Molecular Mechanisms and Treatment Of Autoimmunity In Man And Animal Models

人和动物模型中自身免疫的分子机制和治疗

• 批准号: 8555808
• 负责人: michael j lenardo
• 金额: $ 72.89万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8555808
• 关键词: Age; Animal Model; Animals; Antibodies; Antigen Targeting; Antigens; Apoptosis; Area; Autoantibodies; Autoimmune Diabetes; Autoimmune Diseases; Autoimmune Process; Autoimmunity; Beta Cell; Biological Assay; Blocking Antibodies; Blood Coagulation Factor; Blood Glucose; Cells; Cessation of life; Clinic; Clinical; Clinical Trials; Cooperative Research and Development Agreement; Detection; Development; Diabetes Mellitus; Diagnosis; Diagnostic tests; Disease; Disease model; Early treatment; Epitopes; Factor VIII; Female; Future; Gerda brand of difluprednate; Goals; Gold; Health; Human; Immune; Immune response; Immunology; Immunotherapy; Inbred NOD Mice; Incidence; Individual; Insulin; Insulin Antibodies; Insulin-Dependent Diabetes Mellitus; Intervention; Investigation; Islet Cell; Laboratories; Lymphocyte; Maryland; Measures; Mediating; Medical; Molecular; Multiple Sclerosis; Mus; Myelin Proteins; Nature; Organ; Pathogenesis; Phase; Plasma; Prediabetes syndrome; Process; Proteins; Radiolabeled; Recombinant Proteins; Recombinants; Regulation; Reproducibility; Research; Risk; Role; Sampling; Screening procedure; Severities; Signal Transduction; Specificity; Staging; System; T-Cell Immunologic Specificity; T-Cell Receptor; T-Lymphocyte; Technology; Testing; Therapeutic; Therapy Clinical Trials; Time; Translations; United Kingdom; United States National Institutes of Health; Universities; Variant; Working Women; apoptosis in lymphocytes; base; cohort; diabetic; human subject; improved; insight; instrument; islet; man; mouse model; non-diabetic; prevent; programs; radiotracer; response; success; tool; type I diabetic

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.

我们研究自身免疫性疾病的细胞和分子基础有两个目的。首先，我们想了解导致自身免疫性疾病的T细胞的致病作用和抗原特异性，如多发性硬化症，凝血因子抑制，胰岛素依赖型糖尿病等。其次，我们想测试特异性抗原诱导的细胞凋亡作为治疗这种自身免疫性疾病的一种手段。为此，我们在以下领域取得了进展：1）我们重新启动了对含有多发性硬化症潜在相关抗原的重组分子的研究，目的是建立一个合作研究和开发协议，以在临床试验中测试这种形式的治疗。目前，越来越多的证据表明髓鞘蛋白抗原是自身免疫攻击的靶点。 通过编程识别这些抗原的T细胞死亡，可以证明消除这些细胞对疾病的影响。 2)我们正在研究新的高灵敏度诊断测试，以检测自身免疫性疾病期间的终末器官损伤，以确定这些是否可以提供自身免疫性攻击的早期预警系统; 3）我们正在启动抗原特异性治疗的研究，以防止血友病患者在因子VIII给药后形成阻断抗体。这些研究将采用新的重组蛋白质，构建含有T细胞反应的因子VIII的主要表位区域。我们还将在其他自身免疫性疾病的实验动物中开展研究。特别是，我们专注于I型糖尿病，并一直在研究针对胰岛素的免疫反应，作为糖尿病前期小鼠和人类疾病的先兆。 糖尿病前期状态，称为胰岛炎，涉及以胰岛素为主要抗原的胰岛细胞的细胞和数字反应。在任何明显的表位扩散之前的免疫应答的集中性质可以允许使用胰岛素或其同源物作为治疗实体。 作为这些研究的一部分，我们试图了解T细胞受体刺激抗原诱导死亡的分子调控。 部署高度敏感的早期预警系统作为筛查工具，以识别具有早期免疫介导的器官损伤的个体，并使用抗原特异性治疗方法进行早期干预，我们希望提供靶向治疗，以最大限度地减少终末器官损伤和临床疾病。为此，我们已经制备了非常灵敏的电化学发光分析，可以灵敏和特异地检测FVIII抗体和胰岛素自身抗体。这可以更好地部署我们早期成功干预的努力。我们相信这些研究将为自身免疫性疾病的发病机制提供重要的新见解，并有望刺激新形式的高度特异性免疫治疗的发展。 2012年，我们在开发胰岛素自身抗体（IAA）检测方面取得了显著进展。 IAA的检测有助于预测自身免疫性糖尿病的发展。然而，长期使用的金标准125 I-胰岛素放射性结合试验（RBA）在实验室之间的重现性低，样品处理时间长，并且需要使用新合成的放射性标记胰岛素进行每组试验。因此，快速、非放射性和可重复的测定具有重要的医学意义。我们已经开发了基于电化学发光（ECL）的测定，克服了这些缺陷，可以分别测量非肥胖糖尿病（NOD）小鼠和1型糖尿病个体中的IAA和抗胰岛素抗体（IA）。使用小鼠IAA ECL测定，我们相关的IAA，组织病理学胰岛炎，和血糖在一个队列的雌性NOD小鼠从4至36周龄。我们发现，我们的人IA ECL测定法与传统RBA相比具有优势，并在三个独立实验室中使用来自34名糖尿病患者和59名非糖尿病患者的样本进行了验证。该技术具有快速、灵敏、动态范围宽、背景低等优点。在NOD小鼠模型中，IAA ECL信号与胰岛炎严重程度正相关，并且在8-10周龄时测量的阳性ECL值预测在20周龄时糖尿病发作。使用人血清和血浆样品，我们的IA ECL测定产生了可重复和准确的结果，平均灵敏度为84%，特异性为95%。我们使用位于马里兰州盖瑟斯堡的Wellstat公司总部、位于马里兰州贝塞斯达的NIH和英国剑桥大学的糖尿病免疫学实验室的仪器，对相同的样本进行了检测，发现实验室之间没有统计学显著差异。我们的结论是，高灵敏度，非放射性ECL为基础的测定应有助于可靠和快速检测胰岛素抗体及其前体血清和血浆在实验室之间的标准化方式在研究和临床环境。我们的下一步是评估人类IA测定在糖尿病前期人类受试者或具有赋予1型糖尿病风险的特征的人中检测IAA，并开发针对其他胰岛β细胞抗原的其他自身抗体的类似测定，这些抗体共同预测这种常见疾病的诊断，以改善预测并促进未来的治疗试验。鉴于I型糖尿病的发病率在过去几十年中一直在增加，这些测试可能会允许新的干预措施来预防或阻止这种疾病的发展。