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 我们提出了一个有自身免疫经验的免疫学家参与的合作项目。 糖尿病、质谱学家和内分泌学家--糖尿病专家。我们的共同努力是 从贝塔细胞蛋白中具体鉴定相关多肽 1型糖尿病的免疫发病机制。该项目结合了 易患糖尿病的小鼠，应导致对人类样本进行有针对性的评估。终极的 目标是确定构成自身反应MHC-II反应底物的多肽 启动并使这个过程永久化。重要的是确定相关的糖尿病致病抗原 从糖尿病前期到最初进展的自身免疫反应的不同阶段：它将 包括它们的特征、来源、在疾病过程中的参与和部位 展示的方式。 目的1包括鉴定来源于β细胞颗粒的新多肽 无论是老鼠还是人类。我们检查了从β细胞中分离出的囊泡，主要集中在 肌小体。将在不同的阶段分离出隐小体和胰岛素致密核心颗粒 在糖尿病的发展过程中，他们的多肽将被分离并通过复杂的质量进行检查 采用最先进的仪器和技术进行光谱分析。我们将把重点放在 多肽的独特结构变化或翻译后变化以及它们的全貌 随着β细胞进展为活动性糖尿病而发生变化。我们还将检查水泡和 从新近患病的人的胰岛中排出的产物。 目标2将是通过质谱仪鉴定的新多肽的免疫学验证 光谱分析。将使用免疫学和生化分析来验证贝塔病毒的能力 细胞衍生的多肽与I-Ag7结合并在体外和体内激活T细胞。 目标3将是鉴定和验证胰岛中MHC-II结合的多肽 次级淋巴器官。我们计划检查从MHC-II洗脱出来的多肽 从胰岛、外周淋巴结和脾获得的吞噬细胞表达的分子 并像AIMS 1/2中那样对它们进行表征/验证。这是对 证明了一种免疫原肽，即它是体内与MHC-II结合的阳性鉴定 等位基因。