James W. Thomas - US grants

DESCRIPTION (Adapted from Investigator's abstract): The long term goal of this project is to develop effective strategies that will prevent insulin immunity and autoimmunity. To accomplish this goal, the sites and stages of B lymphocyte development that permit anti-insulin B cells to differentiate and produce antibody will be identified. In contrast to models where the normal immune system deletes or silences autoimmune B cells, autoantibodies to insulin routinely follow administration of autologous hormone. These antibodies may lead to allergic reactions and hormone resistance as well as covert complications that include large birth weight infants and accelerated vascular disease. Spontaneous insulin antibodies may accompany systemic autoimmune disorders and are recognized as part of the autoimmune prodrome of type I diabetes. These observations led to the assertion that the immune system ignores insulin because B cell receptor (BCR) interactions are too few or too weak to induce tolerance. Data on anti-insulin B cell repertoires, however, are not consistent with the concept of true "clonal ignorance". Anti-insulin BCR found in preimmune repertoires are not part of the expressed regions but lineages of insulin binding B cells do not arise, indicating that anti-insulin B cells are censored in stages of differentiation. These observations suggest the hypothesis that insulin autoimmunity arises as a consequence of competing forces that drive clonal expansion of B cells while eliminating self-reactivity. This hypothesis will be tested by using mice that express anti-insulin BCR transgenes that bind insulin with a range of affinities representative of a physiologic repertoire. Three specific aims will (1) determine how the affinity of the preimmune repertoire for endogenous insulin governs the outcome of B cell activation -- tolerance or differentiation; (2) identify the mechanisms that limit expansion of B cells not silenced in the preimmune repertoire; and (3) identify the cell activation events and nuclear transcription pathways that program the phenotypes of B cell differentiation or tolerance. Based on the outcomes of these aims, future strategies may be directed at deletion of low affinity anti-insulin B cells or at inducing clonal elimination in germinal center reactions. These are alternative approaches that may be rationally applied once the stages of B cell development and differentiation that fail to maintain tolerance are identified in normal immune systems and in autoimmune diabetes.

DESCRIPTION: (Adapted from the applicant's abstract) - RA is a systemic inflammatory disease characterized by destructive synovitis that is associated with increased morbidity and early mortality. Immunosuppressive therapy does not halt joint destruction, and the primary invasive lesion is composed of hyperplastic fibroblastoid cells and neovascular tissue termed pannus. These observations and the findings that RA synovium produces growth factors with autocrine potential has led to an alternative hypothesis that the disease is due to a localized tumor-like growth and immunological lesions are secondary. One potent mesenchymal and vascular growth factor, FGF-1, is uniquely overexpresssed in RA synovium and is not detected in non-inflammatory synovium. In addition, a subset of T cells that respond to FGF-1 and express its receptor, FGFR1, is increased in the peripheral blood of RA patients. Therefore, the goal of this project is to understand how FGF-1 expression and regulatory abnormalities of its receptor, FGFR1, contribute to synovial cell proliferation and T cell infiltration in RA. The first specific aim will examine the expression and regulation of FGF-1 and its receptor in inflammatory (RA), non-inflammatory, and normal synovium. Fibroblast explants will be characterized for FGFR1 structures and their signalling via receptor tyrosine kinases. Also, it will be determined whether FGF-1 production is constitutive in outgrown RA synovial fibroblasts. To determine regulation of FGF-1, experiments will examine the capacity of multiple cytokines and growth factors to alter FGF-1 expression and mRNA splicing. The second specific aim is to understand FGFR1-mediated signalling in T cells. This will be accomplished by characterizing FGFR1 number and affinity, identification of isoforms associated with PLC-gamma tyrosine phosphorylation by the receptor, and examining regulation of IL-2 production as well as the AP-1 promoter element. Also, the mechanisms of FGFR1 activation that do not include IL-2 dependent pathways will be assessed by determining if FGF-1 promotes T cell proliferation in the presence of IL-2R blockade and if FGF-1 rescues T cells from apoptotic signals. Finally, the functional status of FGFR+ T cells from RA synvoium will assess receptor affinity, isoform, and status of receptor autophosphorylation.

