-rooted teeth; (2) severe interproximal bone loss and infrabony defects deteriorate clinical results; and (3) there seem to be more defect-associated (tooth, site) factors influencing treatment outcome than patient-associated factors. Introduction The major factor in the etiology of periodontitis is the dental biofilm, i.e., bacterial plaque. Thus, the aim of anti-infective periodontal therapy is the reduction in supra- and subgingival microbial plaque to resolve periodontal inflammation. Subgingival scaling and root planing (SRP) may result in resolution of inflammation, reduced probing pocket depths (PPD), and clinical attachment gain [1, 2, 16]. However, nonsurgical periodontal therapy, i.e., SRP, is not found to be successful at all treated sites. Although not all factors influencing failure of nonsurgical therapy are known, deep periodontal pockets [3, 21, 25, 28] and furcation involvement [6, 9, 23] are some of them. Recent meta-analyses have shown the efficacy of nonsurgical therapy in moderate and deep periodontal pockets. In deep pockets, access flap (AF) techniques result in better reduction in PPD and clinical attachment gain [14, 15]. However, these meta-analyses like many other studies on this issue before [3, 21, 25, 28] use mean values and are not able to reflect the benefit of a particular technique at teeth with certain defects, e.g., infrabony pockets. However, calculation of a mean value, e.g., for PPD reduction, across a patient in fact levels out tooth-related characteristics. Even if separate strata for different baseline PPD are considered, tooth-related factors other than baseline PPD are leveled out also. This may lead to quite similar mean values for nonsurgical and surgical therapy for a patient or across a whole sample because tooth-related differences between both approaches because of different amounts or types of bone loss, jaw, etc. are leveled out. More recent statistical methods such as the multilevel modeling are considering single-tooth or even site characteristics while still regarding the individual patient as a statistical unit. In a structured review of 2002, it was explicitly stated that the included studies showed similar results for nonsurgical and surgical therapy after 1 year. However, insufficient studies are available to evaluate the various treatment procedures, e.g., in angular defects. Further, it is stated that subject characteristics and their possible effect on treatment outcome was not addressed in the studies available at that time [14]. Thus, there is a need for more information on the effect of patient and tooth-related factors on nonsurgical and surgical periodontal therapy. Well known hypotheses and the use of these more recent statistical methods may lead to new information. Clinical measurements in posterior are less reproducible than in anterior teeth [7]. Furcation involvement is a known factor to influence treatment outcome of nonsurgical and surgical therapy. To provide a high level of reproducibility of clinical measurements and to exclude a already known influencing factor, only single-rooted teeth were considered in this study. The hypothesis behind this study is that different tooth-related factors (e.g., tooth type, site, plaque, residual inflammation, horizontal/vertical bone loss) and patient-related factors (e.g., age, sex, diagnosis, smoking) determine the initial outcome of nonsurgical and surgical periodontal therapy in single-rooted teeth. Thus, the aim of the present study was to identify patient and site factors that determine the initial outcome of nonsurgical and surgical periodontal therapy in single-rooted teeth using more recent statistical methods. Materials and methods Patients The study group consisted of 41 patients (22 female) suffering from moderate to severe periodontal disease. These patients had been scheduled for periodontal treatment within a randomized placebo-controlled clinical trial evaluating the effect of a subgingivally delivered 14% doxycycline gel at the Section of Periodontology, Department of Conservative Dentistry, Clinic for Oral, Dental and Maxillofacial Diseases, University Hospital Heidelberg. Each patient had to have three single-rooted teeth that qualified as test teeth for the doxycycline study; that is, only three teeth per patient were included into this study [8]. Inclusion