Brigitte T. Huber - US grants

The aim of the proposed study is the further delineation of B cell differentiation and characterization of B cell triggering in mice. The basic tools in these studies will be antisera specific for the two B cell differentiation markers Lyb3 and Ia.W39 which we have defined previously. Both these antigens are expressed selectively on a functionally defined B lymphocyte subset that is absent in adult mutant mice carrying the xid gene and in newborn normal mice. Since we have previously shown that Lyb3, an isogenic B cell marker, is a receptor for triggering signals, we will now analyze the nature of these signals by studying whether Lyb3 has binding capacity for T cell replacing factor(s). The role of Ia.W39, coded for by a gene in the I-A region of the H-2 complex, as an effector molecule in cell interactions will be tested. In particular, we will examine whether Ia.W39 is essential for optimal presentation of antigens, which are under immune response gene control mapping in the I-A region. Since the membrane expression of both antigens is controlled by a gene on the X-chromosome, there might be a structural or organizational relationship between the two molecules: We will analyze their chronologic appearance during ontogeny. Further we will attempt to in vivo and/or in vitro modulate their expression in order to test whether there is a mutual interdependence in the mechanism of surface expression. We will also study whether a selective loss of receptor/effector function (see above) is seen in these manipulated B cells. We will try to produce hybridomas secreting monoclonal anti-Lyb3 and anti-Ia.W39 antibodies.

The aim of the proposed studies is to analyze the role of the murine Fc receptor complex in B cell differentiation and triggering. Specifically, we will attempt to clone and sequence the gene coding for Lym20.2, a serologically detectable B cell marker that represents an allelic form of the Fc receptor molecule. This gene is either identical or very closely linked to the gene coding for the Mls molecule that has important T cell triggering functions. The precise analysis of the genomic organization and function of this cluster of B cell differentiation antigens will give insights into the molecular mechanism of T cell-B cell interaction. Furthermore, it will provide gene probes that allow the characterization of human B cell malignancies, which represents the long term significance of this project. We will construct genomic transfection libraries in heterologous species of fibroblasts, using chromosomal DNA from Lym20.2+ tissue, and select for stable transfectants that express Lym20. We will then prepare specific subtractive cDNA probes, in order to screen cDNA and genomic cosmid libraries for the Lym20 gene. In parallel, we will use synthetic oligonucleotide probes whose sequence is deduced from a partial amino acid sequence of the Lym20.2 molecule. Having established the identity of the selected cDNA and cosmid clones in chromosomal mapping and transfection experiments, we will analyze the relationship between the Lym20/Fc receptor molecule and the Mls gene product. Finally, we will analyze the mechanism by which Mls alleles control induction of T cell proliferation.

The aim of the proposed studies is to analyze, at the molecular level, the fine specificity of immune response gene function in mice. Specifically, we will attempt to map the hypervariable regions on the T cell receptor Alpha and Beta genes that determine specificity for antigen and MHC restriction, respectively. Furthermore, we will design a model system to test whether an antigen-MHC molecule interaction takes place during antigen presentation. A more precise understanding of the immune response phenomenon is essential in order to gain direct insight in possible mechanisms of immune disorders causing human diseases, such as some types of cancer and AIDS (acquired immune deficiency syndrome). The proposed studies are based on our detailed analysis of the genetic restriction of T cell recognition of heterologous species of insulin at the cellular level. We have already established libraries of T cell hybridomas with precisely defined antigen epitope specificity and Ia epitope restriction. These T cell hybridomas will be used to clone the cDNAs coding for the T cell receptor Alpha and Beta chain and to study the function of specifically mutagenized Ia molecules. We will prepare cDNA libraries from T cell hybridomas with slightly different Ia epitope and/or antigen epitope recognition. We then will sequence the variable region of the Alpha and Beta gene coding for the T cell receptor in the various T cell lines. With gene transfection experiments we will test the requirement for a functional receptor molecule. The possibility exists that MHC restriction and antigen recognition can be scrambled by combining an Alpha chain of a specific T cell clone with a Beta chain from a T cell clone with diffeent antigen and/or Ia epitope recognition. Finally, we will analyze the mechanism of antigen presentation in conjunction with an Ia molecule; namely we will test Ia molecules that have been altered with site specific mutagenesis at a potential Ia-insulin interaction site. Ideally, only insulin presentation should be affected by such an alteration, while recognition by allo- or autoreactive clones should be left intact.