Test the effectiveness and safety of IDEC-151 in patients with rheumatoid arthritis. This study will measure the effect of IDEC-151 on the CD4 antigen present on lymphocytes as a function of a drug dose. We are doing well with regards to recruitment and hope to finish enrolling subjects this year.

The goal of this project is to understand the dysregulated cell growth that is fixed or imprinted on the synovium in rheumatoid arthritis (RA). In contrast to a large body of data on how proinflammatory cytokines impact synovial cells, the actions of nonhematopoietic and angiogenic growth factors in RA are poorly understood, despite their abundance in synovium. Among these factors FGF-1 is markedly over expressed in RA compared to non-inflammatory synovium and the known functions of FGF-1 (angiogenesis and mesenchymal proliferation) are the hallmarks of hyperplastic pannus that causes joint destruction in RA. Recent data support this concept by showing that fibroblasts protect RA T cells from undergoing apoptosis, as usually occurs, when they are removed from the synovium. Therefore, the RA synovium provides an FGF-1 rich environment that may link gene activation in T cells with the intense proliferation and migration of fibroblasts and vascular cells. Studies on the regulation and actions of FGF-1 in cultured synovial cells reveal several unanticipated outcomes. For example, most mesenchymal cells including human dermal fibroblasts, HUVECs and 3T3 cells respond to FGF-1 by vigorous proliferation; synovial cells, however, respond poorly to FGF-1 despite the presence of high affinity receptors. Further, proinflammatory cytokines (e.g. TNF, IL-1, IL-6) have no effect on the expression of FGF-1 in synovial cells, while the "immunosuppressive" cytokine TGFbeta1 plays a key role in inducing FGF-1 production and in the outcome of receptor signaling. In addition, data show that in contrast to its expected antiproliferative effect, TGFbeta synergies with FGF-1 to produce a robust mitotic response even in the absence of serum. This synergy between TGBbeta and FGF-1 is not found in synovial cell lines derived from OA patients or from normals but this phenotype is highly reproducible in RA synoviocytes. In addition, FGF-1 induces gene transcription from an AP-1 promoter (collagenase) only in RA synoviocytes while AP-1 transcription is not activated by the actions of FGF-1 on synovial cells from normal individuals. Cellular correlates of FGF-1 actions are seen as persistent nuclear or perinuclear translocation of FGF-1 in RA synoviocytes but not in synovial cells from normal subjects. These observations suggest that the unchecked production of FGF-1 and its aberrant synergy with TGFbeta1 are linked to alterations in signal transduction and gene expression which are imprinted on RA synoviocytes. Therefore this project will first, identify signal transduction and gene activation pathways that reprogram rheumatoid synoviocytes for a synergistic response to FGF-1 and TGFbeta, and then investigate how these events are coupled to receptor interactions and the intracellular fate of FGF-1. To test the hypothesis that multiple cell types in the synovium may be reprogrammed with an altered growth phenotype, we will extend these studies and examine the molecular programming of endothelial cells from RA synovium. The approaches employed are designed to identify new targets for intervention in progressive joint destruction and a novel technique using cell permeable peptides will be tested in these studies. These studies are relevant not only to understanding progressive joint destruction in RA but are also of importance to a variety of disorders characterized by fibroproliferation including pulmonary fibrosis, interstitial nephritis, and chronic allograft rejection. The project will also provide information on how to utilize the immunomodulatory actions of TGFbeta without its unwanted fibroproliferative actions.