criteria were at least 23 years of age, untreated moderate to severe periodontal disease or recurrent periodontitis without periodontal surgery at least for the last 24 months, effective individual oral hygiene (plaque control record < 35%) [24], and written informed consent. Exclusion criteria were local and/or systemic antibiotic therapy within the last 6 months before the baseline examination of the study, known adverse reactions to any component of the test agent, anticoagulative therapy, liver, kidney, gastroenteral diseases, pregnancy, and women who were lactating. All patients were asked about current and past cigarette consumption. A patient was classified as a current smoker if he smoked one or more cigarettes per day and as a former smoker if he had quit smoking for at least 5 years. Patients who had quit smoking within the last 5 years were classified current smokers. No more than 50% of current smokers were allowed in the sample. All patients were classified as chronic or aggressive periodontitis according to the following criteria: Aggressive periodontitis: Patient is clinically healthy; that is, systemic diseases predisposing for periodontitis are not known; radiographic bone loss greater than or equal to 50% in at least two different teeth and age less that or equal to 35 years at time of diagnosis.Moderate and severe chronic periodontitis: attachment loss greater than or equal to 3 mm and age greater than 35 years [19]. The study was approved by the Institutional Review Boards for Human Studies of the University of Heidelberg. Clinical examination After completion of initial periodontal treatment including oral hygiene instruction and supragingival scaling, PPD were measured at four sites of every tooth. If a patient fulfilled the inclusion criteria, the study protocol, risks, benefits, and procedures were explained, and informed written consent was obtained. One week before test therapy, the Gingival Index (GI) and Plaque Index (PlI) [22] were assessed at six sites (mesiobuccal, midbuccal, distobuccal, distolingual, midlingual, mesiolingual) of every incisor, canine, and premolar. PPD and vertical relative attachment levels (RAL-V) were measured to the nearest 0.5 mm using a straight periodontal probe (PCPUNC 15, Hu Friedy, Chicago, IL) at six sites per tooth. A reference splint gave the location of probing by notches and served as reference for the RAL-V measurements. Bleeding on probing (BOP) was recorded approximately 30 s after probing. After the first course of measurements, all PPD and RAL-V measurements were repeated. The means of the pairs of double measurements were used for analysis. If a pair of PPD double measurements showed a difference greater than 2 mm, a third measurement was performed at the respective site (option 3). Then, the mean of the pair of the closer measurements was taken for analysis at the respective site. All clinical measurements before therapy were performed by one examiner (Eickholz) who also performed active periodontal therapy (subgingival debridement and surgery) in all patients. Within each patient, three test teeth with PPD = 5 mm and BOP or PPD ≥ 6 mm were selected for the doxycycline study. These teeth were excluded from this examination. Three and 6 months after therapy, the clinical examinations were repeated in the same manner by a second examiner (Kim) who did not have any information about baseline data and the therapy [8]. Radiographic examination and evaluation Before therapy, in 36 patients, full-mouth sets of periapical radiographs (Ektaspeed plus; Kodac, Rochester, NY) were obtained in XCP technique (XCP, Kentzler & Kaschner Dental, Ellwangen/Jagst, Germany). Intraoral size 0 (maxillary second incisors and mandibular anteriors) and 2 (all other regions) dental films were exposed to an X-ray source (Heliodent DS®, 60 kV, 7 mA, Sirona, Bensheim, Germany) and developed under standardized conditions (XR 24 Nova®, Dürr Dental, Bietigheim–Bissingen, Germany). All radiographs were viewed in a darkened room using a radiograph screen (67-0420, Dentsply Rinn, Elgin, IL). Each interproximal surface of single-rooted teeth was classified for the type of bone loss (horizontal/vertical). Further, the prevalence of a double contour of the root was noted that was taken as indicative for a mesial or distal groove. Relative bone loss