Recently attention has been newly focused on the histocompatibility antigen, Mls, because of its profound effect on the expressed T cell receptor V-beta gene repertoire. The goal of this research is to characterize Mls-a at the molecular level in order to define the elusive Mls-a gene product. cDNA encoding Mls-a will be identified using transient expression systems that allow functional detection of Mls determinants. Although Mls-a has been described nearly twenty years ago as a single gene trait that induces strong T lymphocyte proliferative responses in mixed lymphocyte cultures matched for the major histocompatibility antigens, the nature of this antigenic determinant has remained an enigma. These studies should provide direct insights into the nature of this "universal" tissue specific ligand that is recognized by the T cell receptor in the context of a class II molecule. This recognition molecule is important because it plays a role in regulation of immune responses by leading to elimination of immature thymocytes and activation of mature peripheral T lymphocytes.

The overall goal of this competitive renewal application is to continue our work on the cellular and molecular characterization of Mls-1, the prototype of the endogenous superantigens (SAGs) which is encoded by the endogenous Murine Mammary Tumor Virus (MMTV) Mtv-7. Furthermore, our study of virally encoded SAGs will be expanded by testing the human pathogens EBV and HIV-1 for this gene trait. The following specific aims are proposed: A. Because SAG expression is critical for successful transmission of infectious MMTV, it is important to understand the control of expression of this molecule. Our preliminary data that MMTV sag transcription is controlled independently of that of the other MMTV genes will be continued. Using transient expression of a reporter construct as read-out, the MMTVenv gene will be tested for promoter/enhancer elements, the octamer sites in the 5' and 3' LTR for B cell specific expression, and the role of the glucocorticoid responsive elements in Mls-1 expression. B. Since it is not known how and when Mls-1 associates with MHC class II molecules, its post- translational pathway will be analyzed by transfecting it into cells that have a general defect in processing class II restricted antigens, as well as cells that vary in their level and form of MHC class II Ii chain expression. Many retroviral proteins, including HIV-1 gp160 and MMTV env, require processing by furin to be functional. Since MMTV SAGs have two potential furin cleavage sites which are highly conserved, the role of this protease for functional Mls-1 expression will be tested by over-expression of furin, as well as by transient expression of a furin inhibitory protein. C. The apparent tri-molecular interaction of Mls-1, MHC class II and the TCR will be analyzed in vitro. Soluble recombinant Mtv-7 sag products and mutants thereof, as well as soluble recombinant TCRs and mutants thereof will be produced. These recombinant products will be tested in functional assays, and the affinity of their interaction with soluble MHC class II molecules will be measured with the help of a Biosensor. D. The human herpesvirus EBV is the causative agent of infectious mononucleosis, which is characterized by a strong self-limited T cell proliferation. In order to investigate whether this is due to a SAG-like response, EBV will be tested for SAG expression. An autologous system will be established for screening wildtype EBV, derived from mononucleosis patients, for T cell proliferation. The Vbeta profile of the responding T cells will be compared to that expressed after mitogen stimulation. If positive results are obtained, well characterized EBV recombinants will be used for mapping the gene responsible for the SAG activity. Finally, the hypothesis will be tested that HIV-1 makes use of a SAG for facilitating viral transmission. Since our laboratory has shown that human T cells respond in a Vbeta specific fashion to the murine retroviral SAG Mls-1, murine T cells will be tested for a Vbeta specific proliferation in response to HIV-1 gene products. The feasibility of all these approaches has already been established, and preliminary data have been collected for the studies involving human pathogens. New insights may be gained for the development of therapeutics and prevention of viral infection.