B lymphocyte; NOD mouse; autoantibody; insulin dependent diabetes mellitus

DESCRIPTION: (provided by applicant) Clone-based physical maps are central to the establishment of an accurate sequence of a complex genome and anchoring the sequence to a chromosomal location. Physical maps produced with large-insert bacterial artificial chromosome (BAC) libraries also provide the cloned DNA necessary for functional studies that are critical for the interpretation of the sequence of the human genome. In order to expand the number of genomes that are amenable to clone-based physical mapping, many new BAC libraries are currently being made from a wide range of vertebrates. Comparative genomics offers a powerful tool for both interpretation of the human genome and improving the efficiency and throughput of building physical maps. Specifically, comparative sequencing can identify functional elements of the human genome through the detection of evolutionarily conserved sequences. In addition, since genome organization is highly conserved among groups of related vertebrates, such as mammals, the map and sequence of the human genome can provide a valuable reference point for 're-mapping' other genomes through the use of orthologous markers. The purpose of the research proposed here is to apply comparative genomics toward the development of practical methods for constructing physical maps from a diverse range of vertebrates as a means of facilitating the use of new BAC libraries by the biomedical research community. To accomplish this, universal probes derived from evolutionarily conserved sequences will be designed and optimized for screening single or multiple vertebrate BAC libraries not associated with extensive sequence resources, and thus provide a basis for building physical maps in species that otherwise would be inaccessible to targeted physical mapping. The experience gained from this work will then be applied to the creation of a novel genome-wide database of universal probes to be made available to the public. The resulting information and technology will be applicable to the simultaneous construction and comparison of clone-based physical maps in multiple species at any genomic locus.

DESCRIPTION (provided by applicant): The goal of this project is to understand the selection and regulation of autoreactive B lymphocytes in Type 1A or insulin dependent diabetes mellitus (IDDM). In the long term this information will be used to identify new targets for the diagnosis and treatment of the disorder. Clinical and experimental data indicate that IDDM results from loss of self-tolerance and subsequent autoimmune destruction of insulin producing beta cells. Although autoantibodies to insulin, GAD and other islet antigens are recognized as early indicators of loss of immunological tolerance, most studies focus on the role of T lymphocytes in the disease. Several recent studies indicate that B lymphocytes also play a critical role in IDDM. NOD mice, a highly relevant animal model of IDDM, are protected from the disease process when B lymphocytes are blocked or eliminated. Further, data indicate that B lymphocytes are uniquely able to present some key beta cell antigens, and their antigen presenting function includes the ability to govern T cell differentiation. To better understand the role of B lymphocytes in IDDM, NOD mice were produced that express immunoglobulin transgenes (Tg) from anti-insulin mab125. While B lymphocytes that carry anti-insulin transgenes are functionally silenced in normal B6 mice, the same transgenes demonstrate breaches of tolerance when expressed in NOD. In addition, when NOD mice are engineered to harbor only the heavy chain (HC) Tg from mab125 (VH 125Tg), these animals develop diabetes at a significantly faster rate than non-Tg controls. In contrast, NOD mice expressing an otherwise identical transgene (VH281) that differs in only two amino acids that are required for insulin binding are protected from developing diabetes. When these HC-Tg NOD were examined for expression of insulin binding B cells, B cell receptors (BCR) from VH125-Tg NOD were observed to combine with endogenous light chains to produce a heterogeneous population of insulin-binding B cells, some of which bind insulin with high affinity. Tracking anti-insulin B cells in VH125Tg NOD shows that changes in BCR avidity for insulin occurs in concert with progression to diabetes development. These observations indicate that in the context of IDDM, HC-Tg mice provide a unique means to track the fate and function of anti-insulin B cells that are usually buried in a large polyclonal repertoire. The cellular and molecular mechanisms that connect B cell actions to IDDM will be examined in the following specific aims: I. To characterize the shifts in structure and function of a B cell repertoire for insulin that accompanies progressive beta cell destruction in NOD mice. II. To understand the diversity and function of B lymphocytes that invade pancreatic islets in the course of T1DM. III. To determine the requirement for B lymphocytes in the initiation of T cell responses to insulin and proinsulin in the context of T1DM. IV. To characterize the antigen presenting function of B cells whose receptors recognize a beta cell autoantigen.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The goal of this proposal is to develop a resource that will provide an effective and reliable means for the primate and biomedical research communities to isolate any specific gene or region of interest from one or all nonhuman primate genomic libraries. To do so, a web-based tool will be developed for the custom design of universal hybridization probes that can be used for the isolation of nonhuman primate bacterial artificial chromosome (BAC) clones. Although whole-genome shotgun sequencing is the best method for discovery-based surveys and comparisons of entire genomes, the whole-genome sequencing efforts in nonhuman primates will not provide the level of high-quality, finished sequence necessary for the detailed functional analysis of specific genes and chromosomal segments. BAC clones are an invaluable experimental substrate for the detailed, hypothesis-driven functional analysis of genomic sequence and can be used in comparative genomic and targeted mapping and sequencing projects focused on a specific gene or