We are studying the cellular and molecular basis of autoimmune diseases with two purposes. First, we want to understand 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. Second, we would like to test specific antigen-induced apoptosis as a means of treating such autoimmune diseases. 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 in a cohort of female NOD mice from 4 up to 36 weeks of age. We found that our human IA ECL assay compared favorably to conventional RBA and validated using samples from 34 diabetic and 59 non-diabetic individuals in three independent laboratories. 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 carried out the assay with identical samples using instruments at the Wellstat company headquarters in Gaithersburg, Maryland, at the NIH in Bethesda, Maryland, and in the diabetes immunology laboratory at the University of Cambridge in the United Kingdom and found no statistically significant variation between laboratories. 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. Our next step is to evaluate the human IA assay in the detection of IAA in prediabetic human subjects or those who harbor features that confer risk of type 1 diabetes 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.

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Zheng, Lixin; Li, Jian; Lenardo, Michael (2017) Restimulation-induced cell death: new medical and research perspectives. Immunol Rev 277:44-60
Grönholm, Juha; Pagni, Philippe P; Pham, Minh N et al. (2017) Metabolically inactive insulin analogue does not prevent autoimmune diabetes in NOD mice. Diabetologia 60:1475-1482
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Lenardo, Michael J (2016) Clinical Genomics - Molecular Pathogenesis Revealed. N Engl J Med 375:2117-2119
Lenardo, Michael; Lo, Bernice; Lucas, Carrie L (2016) Genomics of Immune Diseases and New Therapies. Annu Rev Immunol 34:121-49
Lucas, Carrie L; Lenardo, Michael J (2015) Identifying genetic determinants of autoimmunity and immune dysregulation. Curr Opin Immunol 37:28-33
Grönholm, Juha; Lenardo, Michael J (2015) Novel diagnostic and therapeutic approaches for autoimmune diabetes--a prime time to treat insulitis as a disease. Clin Immunol 156:109-18
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Lucas, Carrie L; Kuehn, Hye Sun; Zhao, Fang et al. (2014) Dominant-activating germline mutations in the gene encoding the PI(3)K catalytic subunit p110? result in T cell senescence and human immunodeficiency. Nat Immunol 15:88-97

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