in percent was assessed at two sites per tooth (mesial and distal) using a Schei ruler [27]. This ruler consisted of six straight lines that divert in the same angle. For each site, the basic line of the Schei ruler was placed to the cementoenamel junction (CEJ) of the respective tooth parallel to the occlusal plane. Then, the ruler was moved until the sixth line was placed tangentially to the apex. Finally, the alveolar crest was located. Location between the two coronal lines meant bone loss up to 20% of root length. Location between the second and third line meant between 20 and 40% bone loss and so forth. If the CEJ was destroyed or overlapped by interproximal restorations, the restoration margin was used as reference. If the alveolar crest could not be determined because of overlapping of adjacent teeth, the interproximal site was classified as “cannot be assessed.” If an interproximal surface showed a double contour, this was classified as an interproximal root groove. All radiographic assessments were performed by an examiner who was blinded to the clinical measurements and therapy assignments (Schenk). Therapy Teeth exhibiting PPD < 3 mm were treated neither by subgingival SRP nor by AF [26]. SRP was performed when PPD ≥ 3 and ≤6 mm were present. If periodontal surgery was necessary at teeth not adjacent to test teeth, this was performed 1 week after SRP: AF was chosen for teeth exhibiting PPD > 6 mm. However, SRP was performed at all test teeth (not included in this analysis) and the respective neighboring teeth under local anesthesia with hand instruments even for baseline PPD > 6 mm. Thus, also some sites with PPD > 6 mm were treated exclusively by SRP. Further, the decision to perform AF was taken per tooth and not per site. Thus, also sites with PPD ≤ 6 mm were treated by AF if the respective tooth exhibited PPD > 6 mm at one site. Thereby, effectiveness of SRP and AF maybe analyzed across the limits of indications as the ranges of PPD for the three treatment groups in Table 4 demonstrate. Maintenance including oral hygiene instruction and professional tooth cleaning was done 3 months after test therapy. Statistical analysis Data handling All data were entered into a personal computer program (Systat™ for Windows version 10, Systat, Evanston, IL; Schenk). Descriptive statistics were then calculated using another program (SPSS™, version 10.0.1, SPSS, Chicago IL) by an independent statistician (Lungeanu). Multilevel analyses were modeled by another independent statistician (Reitmeir) using a third program (SAS® version 6.12, SAS Institute, Cary, NC). Reproducibility The replicate PPD and RAL assessments for all teeth were used to estimate the intraexaminer reproducibility. The intraexaminer reproducibility of PPD and RAL measurements was expressed as the standard deviation of single measurements [4]. Treatment comparison The individual patient was considered as a statistical unit in this analysis. The main outcome variable for the comparison of the therapeutic effects of no further therapy, SRP, or AF was chosen to be the change of PPD. RAL-V was considered as a secondary endpoint. All other clinical parameters (GI, PlI) were control variables. Only sites were included that could be assessed at all three examinations. Additionally, baseline clinical parameters were calculated separately according to therapy (no therapy, SRP, AF), jaw, tooth type (anterior/premolar), and site (interproximal/buccal and oral) and compared using multilevel methodology [10, 11]. A probability p < 0.05 was accepted for a difference to be statistically significant. Because of multiple testing, a Bonferroni correction was performed: We tested 24 independent hypotheses on our set of data. Thus, the statistical significance level that should be used for each hypothesis separately is 1 of 24 times what it would be if only one hypothesis were tested, i.e., p = 0.002. For all analyses, the basic level “site” was nested in the upper level “tooth,” which itself was nested in the next upper level “patient,” and patient effects on the outcome were assumed to be random. This technique allows the identification of single-tooth effects or parameters while still considering the individual patient as a statistical unit and the dependencies of site and tooth data within a patient [10, 11]. Multilevel regressions