region of interest. Universal hybridization probes are a proven technology for the efficient targeted isolation of BAC clones from multiple species in parallel. However, the universal probe technology has not been optimized for the isolation of genomic clones from the eighteen available nonhuman primate BAC libraries. This proposal will establish those optimal parameters and provide them as preset values for the custom design of nonhuman primate universal probes by the public. This custom universal hybridization probe design website will therefore facilitate access to the full spectrum of genetic diversity captured within all current and future nonhuman primate genomic libraries. Nonhuman primates are powerful and unique model systems to study human disease. The research tool to be developed in this proposal will enable the primate and biomedical researchers to isolate specific nonhuman primate genes in a cost-effective manner. As a result, this resource, which is supported by representatives from the primate and biomedical research communities, will provide an important avenue for scientists to readily import nonhuman primate genomic clones into their own laboratory and experimental paradigms. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The identification of genes underlying disease susceptibility is of critical importance to human health. Equally important is the identification of genes that confer resistance to disease. The goal of this R21 proposal is to genetically and molecularly evaluate a candidate gene for the resistance to a human genetic disease, called Lesch-Nyhan disease, in mice. Lesch-Nyhan disease is an inborn error in metabolism caused by a deficiency in the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT1), and leads to severe developmental, neurological, and behavioral disorders for which there is no cure. In contrast to the profound effect HPRT1-deficiency has in humans, loss of HPRT1 activity in mice does not result in the severe clinical phenotypes seen in HPRT1-deficient patients. Thus, because of their resistance to Lesch-Nyhan disease, mice provide a model system for identifying the gene(s) that can suppress the severe clinical symptoms of this human genetic disorder. We recently identified a novel candidate gene which could lead to the suppression of the Lesch-Nyhan disease phenotypes in mice. Specifically, a gene inactivation event in an uncharacterized member of the HPRT1-gene family, PRTFDC1, was found only in mice. Therefore, given the similarity of HPRT1 and PRTFDC1, we hypothesize that this difference in gene content between humans and mice contributes to the differential susceptibility these species have to the severe clinical phenotypes associated with Lesch-Nyhan disease. In order to test this hypothesis, 'humanized' transgenic mice expressing PRTFDC1 will be used to evaluate if expression of this gene in HPRT1-deficient mice leads to the manifestation of the severe clinical phenotypes associated with Lesch-Nyhan disease. In addition, the regulatory interactions between PRTFDC1 and HPRT1 will be characterized by gene expression and protein assays. In summary, there is no adequate animal model of Lesch-Nyhan disease. The experiments proposed here will genetically and molecularly evaluate a candidate gene for modulating the interspecies susceptibility to HPRT1-deficiency as a means to develop a novel animal model of Lesch-Nyhan disease, and dissect the basic etiology of this inborn error in metabolism. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Social behaviors that impact human health, including aggression and parental care, are in part influenced by genetic factors. As the neuroendocrine bases of these behaviors are well conserved across vertebrates, findings in animal models are likely to be applicable to human behavior. Animal models of social behavior with a definitive genetic component are therefore valuable systems in which to identify and study genes that are candidates for influencing human social behavior. The goal of this research project is to develop the genomic resources required to identify the genetic basis of a behavioral polymorphism in an exceptionally promising animal model of social behavior, the white-throated sparrow. The white-throated sparrow has generated a great deal of interest among behavioral biologists because of a plumage polymorphism that predicts many aspects of an individual's social behavior. Dozens of behavioral studies have established that individuals with a white-stripe (WS) on the crown tend to exhibit a more competitive behavior strategy, engaging in more territorial aggression and mate-finding, whereas birds with a tan stripe (TS) exhibit more parental care. The behavioral polymorphism is linked to a polymorphic inversion on chromosome 2, designated 2m, thereby providing a genetic marker for the location of the gene(s) responsible for the behavioral phenotype. Using a comparative genomics approach, we have cytogenetically localized the 2m inversion to a segment of the white- throated sparrow genome orthologous to chicken chromosome 3, and mapped the recombination suppressed region of the 2 (2m) chromosome harboring the gene(s) responsible for the behavioral phenotype. The aims of this proposal are to 1) map the 2m inversion in order to identify candidate genes for the behavioral polymorphism and 2) construct a genomic bacterial artificial chromosome (BAC) library from the white-throated sparrow to support future targeted mapping and sequencing efforts focused on identifying the gene(s) that dictate social behavior in this species. In summary, the white-throated sparrow represents an excellent model for studying the genetics and neuroendocrinology of aggression, parenting, and pair-bonding. This proposal will therefore result in the development of the genomic tools necessary to positionally clone the gene(s) underlying the behavioral polymorphism in a promising animal model of social behavior.Social behaviors that impact human health, including aggression and parental care, are in part influenced by genetic factors. The purpose of this research project is to develop the genomic resources necessary to identify the genetic basis of social behavior in an animal model system, thereby enhancing the ability to model and directly study the genetic basis of human social behavior.