The overall objective of this grant proposal is to further analyze the role of DPIV/CD26 in antigen specific T cell activation, with special emphasis on AIDS related immune dysfunction. The basis is provided by our characterization of the T cell accessory function of CD26. Furthermore, we have recently shown that HIV-1 Tat binds tightly to CD26 and exerts its immunosuppressive effect through this molecule. We now propose to determine the underlying molecular mechanism of the CD26 mediated T cell co-stimulation, and to analyze the role that HIV-1 Tat plays in the antigen specific immunosuppression seen in T cells of HIV-1 infected individuals. 1. We will test the hypothesis that CD26 interacts with a membrane-bound physiological ligand to achieve its accessory function. For this purpose we will produce CHO cells which express a high level of either human or murine CD26 on their cell surface and engineer chimeric CD26 molecules, in conjunction with CD4, CD8 or mutant leucine zipper peptide, respectively. These reagents will be used to screen for specific binding of CD26 to thymic epithelial cell lines and/or conventional APC and peripheral lymphocytes. If binding is achieved, the ligand will be identified either by blocking with known mAbs to various surface molecules, or by cDNA expression cloning. 2. We will test the hypothesis that immuno-incompetence seen in CD4+ T cells early after HIV-1 infection is due to a defect in CD26 mediated signal transduction; namely, we will analyze whether recombinant CD26 molecules reverse the antigen unresponsiveness, and whether HIV-1 Tat blocks the association of adenosine deaminase with CD26. 3. & 4. To test the in vivo significance of the DP IV/CD26 molecule for T cell maturation and activation and the HIV-1 Tat mediated suppression, we will generated chimeric mice which either lack CD26 selectively on lymphocytes or selectively express HIV-1 Tat on these cells. This will be achieved by the injection of blastocysts derived from RAG-2-deficient mice with embryonic stem cells containing a homozygous mutation of the CD26 gene or are transfected with the HIV-1 Tat gene under control of T cell specific regulatory elements. These experiments represent a collaboration with Dr. Fred Alt's group at Harvard Medical School. We will generate a mutant construct of the CD26 gene for the selection of homologous recombinants. ES cells will be transfected with this construct, and cells that have undergone homologous recombination on both chromosomes will be selected. The chimeric offspring form the RAG-2-deficient blastocyst complementation assay will be tested for T cell development and function. If our working hypothesis is correct, these two types of mice should have similar functional profiles. These specific aims represent a combination of basic research on the mechanism of CD26 mediated T cell signaling in vivo and in vitro, as well as a direct analysis of clinical samples, derived from HIV-1 infected individuals, to test the role of CD26 in the observed immunosuppression. They are carried out in collaboration with Dr. W.W. Bachovchin's group who determines the physico-chemical properties of CD26 in association with various binding proteins.

Lyme disease is a multi-faceted illness, initiated upon infection with the spirochete borrelia burgdorferi (Bb). One manifestation of the disease is arthritis, which can result in treatment-resistant chronic arthritis in a small subset of exposed individuals. The prevalence of HLA.DR4 related alleles in these patients is an indication of an autoimmune process. They have explored the role of T helper cells in the development of Lyme arthritis, and the investigators hypothesize that patients with these particular HLA.DR alleles are at risk of developing chronic disease as a result of a vigorous inflammatory Th response to Bb. The interferon gamma production by these activated Thl cells, as well as the presence of Bb, itself leads to upregulation of MHC class II, LFA-1 and ICAM-1 expression predisposing for an autoimmune process by molecular mimicry. Based upon homology to a highly antigenic epitope of outer surface protein A (Osp A) of Bb, the applicants have identified a candidate autoantigen, hLFA-1. The aim of this application is to verify this hypothesis by creating in vitro and in vivo test systems. I. The functional similarity between the homologous epitopes and Osp A Bb and human LFA-1, but not mouse LFA-1, will be analyzed by three in vitro approaches: (a) T cell hybridomas will be generated from DR4 transgenic mice after immunization with Osp A, and the resulting clones will be tested for reactivity to human LFA-1. If double-reactive hybridomas are identified, the antigenic epitopes will be mapped; (b) synovial fluid T cells from chronic Lyme patients will be transformed and tested at the single-cell level for reactivity to Osp A and human LFA-1; (c) Osp A will be tested for binding to ICAM-1. Competition assays will be performed between Osp A and LFA-1. II. Murine models for treatment-resistant chronic Lyme arthritis will be generated by two approaches; (a) LFA-1/DR4 double transgenic mice on an MHC class II-/-background will be created and tested for the development of chronic Lyme arthritis after exposure to Bb. This is based on the observation that mouse LFA-1 does not express the Osp A cross-reactive epitope; (b) Hu-SCID-beige mice will be generated with PBMCs from patients with chronic Lyme arthritis. These mice will contain the non-immune lymphocytes of the susceptible genotype, allowing a detailed analysis of the acquisition of chronic Lyme arthritis after injection with Bb. These investigations, they propose, should provide a vigorous test of their working hypothesis, and will lead to new insights into the mechanism of treatment-resistant chronic Lyme arthritis.