were calculated including only clinical parameters providing six measurements per tooth first (site level: n = 4,146; patient level: n = 40). Further analyses were performed that included also radiographic parameters. Radiographic parameters were only available for those patients contributing full-mouth radiographic examinations and could be related only to interproximal sites (mesiobuccal, mesiolingual, distolingual, mesiolingual), i.e., four sites per tooth (site level: n = 1,792; patient level: n = 36). To identify factors influencing the treatment result, change of RAL-V and PPD were analyzed by application of a multilevel regression model using backward selection. Models were fitted for the dependent variables (1) RAL-V change after 6 months and (2) PPD change after 6 months including the following independent variables: (1) clinical: age, sex, actual smoking (yes/no), pack years, diagnosis (aggressive/chronic periodontitis), jaw (maxilla/mandible), site (interproximal/buccal or oral), tooth type (anterior/premolar), therapy (none/SRP/AF), and PlI and GI at baseline, (2) radiographic: interproximal bone loss in percent, infrabony defect, and double contour of the root. For the analysis including radiographic parameters, the variable “site” (interproximal/buccal and oral) dropped out because only interproximal sites remained in the analysis. A probability p < 0.15 was required for parameters to be kept within the models. Results All 41 patients contributing 700 single-rooted teeth with 4,200 sites finished the 3-month re-examination. The patient characteristics (age, diagnosis, smoking status) are given in Table 1. From baseline to the 3-month examination, two single-rooted teeth were lost: one to an acute periapical periodontitis and another because of prosthodontic planning. After the 3-month re-examination, one patient left the study. Only sites were included into the analysis, which could be assessed at all three examinations: n = 4,146 in 40 patients. Table 1Study population as related to age, sex, smoking habit, and diagnosisVariableNumber (n = 41)PercentAge45.9 ± 10.9SexFemale2253.7SmokingActive1946.3Former1229.3Never1024.4DiagnosisAggressive819.5 Reproducibility The results of the intraexaminer reproducibility of PPD and RAL measurements as standard deviation of single measurement for each examination time point are given in Table 2. Table 2Intraexaminer reproducibility at baseline, 3, and 6 months examinationExaminationMeasurements (n)s (RAL-V)s (PPD)Baseline8,4000.490.493 months8,3760.440.396 months8,2920.530.39s Standard deviations of a single measurement Baseline data The mean clinical parameters (RAL-V, PPD, PlI, GI) at baseline over all sites are given in Table 3. The respective data as related to therapy (no therapy, SRP, AF) are given in Table 4. Whereas no statistically significant differences for PlI were observed between the groups, RAL-V, PPD, and GI of the SRP and AF sites revealed to be significantly (p < 0.001) greater as compared to untreated sites (Table 4). Clinical parameters as related to jaw, tooth type, and site are given in Table 5. Baseline RAL-V and PPD were statistically significantly (p < 0.001) greater in the maxilla than in the mandible, whereas for PlI, it was vice versa (Table 5). For tooth type, just baseline PPD was statistically significantly (p < 0.001) different between premolars and anteriors with deeper pockets at the premolars (Table 5). All baseline clinical parameters were observed to be statistically significantly (p < 0.001) greater at the interproximal than at the buccal and oral sites (Table 5). Table 3Clinical parameters at baseline, 3 and 6 months after therapy (mean ± standard deviation) Baseline3 months6 monthsSites4,1464,1464,146RAL-V (mm)5.12 ± 2.204.87 ± 1.874.92 ± 1.84PPD (mm)3.25 ± 1.832.67 ± 1.192.67 ± 1.20GI0.78 ± 0.970.51 ± 0.860.51 ± 0.86PlI0.16 ± 0.510.32 ± 0.700.37 ± 0.74Table 4Clinical parameters at baseline as related to therapy (mean ± standard deviation)SitesNo therapySRPAF1,440Range2,478Range198RangeRAL-V/mm4.35 ± 1.82*1.25–19.005.55 ± 2.301.75–18.005.39 ± 2.022.25–12.00PPD/mm2.45 ± 1.13*0.25–2.753.66 ± 1.980.50–12.253.98 ± 2.021.00–10.50GI0.48 ± 0.84*0.94 ± 1.001.02 ± 0.98PlI0.15 ± 0.490.16 ± 0.530.14 ± 0.49SRP Scaling and root planning, AF access flap*Statistically significantly different from SRP and AF