In this project, we are investigating the development of new diagnostic and therapeutic approaches to three immunological diseases that share a common pathogenesis: multiple sclerosis, type I diabetes, and the development of inhibitory antibodies against FVIII in the treatment of hemophilia. each of these conditions depends on the action of T cells responding to relevant antigens. Therefore, we would like to use antigen-specific clonal deletion to try to suppress and eliminate the pathogenic T cells. The key feature of this approach is that the antigen itself will be used to program the specific T cells to die through apoptosis. This will provide a therapeutic effect directed specifically at the pathogenic T cells and avoid general immunosuppression which is the conventional approach to these and related disorders. in order to study the feasibility of this approach would like to accomplish two objectives in our research. First, we want to investigate the pathogenic role and antigen-specificity of T cells that cause autoimmune diseases such as multiple sclerosis, clotting factor inhibition, insulin-dependent diabetes, among others. an important focus will be the discovery of what specific antigens trigger the critical pathogenic T cells. Second, we would like to test specific antigen-induced apoptosis as a means of treating such autoimmune diseases. These would first be carried out in experimental animals and, if successful, extended into clinical trials in humans. To these ends, we have made progress in the following areas: 1) we have reinitiated studies of recombinant molecules containing antigens potentially involved in multiple sclerosis with the goal of establishing a Cooperative Research and Development Agreement to test such a form of therapy in a clinical trial. At present there is increasing evidence that myelin proteins antigens are the target of the autoimmune attack. By programmed the T cells that recognize such antigens to die, the effect of eliminating these cells on the disease can be demonstrated. 2) We are studying new highly sensitive diagnostic tests to detect end organ damage during autoimmune diseases to determine if these can provide an early warning system of autoimmune attack;and 3) we are initiating studies of antigen-specific therapy to prevent the formation of blocking antibodies following factor VIII administration to hemophiliacs. These studies will employ new recombinant proteins constructed to contain the principal epitopic regions of Factor VIII to which T cells react. We will also be initiating studies in experimental animals of other autoimmune conditions. In particular, we are focusing on Type I diabetes mellitus and have been studying immune responses against insulin as a harbinger of disease in prediabetic mice and humans. The prediabetic state, known as insulitis, involves both cellular and numeral responses against the islet cells with insulin as the primary antigen. The focused nature of the immune response, which precedes any evident epitope spreading, may allow the use of insulin or congeners thereof as a therapeutic entity. As part of these studies we are trying to understand the molecular regulation of antigen-induced death by T cell receptor stimulation. The deployment of a highly sensitive early warning system as a screening tool to identify individuals with early immune-mediated organ damage with early intervention using antigen-specific treatment approaches, we hope to provide targeted therapy to minimize end-organ damage and clinical disease. to this end, we have prepared extremely sensitive electrochemiluminescence assays that can sensitively and specifically detect FVIII antibodies and insulin autoantibodies. This could better dispose our efforts to intervene early successfully. We believe these investigations will provide important new insights into the pathogenesis of autoimmune diseases and hopefully stimulate the development of new forms of highly specific immune therapy. In 2012, we have made notable progress in developing the test for insulin autoantibodies (IAA). The detection of IAA aids in the prediction of autoimmune diabetes development. However, the long-standing, gold standard 125I-insulin radiobinding assay (RBA) has low reproducibility between laboratories, long sample processing times and requires the use of newly synthesized radiolabeled insulin for each set of assays. Therefore, a rapid, non-radioactive, and reproducible assay is of great medical important. We have developed electrochemiluminescence (ECL)-based assays that over come these deficiencies that can measure IAA and anti-insulin antibodies (IA) in non-obese diabetic (NOD) mice and in type 1 diabetic individuals, respectively. Using the murine IAA ECL assay, we correlated IAA, histopathological insulitis, and blood glucose and found that our IA ECL assay compared favorably to conventional RBA. The ECL assay technology was rapid and sensitive with a broad dynamic range and low background. In the NOD mouse model, IAA ECl signal was positively correlated with insulitis severity, and positive ECL values measured at 8-10 weeks of age were predictive of diabetes onset at 20 weeks of age. Using human serum and plasma samples, our IA ECL assay yielded reproducible and accurate results with an average sensitivity of 84% at 95% specificity. We concluded that highly sensitive, non-radioactive ECL-based assays should facilitate reliable and fast detection of antibodies to insulin and its precursors sera and plasma in a standardized manner between laboratories in both research and clinical settings. we are presently working on evaluating the human IA assay forthe detection of IAA in prediabetic human subjects or those who harbor features that confer risk of type 1 diabetes. This involves showing that the assay has the appropriate sensitivity and specificity in the target population. and to develop similar assays for other autoantibodies directed at other islet beta cell antigens that together are predictive for the diagnosis of this common disorder, in order to improve prediction and facilitate future therapeutic trials. Given that the incidence of type I diabetes has been increasing over the last several decades, these tests might permit new interventions to prevent or forestall the development of this disease. Our therapeutic investigation involves using various recombinant antigens to try to induce clonal deletion in rodent models of multiple sclerosis called """"""""experimental allergic encephalomyelitis"""""""" (EAE) and for type I diabetes called """"""""nonobese diabetic"""""""" (NOD) mice. In both disorders, we can show a potent ability of test antigens to induce T cell apoptosis in vitro. Therefore, these studies are now being extended into in vivo experiments with both models. So far the most promising results have come from EAE. We find that appropriate engine preparations cause marked clonal deletion of T lymphocytes that have infiltrated the spinal cord and brain. It is these lymphocytes, when they have successfully entered the brain, that cause the lesions that show up as plaques on magnetic resonance imaging. The T cells in these disease foci are potentially recognizing key antigens in the myelin sheaths of the central neurons and being triggered cause destructive and inflammatory responses that damage the myelin sheath andmake the neurons vulnerable to degenerative cell death causing further functional impairment. Therefore, eliminating these T cells is likely to have a powerful impact on the disease process. Further studies are in progress to validate and extend these findings.
Showing the most recent 10 out of 23 publications
