PATHOGENESIS AND PREVENTION OF IDDM

IDDM 的发病机制和预防

• 批准号: 2838169
• 负责人: HUGH O MCDEVITT
• 金额: $ 83.64万
• 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/2838169
• 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汇集五个 微生物学和免疫学系的研究人员，以及 病理学制定了一系列合作、研究项目。这些 研究项目旨在应用分子技术， 免疫学来了解CD 4 + T细胞的发育和特异性， NOD小鼠I型糖尿病对胰岛细胞抗原的反应， 表达人IDDM易感基因的NOD转基因模型， 开发改变免疫反应以预防胰岛素依赖型糖尿病的新方法。 Drs.简月秀将开发特异性T细胞染色试剂， 产生具有共价结合的可溶性生物素化的I-A/g7分子， 与荧光链霉亲和素偶联的肽。的肽表位 谷氨酸脱羧酶和前胰岛素原将在Dr. McDevitt的实验室（在DK-51667的支持下）。麦克德维特医生会 利用HLA-DQ 8、7和6转基因NOD小鼠来鉴定 人GAD 65和人PPI的免疫缺陷肽表位 通过IDDM易感（DQ 8）、7和6鉴定免疫显性肽 由IDDM易感者（DQ 8）呈递的人GAD 65和人PPI的表位 和耐药（DQ 7，6）等位基因。相同的T细胞受体染色 将开发用于检测GAD和PPI特异性、DQ 8 限制性T细胞在糖尿病前期和近期发病的糖尿病患者。在 钱博士和马克·戴维斯博士合作， 反应（PCR）将用于对T细胞受体进行表征和测序 来自从胰岛分离的单个T细胞的V α和V β序列 14-18日龄NOD小鼠。这些T细胞受体V β和V α 序列具有可检测的共享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 在转染的T细胞系和转基因小鼠中表达， 确定其功能。将检测转基因T细胞 使用一组重组鼠胰岛细胞测定其抗原特异性 在麦克德维特博士的实验室里生产的抗原克拉布特里博士将制作 表达能够结合和抑制 仅当与修饰的环孢菌素A复合时，钙调神经磷酸酶。改性 环孢菌素A将被用于抑制T细胞中的信号传导，以抑制T细胞的增殖。 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。与博士合作。 麦克德维特，克拉布特里博士将把修改后的亲环素基因导入 NOD小鼠品系。修饰的环孢素A将用于阻断T细胞 在出生后的不同时间点发出信号以诱导对 胰岛细胞抗原韦斯曼博士和朱迪思·静流博士将分离出 造血干细胞分析全骨髓（WBM）的影响 与造血干细胞（HSC）移植在替代 内源性淋巴区室H2匹配或同基因骨髓将 被利用这些实验将探索利用骨骼的潜力 骨髓移植预防I型糖尿病的发展 易受影响的个体。韦斯曼博士还将使用靶向重组 分析NOD小鼠T细胞受体库的发育 自体骨髓和移植骨髓这些研究将依赖于 钱博士和戴维斯博士用同样的技术来追踪 的胰岛细胞特异性T细胞。