The functional identification, biochemical purification and cloning of a novel cytosolic serine protease, QPP (Quiescent Proline di-Peptidase), which prevents quiescent lymphocytes from undergoing programmed cell death (PCD) forms the basis for the working hypothesis that apoptosis is blocked in resting lymphocytes by an active mechanism. QPP seems to be essential for the survival of resting T and B cells, because specific inhibition of this enzyme leads to activation of cellular caspases and PCD. The goals of this proposal are to substantiate these intriguing observations and to define the apoptosis pathway in G/o lymphocytes. I. to directly analyze the role of QPP in lymphocyte, a dominant negative (DN) variant will be constructed by mutating its catalytic site(s) such that it still binds its specific substrate, but no longer cleaves it. Wild type and DN QPP constructs will be expressed in vitro in cell lines and primary T cells, as well as in vivo by the use of the RAG-2 blastocyst ES complementation system. II. To understand the functional significance of QPP in the protection of lymphocytes from PCD, its physiological substrate(s) has to be identified. Two approaches will be used: i) rDN QPP protein as affinity matrix to extract the substrate from lymphocyte lysate; and ii) the yeast-two hybrid system with the DN QPP as bait for screening a lymphocyte cDNA library. The proteins identified by these methods will be verified as substrates of QPP by their susceptibility to cleavage by wild type enzyme, followed by N- terminal sequence analysis. III. Preliminary data indicate that the caspase cascade initiated in quiescent lymphocytes by blocking QPP differs significantly from other well characterized apoptotic pathways in these cells, such as irradiation- or Fas-induced PCD. Radioactively labeled zVADfmk, an irreversible caspase inhibitor, will be used as an active site-directed affinity reagent, in conjunction with 2D gel analysis, to identify the caspase(s) involved. IV. All cells tested so far contain a protease activity that resembles QPP in its blots, namely, a band of 1.7 kB that is expressed in all tissues and a band of 2.5 kB that is seen mainly in lymphoid cells and pancreas. Furthermore, the sequencing of QPP ESTs revealed clones that contain internal sequence gaps. Thus, it will be determined whether this enzyme is differentially spliced and/of differentially modified in a tissue specific manner. Taken together, these studies will provide new insights into the maintenance of homeostasis of resting lymphocytes in the immune system.

This project is a request for funds for an expanded Core Flow Laboratory by an existing user group at Tufts University School of Medicine: Drs. Brigitte T. Huber (P.I.), Brent Corhan (Co-P.I.), Claire Moore (Co- P.I.), Naomi Rosenberg (Co-P.I.), David A. Thorley-Lawson (Co-P.I.), and Henry H. Wortis (Co-P.I.). The current facility, consisting of a FACStar Plus and a FACScan, has been in operation for the past 10 years and has been self-supported and upgraded through fees charged to users, most of which are paid from NIH-funded grants. Thus, the technical expertise is in place. An expansion is requested for two reasons: a) the current equipment is in need of substantial improvements to remain current, and b) state of the art techniques in cell cycle analysis and phenotyping require the use of a 4 color instrument and a HeNe laser. The projected use by this core group, all funded through RO1 grants, is 75%, which leaves 25% for other investigators at Tufts University School of Medicine. The facility will be run under the overall direction of the P.I., with direct supervision by the Flow Laboratory manager and a full time operator. The overall benefit of the expanded Core Flow Laboratory for the user group in particular, and the Basic Sciences faculty at Tufts University School of Medicine in general, will be considerable, because it will allow the full faculty at Tufts University School of Medicine in general, will be considerable, because it will allow the full benefit of the NIH sponsored research commitment. The institutional support has been guaranteed.