with p < 0.001Table 5Clinical parameters at baseline as related to jaw (maxilla/mandible), tooth type (anterior/premolar), and site (interproximal/buccal and oral) (mean ± standard deviation) Maxillamandiblepremolaranteriorinterproximalbuccal/oralSites1,9262,2201,5902,5662,7641,382RAL-V/mm5.24 ± 2.14*5.00 ± 2.265.19 ± 2.20*5.07 ± 2.225.37 ± 2.26*4.60 ± 2.01PPD/mm3.55 ± 1.88*3.00 ± 1.783.47 ± 1.88*3.12 ± 1.813.67 ± 1.82*2.43 ± 1.59GI0.80 ± 0.98*0.76 ± 0.960.83 ± 0.97*0.75 ± 0.960.89 ± 0.98*0.56 ± 0.89PlI0.11 ± 0.43*0.19 ± 0.570.14 ± 0.48*0.17 ± 0.530.18 ± 0.54*0.11 ± 0.44*Statistically significantly different with p < 0.001 Treatment effect The healing phase passed uneventfully for all patients. Clinical parameters as related to therapy after 3 and 6 months are given in Table 6, and their change after 3 and 6 months is given in Table 7. Whereas in untreated sites, the study failed to observe any significant change, SRP and AF resulted in PPD reduction and PAL-V gain after 3 and 6 months (p < 0.001; Table 7). Table 6Clinical parameters at 3 and 6 months after therapy as related to therapy (mean ± standard deviation) 3 months after therapy6 months after therapyNo therapySRPAFNo therapySRPAFSites1,4402,4781981,4402,478198RAL-V (mm)4.37 ± 1.765.13 ± 1.905.14 ± 1.644.47 ± 1.765.17 ± 1.855.11 ± 1.61PPD (mm)2.33 ± 0.962.84 ± 1.253.09 ± 1.342.35 ± 1.052.83 ± 1.242.99 ± 1.27GI0.36 ± 0.760.58 ± 0.900.69 ± 0.940.36 ± 0.760.58 ± 0.900.65 ± 0.93PlI0.22 ± 0.560.36 ± 0.740.52 ± 0.890.31 ± 0.680.41 ± 0.780.27 ± 0.65SRP Scaling and root planing, AF access flapTable 7Change of clinical parameters at 3 and 6 months after therapy as related to therapy (mean ± standard deviation) 3 months after therapy6 months after therapyNo therapySRPAFNo therapySRPAFSites1,4402,4781981,4402,478198RAL-V (mm)0.03 ± 0.740.41 ± 1.11*0.26 ± 1.06*0.12 ± 0.940.38 ± 1.20*0.28 ± 1.18*PPD (mm)−0.11 ± 0.72−0.82 ± 1.33*−0.89 ± 1.25*0.09 ± 0.87−0.82 ± 1.40*−0.99 ± 1.41*GI−0.13 ± 0.97−0.36 ± 1.14*−0.33 ± 1.14*−0.13 ± 1.03−0.36 ± 1.18*−0.36 ± 1.44*PlI0.08 ± 0.690.20 ± 0.82*0.37 ± 0.97*0.16 ± 0.750.25 ± 0.87*0.13 ± 0.68*SRP Scaling and root planing, AF access flap*Statistically significantly different from no therapy with p < 0.001 Prognostic factors RAL-V gain was less favorable in current smokers, in aggressive periodontitis, and at interproximal sites (Table 8). PPD reduction was poorer in current smokers, at premolars, in the maxilla, and at interproximal sites (Table 9). Whereas baseline PPD was related to better PPD reduction and RAL-V gain (p < 0.0001), baseline RAL-V was related to better RAL-V gain but to less favorable PPD reduction (Tables 8 and 9). Baseline PlI was associated with poorer PPD reduction (p < 0.15; Table 9). AF and SRP (p = 0.0114) were associated with better PPD reduction (Table 9). Table 8Backward multilevel linear regression analysis—dependent variable: RAL-V reduction 6 months after therapy, n = 40 patients, 4,146 sites EstimateSEdfF valuep valueIntercept−1.87760.106837Current smoking−0.28750.11063,4276.760.0094Site (interproximal)−0.23410.02773,42771.28<0.0001Aggressive periodontitis−0.29030.13083,4274.930.0265Baseline PPD0.27460.01583,427303.72<0.0001Baseline RAL-V0.18320.01413,427169.61<0.0001Table 9Backward multilevel linear regression analysis—dependent variable: PPD reduction 6 months after therapy, n = 40 patients, 4,146 sites EstimateSEdfF valuep valueIntercept−1.64870.103738Current smoking−0.33120.10553,4519.860.0017Jaw (maxilla)−0.12990.03243,45116.05<0.0001Site (interproximal)−0.36640.02623,451195.10<0.0001Tooth type (premolar)−0.05010.03383,4512.200.1381Baseline PPD0.61620.01463,4511774.31<0.0001Baseline RAL-V−0.03540.01283,4517.630.0058Baseline Plaque Index−0.03680.02443,4512.270.1318Scaling and root planing0.11710.03923,4514.480.0114Access flap0.09970.08613,4514.480.0114 Taking radiographic parameters into consideration, current smoking resulted in less favorable and baseline PPD in better RAL-V gain and PPD reduction, respectively (Tables 10 and 11). Further, tooth type (premolar) and aggressive periodontitis were associated with poorer RAL-V gains (Table 10). Location in the maxilla and baseline PlI resulted in less favorable PPD reduction (Table 11). Generally, sites with greater interproximal bone loss demonstrated poorer RAL-V gain (p = 0.0001), and sites with infrabony defects were associated