DESCRIPTION (provided by applicant): Nonhuman primates offer a unique model for studying human biology, disease, and evolution. Nevertheless, 95% of nonhuman primates lack the basic genomic sequence resources required for modern genetic and comparative genetic studies of disease, including AIDS. Thus, there is a need to develop affordable and efficient methods that use the available nonhuman primate genomic resources as a springboard for population- based genomic sequencing in a greater diversity of species. The focus of this R21 proposal is to test and to demonstrate the capability of a newly developed genomic technology, microarray-based genomic selection (MGS), as such a method. We and others have shown that microarrays can be used to selectively enrich for targeted regions of a complex genome from total genomic DNA, thereby providing the templates necessary for population-based resequencing of the targeted intervals. However, if MGS could be used in a cross-species format, its utility would increase exponentially. Though the cross-species application of MGS is untested, analogous studies of cross-species hybridization between nonhuman primates using gene expression microarrays have already proved successful. The first goal of this proposal is therefore to determine the capability of cross-species MGS to enrich for orthologous targeted genomic regions of nonhuman primates at divergence levels from 1-10% (Aim 1). The second goal of this proposal is to directly demonstrate the utility of cross-species MGS for nonhuman primate and AIDS research. To do so we will apply the cross-species MGS approach toward sequencing 125-250 candidate genes for the benign nature of SIV infection in the sooty mangabey, an exceptional nonhuman primate model for AIDS research that lacks genomic resources (Aim 2). In summary, a method that can simultaneously produce both comparative and population-based sequence data from targeted regions in multiple species will be tested and applied toward the identification of genetic factors that suppress progression to AIDS. PUBLIC HEALTH RELEVANCE: As our closest living relatives, nonhuman primates are a unique genetic resource for studying disease progression to AIDS. The goal of this research project is to apply a new DNA sequencing technology toward sequencing genes in nonhuman primates, including a biomedical model for AIDS research.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (01): Behavior, Behavioral Change, and Prevention and specific Challenge Topic, 01-GM-102: Model organisms for social behavior studies. Identification and development of model organisms that allow for integrative analyses of the genetic, biochemical, physiological, and environmental components of social behavior. Mental health disorders, including autism spectrum disorders, schizophrenia and depression, are characterized in part by severe impairments in social behaviors including social reciprocity, social cognition and social motivation. Unfortunately, because of the limited social repertoire of traditional animal models such as mice and rats, our understanding of the genetic and neurobiological mechanisms that govern social behaviors are poorly understood. Consequently, pharmacological interventions to treat deficits in social behaviors have met with little success. However, the prairie vole (Microtus ochrogaster) is rapidly emerging as a premier model organism for understanding the fundamental neurobiology of social behaviors. In contrast to more established rodent models such as mice and rats, voles demonstrate a much richer and diverse range of complex social behaviors. For instance, socially monogamous prairie voles are highly affiliative, form life-long pair bonds with their mates, and both parents provide extensive care of their young. In contrast, meadow voles (M. pennsylvanicus) are relatively asocial, do not form social attachments of any sort, and are uniparental. Comparative