DESCRIPTION (provided by applicant): Lyme disease is a multifaceted illness, initiated upon infection with the spirochete Borrelia burgdorferi (Bb). One manifestation of the disease is arthritis that can become a debilitating, chronic disease in genetically susceptible individuals. During the tenure of this grant, a candidate auto-antigen, hLFA-1, was identified which may elicit an autoimmune response in T cells by molecular mimicry with outer surface protein A (OspA) of Bb. These data form the basis for the current proposal. I. The analysis of the inflammatory T cell response will be expanded and refined by: a) single cell sorting of OspA-reactive T cells from Lyme arthritis patients, using cytokine secretion/capture assays to identify and clone OspA-reactive T cells, regardless of epitope fine specificity. The T cell response of patients in the acute and the chronic phase of the disease will be compared that will provide insights into the mechanism of treatment resistant Lyme arthritis. b) Analyses of OspA responses in HLA.DRB1 transgenic mice. DRB1*0401 and *0101 are associated with susceptibility to chronic Lyme arthritis, while the presence of *1101 seems to protect from this disease. To study the underlying mechanism, the OspA immune response in Bb infected C3H mice expressing these human DRB chains will be mapped and compared. c) testing hLFA-1/DRIB 1*0401 transgenic mice as an animal model for chronic Lyme arthritis. These mice will be infected with Bb and screened for the development of a chronic arthritic disease. II. The contribution of Abs to the inflammatory joint disease will be assessed. A prominent OspA-specific Ab response is observed during the chronic phase of the disease, correlating with the onset of prolonged bouts of arthritis. These Abs will be tested for an autoimmune reaction, and their involvement in joint inflammation will be determined by: a) the identification of immune complexes in synovial lesions of Lyme arthritis patients; b) the use of OspA Abs from chronic Lyme arthritis patients as probe for autoantigen; c) Bb infection in FcRn-deficient, hLFA-1/ DR*0401 transgenic mice: FcRn-/- mice clear serum IgG fast and are protected from Ab-mediated autoimmune diseases. III. A new candidate for molecular mimicry of OspA, hMAWD-BP, will a) be tested for cross reaction with OspA specific T cells and Abs, b) its aa sequences in the OspA-homologous regions of human and mouse will be compared; c) if significant differences are observed, hMAWD-BP transgenic mice will be prepared; finally, d) Genebank searches will be carried out for the identification of other human homologues of bacterial OspA. These experiments are designed to elucidate the mechanism of treatment resistant Lyme arthritis.

[unreadable] DESCRIPTION (provided by applicant): This is a new application to establish a biodefense training program to produce predoctoral trainees who will aid the nation in combating the new challenges of the bioterror threat. The program has a faculty of 19, drawn from the Genetics, Immunology and Molecular Microbiology Graduate Programs of the Sackler School of Graduate Biomedical Sciences of Tufts University School of Medicine and New England Medical Center Hospitals. Focused training in the pathogenesis and genetics of microorganisms that are relevant to biodefense will be integrated with training in understanding host responses to these microorganisms. In this way, our trainees will be prepared to attack biodefense-related problems with an in-depth understanding of pathogen-host interaction. A newly developed curriculum integrates courses offered by Genetics, Immunology and Molecular Microbiology. In addition, a newly-developed course presents the pathogenesis of the most potent biological agents together with scientific, medical, social, and political issues relevant to bioterrorism and biowarfare. The Biodefense Training Program also interfaces with the newly conceived Study Center for the Immunogenetics of Infectious Disease (SCIID), an organization that focuses on genetic, immunological and microbiological aspects of infectious disease. The SCIID will be located in the newly constructed Jaharis Family Center, and will provide an intellectual and physical home for our trainees. This Center will also give students the opportunity to work at the BL-3 level of containment, use gene array technology to study host and pathogen gene expression, and have access to BL-3-level cell sorting. Predoctoral students enrolled in the Genetics, Immunology or Molecular Microbiology Graduate Programs may apply to enter the Biodefense Training Program after their first year of graduate school. Selection for our program will be based on high achievement in lab rotations and coursework during the first year, selection of one of the Biodefense Training Program faculty as a thesis mentor, and commitment to work on a dissertation topic relevant to biodefense concerns. Three students will be selected for two years of support (six positions). Benefits of our program include: 1) students are already enrolled who are working on biodefense related issues; 2) a highly experienced training faculty is in place; 3) a strong curriculum already exists; and 4) development of the SCIID shows institutional commitment to this program and provides an outstanding training setting. [unreadable] [unreadable] PARTICIPATING FACULTY: The 19 training faculty include 11 professors, 3 associate professors, and 5 assistant professors. Six of the training faculty are women. Fourteen of the training faculty have primary appointments at Tufts University and five training faculty are located at the New England Medical Center Hospital. The primary areas of research include genetics, immunology and molecular microbiology. [unreadable] [unreadable]

Abstract Not Provided.