with inferior PPD reduction (p < 0.0001; Tables 10 and 11). Table 10Multilevel linear regression analysis—dependent variable: RAL-V reduction 6 months after therapy, n = 36 patients, 1,796 sites EstimateSEdfF valuep-valueIntercept−1.82900.138533Current smoking−0.37580.13341,3137.940.0049Tooth type (premolar)−0.07630.05021,3132.310.1292Aggressive periodontitis−0.33110.16351,3134.100.0431Baseline PPD0.23710.02561,31385.86<0.0001Baseline RAL-V0.26370.02421,313118.36<0.0001Bone loss in %−0.01150.00161,31350.58<0.0001Table 11Multilevel linear regression analysis—dependent variable: PPD reduction 6 months after therapy, n = 36 patients, 1,796 sites EstimateSEdfF valuep valueIntercept−2.07710.136434Current smoking−0.46460.11971,31315.080.0001Jaw (maxilla)−0.14690.04331,31311.510.0007Baseline PPD0.62860.01191,3132791.95<0.0001Baseline Plaque Index−0.05500.03321,3132.750.0097Infrabony defect−0.29320.06711,31319.10<0.0001 Discussion Study population Patients treated in this study ranged from 23 to 71 years of age with chronic and aggressive periodontitis. Original measurements were performed in the setting of a randomized placebo-controlled clinical trial evaluating the effect of a subgingivally delivered 14% doxycycline gel at the Section of Periodontology, Department of Conservative Dentistry, Clinic for Oral, Dental and Maxillofacial Diseases, University Hospital Heidelberg. Each patient had to provide three single-rooted teeth that qualified as test teeth for the doxycycline study. To blind the examiner for the test teeth, not only those but all single-rooted teeth were assessed in all patients [8]. Despite the test teeth and their direct neighbors, all single-rooted teeth were treated according to the following criteria: Teeth exhibiting PPD < 3 mm were treated neither by subgingival SRP nor by AF. SRP was performed when PPD ≥ 3 mm was present. AF was chosen for teeth exhibiting PPD > 6 mm [21]. However, teeth directly adjacent to test teeth were treated only by SRP irrespective of baseline PPD. Thus, also some sites with PPD > 6 mm were treated exclusively by SRP. Further, the decision to perform AF was taken per tooth and not per site. Thus, also sites with PPD ≤ 6 mm were treated by AF, if they were located at a tooth with one or more sites with PPD > 6 mm. Thereby, effectiveness of SRP and AF may be analyzed across the limits of indications as the ranges of PPD for the three treatment groups in Table 4 demonstrate. How could we exclude that the topically delivered doxycycline had any influence on the treatment results of the sites that were evaluated in this study? The subgingivally delivered agent or vehicle control may be partially absorbed and thereby have systemic effects at the other test teeth. A pharmacokinetic study had found doxycycline only in the blood of 1 of 16 patients [18]. Thus, a systemic additional effect on the other teeth is quite unlikely. It has to be kept in mind that in the sample investigated in this study, only one tooth of the whole dentition per patient received the active agent, i.e., doxycycline [8]. Further, amounts of the subgingivally delivered substance may leave the pocket via sulcus fluid and affect the other test teeth. Split-mouth studies designed to compare active agent and vehicle of topical antibiotics have made the provision that teeth selected as test sites should be separated by at least one tooth [8, 12], minimizing the interaction between therapy modes. In a pharmacokinetic study from control sites, much smaller amounts of doxycycline could be detected in the gingival crevicular fluid than from another site that had received the particular doxycycline gel subgingivally in the same dentition [20]. Thus, it is unlikely that the topical doxycycline had any additional effect on the treatment results of all teeth but the test teeth. Treatment effect The results observed in this study after SRP are in agreement with observations reported by other authors 3 and 6 months after nonsurgical debridement of single-rooted teeth [2, 6]. Our PPD reductions and RAL-V gains achieved 6 months after therapy confirm data by a meta-analysis on nonsurgical vs surgical periodontal therapy that reports PPD reduction of 1.07 mm and vertical attachment (PAL-V) gains of 0.6 mm at sites with baseline PPD 4–6 mm 6 months after SRP. However, PPD reductions 6 months