Identification of relevant peptides involved in the initiation and progression of autoimmune diabetes

鉴定参与自身免疫性糖尿病发生和进展的相关肽

• 批准号: 10246429
• 负责人: EMIL Raphael UNANUE
• 金额: $ 43.48万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-20 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10246429
• 关键词: Alleles; Antigens; Autoimmune; Autoimmune Diabetes; Autoimmune Process; Beta Cell; Binding; Biochemical; Biological Assay; Blood; CD4 Positive T Lymphocytes; Cells; Cellular biology; Cytoplasmic Granules; Dendritic Cells; Development; Diabetes Mellitus; Disease; Endocrinologist; Evaluation; Glucose; Goals; Human; Hybridomas; Immunologics; Immunologist; In Vitro; Inbred NOD Mice; Individual; Insulin; Insulin-Dependent Diabetes Mellitus; Investigation; Islets of Langerhans; Lead; Lymphoid Tissue; Mass Spectrum Analysis; Measures; Mouse Strains; Mus; Non obese; Pathogenesis; Peptides; Peripheral; Phagocytes; Population; Prediabetes syndrome; Process; Proteins; Reaction; Sampling; Seminal; Site; Source; Spleen; Structure; T-Lymphocyte; Techniques; Technology; Testing; Thymus Gland; Tissues; Urine; Validation; Vesicle; autoreactivity; diabetic; diabetogenic; experience; experimental study; granule cell; immunogenic; in vivo; instrumentation; islet; lymph nodes; macrophage; non-diabetic; novel; response; secondary lymphoid organ

Project Summary We propose a collaborative project involving immunologists experienced in autoimmune diabetes, mass-spectrometrists, and endocrinologists-diabetologists. Our combined efforts are to specifically identify relevant peptides from beta cell proteins involved in the immunopathogenesis of type 1 diabetes. The project combines experimental studies in diabetes-prone mice that should lead to targeted evaluation of human samples. The ultimate goal is to identify peptides that form the substrate for the autoreactive MHC-II response that initiates and perpetuates the process. Important is to identify relevant diabetogenic antigens at different stages of the autoimmune reaction from pre-diabetes to the initial progression: it will include their characterization, source in beta cells, involvement in the disease process, and sites of presentation. Aim 1 consists of an identification of novel peptides derived from beta cell granules of both mice and humans. We examine vesicles isolated from beta cells with a major focus on crinosomes. Crinosomes and insulin dense core granules will be isolated during different stages of diabetes development, their peptides will be isolated and examined by sophisticated mass spectrometry with state of the art instrumentation and technology. We will place emphasis in unique structural or post translational changes in peptides and whether the panoply of them changes as the beta cell progresses into active diabetes. We also will examine vesicles and exocytosed products from human islets from recently diseased individuals. Aim 2 will be an immunological validation of novel peptides identified by mass spectrometry. Immunological and biochemical assays will be used to validate the ability of beta cell derived peptides to bind to I-Ag7 and activate T cells in vitro and in vivo. Aim 3 will be the Identification and validation of the MHC-II-bound peptidome in islets and secondary lymphoid organs. We plan to examine the peptidome eluted from MHC-II molecules expressed by phagocytes obtained from islets, peripheral lymph nodes, and spleens of the NOD mouse and to characterize/validate them as in Aims 1/2. This is the seminal test to prove an immunogenic peptide, that is, it's a positive identification bound in vivo to MHC-II alleles.

项目摘要 我们提出了一个合作项目，涉及自身免疫中经历的免疫学家 糖尿病，群众光谱师和内分泌学家 - 糖尿病学家。我们的综合努力是 从涉及的β细胞蛋白中明确鉴定相关的肽 1型糖尿病的免疫致病发生。该项目结合了实验研究 易糖尿病的小鼠应导致对人类样本的靶向评估。最终 目标是识别形成自动反应性MHC-II响应底物的肽 启动并永久化过程。重要的是要鉴定相关的糖尿病性抗原 自身免疫反应的不同阶段从糖尿病前期到初始进展：它将 包括它们的表征，β细胞中的来源，参与疾病过程和部位 演讲。 AIM 1由鉴定来自β细胞颗粒的新肽的鉴定 老鼠和人类。我们检查从β细胞中分离出的囊泡，主要关注 灌木体。在不同阶段将隔离沟以及胰岛素密集的核颗粒 在糖尿病发育中，它们的肽将通过复杂的质量隔离和检查 具有最先进的仪器和技术状态的光谱法。我们将重点放在 肽的独特结构或后翻译变化以及它们的全盘是 随着β细胞发展为活性糖尿病的变化。我们还将检查囊泡和 来自最近患病个体的人类胰岛的胞外产品。 AIM 2将是对质量鉴定的新肽的免疫学验证 光谱法。免疫学和生化测定将用于验证β的能力 细胞衍生的肽与I-AG7结合并在体外和体内激活T细胞。 AIM 3将是胰岛中MHC-II结合肽组的识别和验证，并且 继发性淋巴器官。我们计划检查从MHC-II洗脱的肽组 从胰岛，外周淋巴结和脾脏获得的吞噬细胞表达的分子 点头鼠标的表征/验证它们如AIMS 1/2所示。这是开创性的测试 证明一种免疫原性肽，也就是说，它是在体内与MHC-II结合的阳性鉴定 等位基因。