Identification of Islet Proteins Stimulatory for T Cells from Diabetes Patients

糖尿病患者 T 细胞刺激性胰岛蛋白的鉴定

• 批准号: 7780170
• 负责人: JERRY P PALMER
• 金额: $ 46.08万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-03 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7780170
• 关键词: Adult; Affect; Age; Antigens; Arts; Autoantibodies; Autoimmune Diabetes; Autoimmune Diseases; Autoimmune Process; Autoimmunity; Beta Cell; Biochemical; Biological Assay; Cells; Cellular biology; Charge; Collaborations; Data; Development; Diabetes Mellitus; Educational workshop; Europe; Failure; Human; Hydrophobicity; Hyperglycemia; Immunoblotting; Individual; Insulin; Insulin-Dependent Diabetes Mellitus; Intervention; Islet Cell; Ketoses; Ketosis; Lead; Masks; Mediating; Methods; Molecular Weight; Monitor; Non-Insulin-Dependent Diabetes Mellitus; North America; Obesity; Pathogenesis; Patients; Population; Protein Analysis; Protein Region; Proteins; Proteomics; Sensitivity and Specificity; Shotguns; Symptoms; T-Lymphocyte; Techniques; Therapy Clinical Trials; United States National Institutes of Health; abstracting; autoreactive T cell; base; cell type; innovation; insight; interest; islet; novel; novel therapeutics; physical property; public health relevance; two-dimensional

DESCRIPTION (provided by applicant): Identification of Islet Proteins Stimulatory for T cells from Diabetes Patients. Abstract. Type 1 Diabetes Mellitus (T1DM) is a cell-mediated autoimmune disease directed against the beta cells whereas Type 2 diabetes (T2DM) is not autoimmune. In recent years, a group of autoimmune phenotypic T2DM patients have been described suggesting that T cell mediated autoimmunity may be associated with the pathogenesis and progression of beta cell failure in both T1DM and T2DM. In two masked NIH sponsored workshops, our cellular immunoblotting T cell assay, which uses isolated human islets, has been validated to distinguish T1DM patients from controls with excellent specificity and sensitivity. Interestingly, T cells from T1DM patients respond to select molecular weight regions of islet proteins (preliminary data) as identified using cellular immunoblotting. Therefore, we hypothesize that individual islet proteins are responsible for the T cell reactivity observed in autoimmune diabetes patients and that identifying these proteins will provide insight into T cell reactivity in autoimmune diabetes. Previously, islet proteins utilized in T cell assays were identified based on autoantibody reactivity and not direct T cell reactivity. What is currently needed is the identification of islet proteins that directly stimulate T cells from autoimmune diabetes patients. In collaboration with experts in proteomics, we propose to isolate and identify islet proteins stimulatory to T cells from autoimmune diabetes patients using the following three approaches: a). Isolation of T cell stimulatory proteins from molecular weight regions of interest, identified by cellular immunoblotting, using electro-elution of one and two-dimensional SDS-PAGE followed by tandem mass spectrometric (MS/MS) identification of proteins. b). Sequential biochemical approaches to isolate proteins to near homogeneity followed by MS/MS identification of the purified proteins. c). A novel orthogonal separation strategy to identify proteins that activate T cells. Once the T cell stimulatory islet proteins are isolated and identified, we will proceed to verify the isolated islet proteins by analysis of T cell reactivity in autoimmune diabetes patients. Identification of individual antigenic islet cell proteins that directly stimulate T cells from diabetes patients could lead to the development of new antigen based assays and intervention strategies, and provide innovative methods to follow and assess new therapeutic trials. PUBLIC HEALTH RELEVANCE: Autoimmune diseases affect 5-7% of the adult population in North America and Europe. We propose to identify and isolate the islet proteins which stimulate the autoreactive T cells in patients who develop autoimmune diabetes (type 1 diabetes and a subset of type 2 diabetes). The results of these studies will have major implications toward furthering our understanding of the development of autoimmune disease. Subsequent identification of potential islet proteins will be useful in the development of new antigen based intervention strategies and new assays to monitor immunomodulatory therapies for treating autoimmune diabetes.

描述（由申请人提供）：从糖尿病患者中鉴定刺激T细胞的胰岛蛋白。抽象的。1型糖尿病（T1 DM）是一种针对β细胞的细胞介导的自身免疫性疾病，而2型糖尿病（T2 DM）不是自身免疫性的。近年来，已描述了一组自身免疫表型T2 DM患者，表明T细胞介导的自身免疫可能与T1 DM和T2 DM中β细胞衰竭的发病机制和进展相关。在两个由NIH赞助的掩蔽研讨会中，我们的细胞免疫印迹T细胞测定法（使用分离的人胰岛）已被验证可区分T1 DM患者和对照组，具有出色的特异性和灵敏度。有趣的是，来自T1 DM患者的T细胞对胰岛蛋白的选择分子量区域（初步数据）有反应，如使用细胞免疫印迹鉴定的。因此，我们假设，个别胰岛蛋白是负责在自身免疫性糖尿病患者中观察到的T细胞反应性，并确定这些蛋白质将提供深入了解自身免疫性糖尿病中的T细胞反应性。以前，T细胞测定中使用的胰岛蛋白是基于自身抗体反应性而不是直接T细胞反应性来鉴定的。目前需要的是鉴定直接刺激自身免疫性糖尿病患者T细胞的胰岛蛋白。与蛋白质组学专家合作，我们提出使用以下三种方法从自身免疫性糖尿病患者中分离和鉴定刺激T细胞的胰岛蛋白质：从感兴趣的分子量区域分离T细胞刺激蛋白，通过细胞免疫印迹鉴定，使用一维和二维SDS-PAGE的电洗脱，然后进行蛋白质的串联质谱（MS/MS）鉴定。B）。连续的生物化学方法将蛋白质分离至接近同质，然后对纯化的蛋白质进行MS/MS鉴定。（c）。一种新的正交分离策略来识别激活T细胞的蛋白质。一旦T细胞刺激性胰岛蛋白被分离和鉴定，我们将通过分析自身免疫性糖尿病患者的T细胞反应性来验证分离的胰岛蛋白。直接刺激糖尿病患者T细胞的单个抗原性胰岛细胞蛋白的鉴定可能导致新的基于抗原的测定和干预策略的开发，并提供创新方法来跟踪和评估新的治疗试验。 公共卫生相关性：自身免疫性疾病影响北美和欧洲5-7%的成年人口。我们建议识别和分离刺激自身免疫性糖尿病患者（1型糖尿病和2型糖尿病的一个子集）的自身反应性T细胞的胰岛蛋白。这些研究的结果将对进一步了解自身免疫性疾病的发展产生重大影响。潜在的胰岛蛋白的后续鉴定将有助于开发新的基于抗原的干预策略和新的检测方法，以监测用于治疗自身免疫性糖尿病的免疫调节疗法。