after AF for mean baseline PPD of 5.4 mm were 1.0 mm which is less than the meta analysis lists for baseline PPD 4–6 mm: PPD reduction 1.37 mm. PAL-V gain after AF was 0.3 mm which comes quite close to the 0.31 mm reported by the meta analysis [15]. Serino et al. report results 12 months after treatment [28]. For baseline PPD 4–5 mm they report PAL-V gains of 0.31 mm and PPD reduction of 1.1 mm after non-surgical as well as 0.21 mm and 1.6 mm after surgical therapy. This is quite similar to the results of this study regarding PAL-V gain and PPD reduction after SRP. However, the PPD reduction after surgical therapy is more favorable than ours [28]. Prognostic factors Current smoking was generally associated with less favorable PPD reduction and RAL-V gain in all models. Smoking is the most significant external risk factor for periodontitis [5, 13, 29] and is known to deteriorate the results of periodontal therapy in general [7, 30]. However, smoking was the only patient-associated factor that was kept in all models. The diagnosis of aggressive periodontitis was associated with less favorable RAL-V gains, whereas it was not kept in the models to explain PPD reduction. It is interesting to note that the other patient-associated factors, i.e., age and sex, were kept in neither model. This confirms observations of D’Aiuto et al. [5]. It is interesting to note that the diagnosis of aggressive periodontitis affected RAL-V gains but not PPD reduction. It seems that patients suffering from aggressive periodontitis have a higher risk for recessions than patients with chronic periodontitis. Several tooth- and site-associated factors were identified by multilevel analysis to influence clinical outcomes. Generally, baseline PPD was related to better PPD reduction and RAL-V gain (p < 0.0001). PPD reduction and RAL-V gain was less pronounced in the premolars and at interproximal sites (p < 0.15), and PPD reduction was less pronounced in the maxilla (p < 0.001). These results are plausible because compared to anterior teeth, premolars are less accessible and interproximal sites are more difficult to debride than oral and lingual sites. Less favorable PPD reduction in premolars than in incisors was also recently reported by other authors [5]. However, D’Aiuto et al. observed more favorable PPD reduction at interproximal sites, whereas this study observed the contrary. The multilevel analysis performed in this study was adjusted for baseline PPD. Baseline pockets were deeper interproximally than at buccal or palatal/lingual sites. This might explain more favorable PPD reduction at interproximal sites without adjusting for baseline PPD. Considering the different therapies, SRP and AF provided better PPD reduction than no therapy. However, the analysis failed to reveal statistically significant differences between both therapies in single-rooted teeth. It seems that AF provides no advantages over SRP in single-rooted teeth. Taking radiographic parameters into consideration, current smoking and baseline PPD were kept in the models. However, the greater the interproximal bone loss, the poorer the RAL-V gain (p < 0.0001), and infrabony defects were associated with inferior PPD reduction (p < 0.0001). Total amount of bone loss seems to be associated with poor prognosis of the respective tooth generally irrespective of the type of treatment. This has also been observed for long-term stability [17]. D’Aiuto et al. [5] did not assess radiographic parameters. However, they considered tooth mobility and found mobility was correlated with less favorable PPD reduction. It is interesting to note that interproximal grooves that had been assessed as root double contours failed to influence the outcomes of therapy. Up to our best knowledge, this is the first analysis on the influence of baseline bone loss on the outcome of nonsurgical or surgical periodontal therapy that does not use patient means but individual tooth parameters. Within the limitations of the present study the following conclusions may be drawn: (1) Nonsurgical and surgical periodontal therapies are effective in single-rooted teeth; (2) severe interproximal bone loss and infrabony defects deteriorate the clinical results, and (3) there seem to be more defect-associated (tooth, site) factors influencing treatment outcome than patient-associated factors.