studies between vole species have already led to the identification of several genes and neural pathways that contribute to variation in social behaviors, yet, largely because of a lack of genomic resources for these species, research has been limited to the study of a handful of candidate genes. The goal of this research proposal is to advance the development of the prairie vole as an extraordinary model organism for understanding the biological underpinnings of social behaviors by developing a key genomic resource, a catalog of vole gene sequences. Specifically we will 1) generate partial or complete gene sequences for 18,000 vole genes that encompass >50% of the transcriptome, and 2) for the first time examine the nature of social bond formation on a genome-wide scale. In summary, this research project will generate a critical resource for an emerging model of human social behavior and will likely lead the discovery of novel gene pathways involved in the regulation of complex social behavior such as social bonding. These discoveries may ultimately lead to novel pharmacological interventions to enhance social cognitive function in autism, schizophrenia, or depression. PUBLIC HEALTH RELEVANCE: Animal models are critical for discovering the genetic, biochemical, physiological and environmental components of social behavior. The goal of this research project is to develop a key resource needed to exploit an emerging animal model system that has proven utility for understanding the biological and environmental mechanisms of social behavior in humans related to alcoholism, autism, parental care, and pair-bonding.

? DESCRIPTION (provided by applicant): This amended application seeks continued support for the T-32, Interdisciplinary Training in Rheumatic Diseases at Vanderbilt University. The program has completed its first 5 year cycle. The program seeks support for 2 predoctoral and 3 postdoctoral positions. The goal of the program is to provide the next generation of investigators with the tools necessary to make critical discoveries and to advance our understanding and treatment of complex rheumatic disorders. Our faculty have created an interactive environment of discovery that bridges both clinical and basic research for pre and post-doctoral trainees. An interdisciplinary approach is coupled with a carefully orchestrated mentoring process so that trainees are provided with a broad perspective and they are exposed to opportunities for clinical translation of problems that cannot be solved by a single laboratory. The preceptors in the program are highly interactive and maintain extensive collaborative efforts between and among the research groups. These interactions create four Research Interest Groups within the program: Innate and Adaptive Immunity; Vascular Biology and Inflammation; Clinical and Health Services Research; and Musculoskeletal Biology. The program is organized to meet the career goals of individuals who want to apply advanced technologies, such as genomics or proteomics to rheumatic disease as well as to support the careers of individuals who want to translate these advances to improve health care. In addition to Departmental and preceptor-specific laboratory instruction, each trainee receives rheumatic disease research training through an interdisciplinary curriculum. Combined research forums, journal clubs and shared core facilities promote interactions beyond individual laboratories or divisions. Trainees in the program are actively mentored by both faculty members and through peer mentoring by former students and senior fellows in the program. Revisions in the application address recruiting, selection and monitoring progress of trainees. Degree granting pathways for MSCI and MPH will be available to the program. Significant institutional investment in the Division's facilities and the recruitment of outstanding new facult further strengthen the program for the future.