DESCRIPTION (provided by applicant): This competitive renewal application is based on the discovery that the env gene of HERV-K18 (Human Endogenous Retrovirus) encodes a superantigen, which is transcriptionally activated by EBV (Epstein-Barr Virus) and IFN-alpha (type I interferon). The working hypothesis is that the T cell stimulation elicited by the superantigen is not only essential for establishing life-long persistent infection with EBV in healthy individuals, but also plays a crucial role in EBV-associated diseases and malignancies. The following specific aims are proposed to test this model: I) The control of HERV-K18 expression will be defined. The role of CD21 engagement in the initiation of superantigen expression will be analyzed, based on the observation that IFNalpha and EBV infection lead to superantigen expression, both acting through CD21 on resting B cells. Furthermore, the role of EBV LMP-2A in sustained superantigen expression will be evaluated, based on the finding that this EBV latent gene product is sufficient to transactivate HERV-K18 env, leading to superantigen activity. II) HERV-K18 superantigen-reactive cells will be delineated in vivo and in vitro. For this purpose, an HLA.DR/HERV-K18 Env tetramer will be constructed to identify and stimulate superantigen-reactive T cells. The recent discovery of the murine TCR (T cell receptor) Vbeta specificity for the human superantigen will be exploited to define and map the TCR-superantigen interaction site. In addition, the role of CD48 in the superantigen-induced T cell activation will be tested, since EBV upregulates expression of this co-stimulatory molecule. III) The role of the HERV-K18 superantigen in EBV-infection will be determined, because the central dogma of this proposal is that the superantigen activity is required for the successful EBV life-cycle in the human host. In vitro and in vivo EBV-infection/ outgrowth/ persistent latency/lytic cycle/reactivation models will be used to address this aim. IV) The DNA of patients suffering from EBV-associated diseases will be typed for preferential expression of certain HERV-K18 alleles, compared to their respective healthy controls. This aim is based on the observation that the 3 HERV-K18 env alleles defined so far are unequally distributed in the Caucasian population and differ in their superantigen expression level. Collectively, these studies will further the general understanding of how viruses exploit the immune system of their hosts to their own advantage.

mass spectrometry

[unreadable] DESCRIPTION (provided by applicant): The etiology of Chronic Fatigue Syndrome (CFS) is far from understood and is likely due to multiple genetic components. Infection with EBV and treatment with IFN-a have been implicated in the pathogenesis. Our laboratory has shown that EBV-infection, and exogenous IFN-a?, activate transcription of the env gene of a Human Endogenous Retrovirus, HERV-K18. This provirus is normally silent, but when induced it encodes a superantigen (SAg), which is a class of proteins that is capable of deregulating the immune system. Three alleles of HERV-K18 env have been documented, K18.1, K18.2, K18.3, whose gene products have SAg activity, but are predicted to differ biochemically and functionally. Our working hypothesis is that HERV-K18 is a risk factor for CFS. In a pilot study, the allele and genotype distributions of the HERV-K18 env gene were compared between various groups of CFS patients and healthy controls. Although only a limited number of samples were available in the various cohorts, the odds ratios that were obtained were statistically significant. The most intriguing interpretation of these data are that they provide genetic evidence for the unique etiology of at least one group of CFS patients. Thus, it may be possible to delineate different subtypes of CFS, depending on the clinical history of the patients. It is now proposed to substantiate these pilot results, using a much larger cohort of 400 CFS patients associated with EBV that has been assembled by the co-investigator, Dr. Renee Taylor. Dr. Ben Katz, board certified in both Pediatrics and Pediatric Infectious Diseases, will clinically evaluate the patient cohort, and Dr. Inga Peter, a genetic epidemiologist and biostatistician, will oversee the statistical analyses. In addition, the expression pattern of the HERV-K18 SAg during active disease versus intermission will be measured. Furthermore, T cell stimulatory activity of this SAg, expressed on peripheral blood lymphocytes of patients during the course of the disease, will be tested ex vivo, using a T cell hybridoma reporter assay that has been developed in our lab. Since SAg-activated T cells produce massive quantities of chemokines, lymphokines and neurokines, the expression of the HERV-K18 SAg could influence not only the immune system, but other organs as well. A positive association between CFS and either HERV-K18 alleles or expression patterns would open new avenues for the development of clinical treatments of this chronic disease. CFS is a disease that affects a significant number of people worldwide, yet the underlying mechanism(s) of pathogenesis remains unclear. The herpesvirus EBV and IFN-a have been suggested to be associated with CFS, although these concepts are far from accepted. We propose a novel genetic aspect in the EBV/ CFS association, namely the presence of certain HERV-K18 alleles that differ in their superantigen activity. [unreadable] [unreadable] [unreadable]

