PATHOGENESIS AND PREVENTION OF IDDM

IDDM 的发病机制和预防

• 批准号: 6329417
• 负责人: HUGH O MCDEVITT
• 金额: $ 87.7万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-15 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6329417
• 关键词: NOD mouse; disease /disorder etiology; disease /disorder prevention /control; genetically modified animals; insulin dependent diabetes mellitus

The purpose of this research program is to bring together five investigators in the Departments of Microbiology and Immunology, and Pathology to develop a series of collaborative, research projects. These research projects are designed to apply the techniques of molecular immunology to understanding the development and specificity of the CD4+ T responses to islet cell antigens in type I diabetes in the NOD mouse, in NOD transgenic models expressing human IDDM susceptibility genes, and to develop novel methods of altering the immune responses to prevent IDDM. Drs. Yueh-Hsiu Chien will develop specific T cell staining reagents by producing soluble biotinylated I-A/g7 molecules with a covalently bound peptide coupled to fluorescent streptavidin. The peptide epitopes of glutamic acid decarboxylase and pre pro-insulin will be identified in Dr. McDevitt's laboratory (under the support of DK-51667). Dr. McDevitt will utilize NOD mice transgenic for HLA-DQ8, 7, and 6 to identify the immunodeficient peptide epitopes of human GAD 65 and human PPI presented by IDDM susceptible (DQ8), 7, and 6 to identify the immunodominant peptide epitopes of human GAD 65 and human PPI presented by IDDM susceptible (DQ8) and resistant (DQ7,6) alleles in man. The same T cell receptor staining reagents will be developed for detection of GAD and PPI specific, DQ8 restricted T cells in pre-diabetic and recent onset diabetic patients. In a collaboration between Dr. Chien and Dr. Mark Davis, the polymerase chain reaction (PCR) will be used to characterize and sequence T cell receptor V alpha and V beta sequences from single T cells isolated from the islets of 14-18 day old NOD mice. These T cell receptor V beta and V alpha sequences have detectable shared CDR 3 sequence motifs, suggesting an antigen specific T cell response. T cell receptors from single cells will be expressed in transfected T cell lines and transgenic mice to permit a determination of their function. Transgenic T cells will be tested for their antigenic specificity using a panel of recombinant murine islet cell antigens produced in Dr. McDevitt's laboratory. Dr. Crabtree will produce transgenic mice expressing a mutant cyclophlin able to bind and inhibit calcineurin only when complexed to a modified cyclosporin A. Modified cyclosporin A will be used to inhibit signaling in T cells, to inhibit T cell activation and T cell development, and to induce tolerance to transplanted tissues and islet cell antigens. In collaboration with Dr. McDevitt, Dr. Crabtree will introduce the modified cyclophilin gene into the NOD mouse strain. Modified cyclosporin A will be used to block T cell signaling at various time points following birth to induce tolerance to islet cell antigens. Dr. Weissman and Dr. Judith Shizuru will isolate of hematopoietic stem cells to analyze the effects of whole bone marrow (WBM) versus hematopoietic stem cell (HSC) transplantation in replacing the endogenous lymphoid compartment. H2 matched or syngeneic bone marrow will be used. These experiments will explore the potential for utilizing bone marrow transplantation to prevent the development of type I diabetes in susceptible individuals. Dr. Weissman will also use targeted recombination to analyze the development of the T cell receptor repertoire in NOD mice with endogenous and transplanted bone marrow. These studies will rely on the same techniques used by Drs. Chien and Davis to trace the development of islet cell specific T cells in the NOD mouse.

这项研究计划的目的是将五个 微生物学和免疫学系的研究人员，以及 开展一系列的病理学合作、研究项目。这些 研究项目旨在应用分子生物学技术 免疫学对了解CD4+T细胞的发育和特异性的作用 NOD小鼠对I型糖尿病胰岛细胞抗原的反应 表达人IDDM易感基因的NOD转基因模型 开发改变免疫反应的新方法来预防IDDM。 钱越秀博士将通过以下方式开发特异性T细胞染色试剂 制备共价结合的可溶性生物素化I-A/G7分子 与荧光链霉亲和素偶联的多肽。的多肽表位 谷氨酸脱羧酶和前胰岛素原将在DR中被鉴定。 麦克德维特实验室(在DK-51667的支持下)。麦克德维特医生会 利用转人类白细胞抗原-DQ8、7和6基因的NOD小鼠鉴定 人GAD 65和人PPI免疫缺陷肽表位公布 用IDDM易感多肽(DQ8)、7、6鉴定免疫优势肽 IDDM易感基因(DQ8)呈现的人GAD 65和人PPI表位 和男性的抗性(DQ7，6)等位基因。同样的T细胞受体染色 将开发检测GAD和PPI特异性DQ8的试剂 糖尿病前期和新发糖尿病患者的限制性T细胞。在……里面 钱博士和马克·戴维斯博士的合作，聚合酶链 利用聚合酶链式反应(PCR)对T细胞受体进行鉴定和测序 从胰岛分离的单个T细胞的Vα和Vβ序列 14-18日龄NOD小鼠。这些T细胞受体Vβ和Vα 序列具有可检测到的共享CDR 3序列基序，表明 抗原特异性T细胞反应。来自单个细胞的T细胞受体将 在转基因T细胞系和转基因小鼠中表达，以允许 它们的功能的确定。转基因T细胞将接受检测 用一组重组小鼠胰岛细胞研究其抗原性 麦克德维特博士实验室生产的抗原。克拉布特里博士将制作 表达能够结合和抑制突变型环磷脂的转基因小鼠 钙调神经磷酸酶仅在与修饰的环孢素A复合时。 环孢菌素A将用于抑制T细胞中的信号转导，以抑制T细胞 细胞活化和T细胞发育，并诱导对 移植组织和胰岛细胞抗原。与Dr. 麦克德维特，克拉布特里博士将把改良的亲环素基因引入 诺德老鼠的品系。改良的环孢素A将用于封闭T细胞 在出生后的不同时间点发出信号以诱导对 胰岛细胞抗原。魏斯曼博士和朱迪思·静鲁博士将分离出 全骨髓(WBM)对造血干细胞的影响分析 与造血干细胞(HSC)移植比较 内源性淋巴间隔室。H2相合或同基因的骨髓将 被利用。这些实验将探索利用骨骼的潜力 骨髓移植预防儿童I型糖尿病的研究 易感人群。魏斯曼博士还将使用定向重组 NOD小鼠T细胞受体谱系的发育分析 用内源性和移植的骨髓。这些研究将依赖于 钱博士和戴维斯博士用来追踪疾病发展的相同技术 NOD小鼠胰岛细胞特异性T细胞的表达。