DESCRIPTION (provided by applicant): An estimated 1 million Americans are infected with HIV and another ~50,000 are infected each year. Despite progress in the treatment of AIDS, in 2007 ~2 million people around the world died from this disease. The primary nonhuman primate model in HIV/AIDS research is the rhesus macaque which develops simian-AIDS after infection with SIV. In both humans and rhesus macaques it has been demonstrated that host genetic variation can provide resistance or susceptibility to the progression to AIDS. However, as non-natural hosts of HIV/SIV, humans and rhesus macaques have not had time to adapt specific mechanisms to prevent AIDS, and those protective genetic variants that are present in the human and macaque populations are likely relics of past selection related to other infectious diseases. In contrast, the genomes of natural hosts of SIV have co-evolved with the virus and been relentlessly shaped by natural selection to acquire robust genetic mechanisms to suppress disease progression after SIV infection. Thus, identification of those robust genetic mechanisms in a natural host SIV would reveal a proven solution for how to suppress AIDS. The goal of this R21 is to identify candidates for the genetic mechanisms that have evolved to suppress progression to AIDS in a natural host of SIV, the sooty mangabey. To do so we will characterize and compare the sooty mangabey and rhesus macaque with respect to two compelling but unexplored candidate mechanisms for the difference in disease progression after SIV infection in these two nonhuman primates: micro RNA (miRNA) expression profiles during the course of SIV infection, and differences in gene copy number between these species. Specifically, we will: 1) characterize and compare the miRNA expression profiles between SIV- infected and uninfected sooty mangabeys and rhesus macaques, and 2) use array CGH to identify genes that differ in copy number between sooty mangabey and rhesus macaque as the means to identify specific miRNAs and genes that are candidates for the genetic basis of susceptibility/resistance to the progression to AIDS in these species. In summary, a comprehensive genomic study will be used to identify candidate genetic factors that have evolved to suppress AIDS in a nonhuman primate model of this disease. PUBLIC HEALTH RELEVANCE: Nonhuman primates are a unique genetic resource for studying disease progression to AIDS. The goal of this project is to characterize and compare the genomic properties of two closely-related model species used in AIDS research as a means to identify genetic mechanisms that can suppress disease progression in HIV- infected individuals.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Technological advances and the proven value of comparative genomics have led to the sequencing of a diverse sample of vertebrate genomes. Even so, only a tiny fraction of all species have been selected for genome sequencing. The focus of this R21 proposal is to test and to demonstrate the capability of a newly developed genomic technology, microarray-based genomic selection (MGS), as a method that uses the available nonhuman primate genomic resources as a springboard for population-based genomic sequencing in a greater diversity of species. To date we have developed an MGS array based on the sequence of the human genome and have tested the ability of this array to capture orthologous fragments from 4 nonhuman primates (chimpanzee, orangutan, rhesus macaque and marmoset) at increasing evolutionary distances to human (1-10%). As expected the efficiency of MGS was correlated was negatively correlated with the divergence from human. That is, while the MGS efficiency was basically equivalent between human and chimpanzee and orangutan (which are 97-99% identical to human), the efficiency of MGS was 2.4-fold lower in rhesus (94% identical to human), and 8-fold lower in the marmoset (90% identical to human). In summary, our results to date suggest that the MGS methodology is quite tolerant of mismatches between the probe and target sequence when they are 97.5% identical, but the efficiency of this method drops off significantly as the divergence increases. Moreover, these initial results demonstrate that we are well on our way to quantifying the cross-species efficiency of this method.

DESCRIPTION (provided by applicant): Type IA or insulin dependent diabetes (T1D) results from of an autoimmune process that destroys insulin producing beta cells in the pancreatic islets. Clinical trials demonstrate that targeting the adaptive immune system can preserve 2 cell function, but a major road block to intervention in T1D is limited access to critical early stages of the disorder. The goal of this project is to validate the position of a novel CD4+ T cell subset, T follicular helper cells (TFH) as critical early mediators of the breach of tolerance in T1D. Recent advances in vaccine biology reveal that high affinity IgG antibodies require the action of TFH. Accordingly, we propose the high affinity autoantibodies in T1D reflect the actions of TFH at a critical early checkpoint in the disease. To investigate TFH differentiation and function in the progression of T1D in NOD mice, we developed a new model that uses a targeted (knock-in) anti-insulin VH gene to detect TFH driven class switch recombination (IgG) in T1D. This model will be tested in Specific Aims that 1) identify the sites and stages in T1D when TFH initiate loss of immune tolerance, and 2) determine the contribution of regulatory T cells to the genesis of TFH in T1D. The model will validate a new early checkpoint in T1D progression and will provide better targets for early diagnosis and intervention in the future. . PUBLIC HEALTH RELEVANCE: Over the past decade the incidence of T1D has increased at an alarming rate of 3% per year in a world-wide distribution and in multiple ethnic groups. Despite improved metabolic control with insulin delivery systems, T1D remains a major cause of renal failure, blindness, amputations, and cardiovascular disease. This project offers the prospect to identify a previously unrecognized target for diagnosis and therapy.