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. The control of glucose metabolism is a complex process, and dysregulation at any level can cause impaired glucose tolerance and insulin resistance. These two defects are well-known characteristics associated with obesity and onset of type 2 diabetes. We have recently discovered a novel regulator of glucose metabolism, the N-terminal dipeptidase, DPP2, a serine protease that had been identified and cloned by the Huber lab. We have generated a conditional DPP2 knock down (kd) mouse and crossed it with a neurogenin-3 (NGN3)-Cre transgenic (tg) mouse that led to specific kd of DPP2 in the hypothalamus of the brain and the gastrointestinal (GI) tract (NGN3-DPP2 kd). These mice spontaneously develop hyperinsulinemia, glucose intolerance and insulin resistance by 4 months of age. In addition, we observed an increase in food intake in NGN3-DPP2 kd mice, which was associated with a significant increase in adipose tissue mass. This phenotype was exacerbated with age and when challenged with a high fat diet. We conclude, therefore, that DPP2 enzyme activity is essential for maintaining glucose homeostasis. This work constitutes an ongoing collaboration between two scientists on the Boston campus of Tufts: Brigitte T. Huber, PI, Department of Pathology, an immunologist who is an expert on DPP2 and has produced the conditional DPP2 kd mouse, and Ronald M. Lechan, co-PI, Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, a neuroendocrinologist and neuroanatomist who has characterized the DPP2 expression pattern in the brain. A joint publication has already resulted from the characterization of this new animal model for type 2 diabetes. In order to define whether the DPP2 enzyme activity controls glucose metabolism over a central (brain) or peripheral (GI tract) mechanism, we have recently crossed the conditional DPP2-loxP mice with Sf1-Cre tg mice, which leads to DPP2 kd selectively in the ventromedial nucleus (VMN) of the hypothalamus, but not the GI tract (Sf1-DPP2 kd). These mice develop similar glucose intolerance as the NGN3-DPP2 kd mice. Thus, we are now in an excellent position to identify the specific substrate(s) of DPP2 in the VMN that prevents hyperinsulinemia and obesity. We are applying for a Russo grant to lay the groundwork for this project, providing a solid basis for a dual investigator RO1 application to NIDDK that is planned for fall of this year (the specific aims are outlined in the summary section). Our main goal is to assess the logistics of VMN dissection in the hypothalamus in terms of yielding sufficient material for eventual DPP2 substrate identification. It is highly likely that this substrate is a secreted neuropeptide. Thus, for our initial approach, we propose to use shotgun proteomics to analyze the proteins secreted by the dissected VMN after short culture at 37 ?C. This work will be performed in collaboration with John R. Yates, 3rd, at Scripps Research Institute in La Jolla, one of the top experts in the field of identifying neuropeptides by this approach. Since the N-terminus controls protein turnover, we expect to see a quantitative difference in the level of a DPP2-subsrate protein in the two strains. In addition, we will look for N-terminal peptides that are modified by DPP2 in wild type, but not mutant mice. Candidate substrates will then be analyzed by in situ hybridization of sections of the VMN. The special strength of this application is the unique expertise of the two PIs, which is highly complementary.