GAD ANTIBODY MEDIATED CONTROL OF EPITOPE SPECIFIC ANTIGEN PRESENTATION

GAD 抗体介导的表位特异性抗原呈递控制

• 批准号: 6301167
• 负责人: AKE LERNMARK
• 金额: $ 17.91万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6301167
• 关键词: antigen presentation; autoantibody; clinical research; complementary DNA; diabetes mellitus genetics; gene induction /repression; glutamate decarboxylase; human genetic material tag; human subject; immunogenetics; immunoregulation; insulin dependent diabetes mellitus; molecular cloning; nucleic acid sequence; pathologic process

Before and at the clinical onset, insulin-dependent diabetes mellitus (IDDM) is closely associated with islet auto-immunity, in particular with the presence of auto-antibodies (Ab) against GAD65 - the Mr 65,000 isoform of glutamic acid decarboxylase. GAD65Ab have a high diagnostic sensitivity, specificity and predictive value for IDDM. The now standardized GAD65Ab radiobinding assay that we successfully developed after cloning human islet GAD65 using our GAD65 cDNA plasmid to prepare radioactive GAD65 by coupled in vitro transcription and translation was used to demonstrate that epitope-specific GAD65Ab have a higher diagnostic sensitivity for IDDM than non-epitope analyzed GAD65Ab. The objective is to determine the mechanisms of GAD65 antibody-associated auto-immunity in IDDM in two Specific Aims. 1) To test the hypothesis that epitope-specific GAD65Ab determine progression to IDDM in intermediary phenotypes. Specifically, we will construct combinatorial antibody libraries from: 1) new onset IDDM patients (Project 3); 2) GAD65Ab- positive healthy individuals not progressing to IDDM (Project 4); 3) GAD65Ab-positive patients who are classified and treated for type 2 (NIDDM) diabetes (Project 3) in order to isolate Fab reactive with IDDM-associated GAD65 epitopes; b. determine the cDNA sequences of heavy and light chain anti- GAD65Ab selected by GAD65 epitope-specific panning. 2) To test the hypothesis that GAD65 antibody-mediated uptake of GAD65 deviate HLA- restricted clonal T cell responses. Specifically we will: a. transfect epitope-specific GAD65Ab to human B cells to determine uptake, processing and presentation; b. explore the role of antibody fine specificity in influencing GAD65 epitope presentation using GAD65 peptide and HLA restricted T cell clones in collaboration with Project 1. Project 2 will collaborate with project 1 to define the mechanisms by which membrane-bound GAD65Ab on B lymphocytes may deviate antigen- presentation on MHC Class II antigens and subsequent T-cell responses. GAD65-peptide specific and DRB1 or DQB1-restricted T cells from project 1 will be used to determine deviation responses. The collaboration with Project 3 is to study new onset IDDM patients and GAD65Ab-positive patients who are classified and treated for type 2 (NIDDM) diabetes. These patients will be compared with non-progressing marker-positive healthy subjects individuals from Project 4.

临床发作之前和处于胰岛素依赖性糖尿病 （IDDM）与胰岛自动免疫密切相关，特别是 针对GAD65的自动抗体（AB）的存在-MR 65,000同工型 谷氨酸脱羧酶的。 GAD65AB具有很高的诊断 IDDM的灵敏度，特异性和预测价值。现在 我们成功开发的标准化GAD65AB射射射测定法 在使用我们的GAD65 cDNA质粒进行克隆后，将人类胰岛GAD65进行准备 通过体外转录和翻译结合的放射性GAD65是 用于证明表位特异性GAD65AB具有更高的诊断 IDDM的敏感性比分析的GAD65AB分析的敏感性。目标是 确定GAD65抗体相关的自身免疫的机制 IDDM以两个具体目标。 1）检验表位特异性的假设 GAD65AB确定中间表型中的IDDM的进展。 具体而言，我们将从：1）构建组合抗体库 新发作IDDM患者（项目3）； 2）GAD65AB-阳性健康 个人未发展到IDDM（项目4）； 3）GAD65AB阳性 对2型（NIDDM）糖尿病进行分类和治疗的患者 （项目3）为了与IDDM相关的GAD65隔离反应性。 表位； b。确定重链和轻链抗的cDNA序列 GAD65AB由GAD65表位特异性平移选择。 2）测试 假设GAD65抗体介导的GAD65的摄取偏离HLA- 克隆T细胞反应受限。具体来说，我们将：转染 表位特异性GAD65AB到人B细胞以确定摄取，加工 和演示； b。探索抗体精细特异性的作用 使用GAD65肽和HLA影响GAD65表位表现 与项目1合作的限制T细胞克隆。 项目2将与项目1合作，以定义机制 B淋巴细胞上哪些膜结合的GAD65AB可能会偏离抗原 关于MHC II类抗原和随后的T细胞反应的介绍。 Project 1的GAD65肽特异性和DRB1或DQB1限制的T细胞 将用于确定偏差响应。与之合作 项目3将研究新的发病IDDM患者和GAD65AB阳性 对2型（NIDDM）糖尿病进行分类和治疗的患者。这些 将将患者与非促进标记阳性健康进行比较 受试者来自项目4。