Identification of Biomarkers of Autoimmunity in T1D by Novel Tools

通过新工具鉴定 T1D 自身免疫生物标志物

• 批准号: 7224767
• 负责人: Mikael Knip
• 金额: $ 13.5万
• 依托单位: UNIVERSITY OF HELSINKI
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7224767
• 关键词: Scandinavian country; autoantigens; autoimmunity; biomarker; cell line; clinical research; gene expression; genetic library; histocompatibility typing; human tissue; insulin dependent diabetes mellitus; microarray technology; molecular biology information system; pancreatic islets; polymerase chain reaction; protein sequence; protein structure; serum; transfection /expression vector

DESCRIPTION (provided by applicant): This proposal aims at the identification of novel biomarkers in type 1 diabetes (T1D) through innovative approaches. The approach of screening a phage cDNA expression library constructed from human pancreatic islets with sera from subjects with preclinical and clinical T1D followed by validation of the isolated clones on microarrays is a useful strategy for the identification of a high number of putative autoantigens. The research proposal comprises the following steps: (a) The construction of antigen phage display libraries from cDNAs generated from human pancreatic islets obtained from organ donors. A new phage display vector (pPAO) developed in one of the participating laboratories will be used for this purpose. This vector has been designed to optimize the rescue of antigenic peptides by selecting in frame coding sequences. (b) The selection of the cDNA phage library based on antibodies present in sera obtained from subjects with various stages of preclinical and clinical T1D. (c) The characterization of a large number of the isolated antigen fragments by (i) confirmation of the reactivity of the screened antigen fragments using serum antibodies from subjects with signs of beta-cell autoimmunity by protein microarray and classical immunoassays; (ii) the automated sequencing and data bank screening for the identification of the original cDNA clones from which all the positive fragments derive. (d) The validation of the general antigenic properties of the isolated antigens by challenging with sera from well characterized subjects at different stages of the disease. This step will identify the peptides providing the best profile of immune recognition. (e) The construction of a protein microarray with the selected set of antigens with diagnostic and prognostic capacity. T1D is a chronic disease affecting an increasing number of young people. This research proposal aims at the construction of a protein microarray for the detection of autoantibodies relevant for T1D. Such a microarray is expected to improve the diagnosis of T1D, to identify subtypes of the disease based on differences in the specificity of the autoimmune response, to define autoantibody signatures that may have prognostic value, and to facilitate the monitoring of disease progression or response to therapy.

描述（由申请人提供）：该提案旨在通过创新方法鉴定 1 型糖尿病 (T1D) 的新型生物标志物。使用来自临床前和临床 T1D 受试者的血清筛选由人胰岛构建的噬菌体 cDNA 表达文库，然后在微阵列上验证分离的克隆的方法是鉴定大量假定自身抗原的有用策略。该研究计划包括以下步骤： (a) 利用从器官捐献者获得的人胰岛产生的 cDNA 构建抗原噬菌体展示文库。其中一个参与实验室开发的一种新的噬菌体展示载体（pPAO）将用于此目的。该载体被设计为通过选择框内编码序列来优化抗原肽的拯救。 (b) 根据从临床前和临床 T1D 各个阶段的受试者获得的血清中存在的抗体选择 cDNA 噬菌体文库。 (c) 通过以下方式表征大量分离的抗原片段：(i) 使用来自具有β细胞自身免疫迹象的受试者的血清抗体，通过蛋白质微阵列和经典免疫测定法确认筛选的抗原片段的反应性； (ii) 自动测序和数据库筛选，用于鉴定所有阳性片段所源自的原始 cDNA 克隆。 (d)通过用来自疾病不同阶段的明确特征受试者的血清进行攻击来验证分离抗原的一般抗原特性。此步骤将鉴定提供最佳免疫识别特征的肽。 (e)用选定的一组具有诊断和预后能力的抗原构建蛋白质微阵列。 T1D 是一种影响越来越多年轻人的慢性疾病。该研究计划旨在构建蛋白质微阵列，用于检测与 T1D 相关的自身抗体。这种微阵列有望改善 T1D 的诊断，根据自身免疫反应特异性的差异识别疾病的亚型，定义可能具有预后价值的自身抗体特征，并促进疾病进展或治疗反应的监测。