Effect of early diet and virus infections on immune regulation and the development islet autoimmunity -Trial to Reduce IDDM in the Genetically at Risk (TRIGR)

描述（由申请人提供）：早期饮食和病毒感染对免疫调节和胰岛自身免疫发育的影响——降低遗传风险（TRIGR）中IDDM的试验。该研究将利用TRIGR（饮食干预）的设置，为1型糖尿病（T1D）发病机制中的主要环境候选危险因素提供重要的新信息。目的是评估n-3脂肪酸、维生素D、牛奶暴露和病毒感染与免疫调节和炎症指标之间的关系，以及胰岛自身免疫的诱导。胰岛自身免疫定义为在测量的四种糖尿病相关自身抗体中至少有两种重复呈阳性。目前的研究将以TRIGR队列中收集的生物样本为基础。TRIGR研究是一项国际双盲随机临床试验，2002-2007年间在15个国家招募了2159名具有hla相关疾病易感性和一级亲属患有T1D的婴儿。在TRIGR中，广泛水解酪蛋白配方与普通牛奶配方相比。所有的研究对象都会被跟踪到最小的孩子10岁。每隔3至12个月采集一次血样。我们在队列或巢式病例对照设计中测量血清：用气相色谱法测定脂肪酸组成；25- oh -维生素D浓度化学发光微球免疫分析法；病毒抗体与斑块中和和酶免疫测定；酶联免疫吸附法测定牛奶抗体用放射性胰岛素和冷胰岛素竞争放射免疫法测定日粮牛胰岛素抗体使用Milliplex MAP试剂盒检测细胞因子和趋化因子。在研究细胞对柯萨奇病毒B、ß-乳球蛋白和牛胰岛素的应答时，我们使用冷冻外周血单个核细胞（PBMC），利用RT-qPCR方法检测牛奶蛋白刺激的外周血单个核细胞中Treg Th1、Th2和Th17标记物FoxP3、CTLA-4、IFN-g、IL-4、IL-5、IL-13、IL-17和IL-22。我们还将分析冷冻pbmc中负责调节性t细胞发育的两个基因（FOXP3, IL-2）的表观遗传调控。这些方法在我们的实验室中已经非常完善和运行良好。从TRIGR收到样品后，可以直接开始分析。此应用程序涵盖了血清25- oh -维生素D浓度，血清脂肪酸，免疫调节，炎症标志物和病毒感染分析的费用。它还包括协调、数据收集、管理和统计分析以及本研究的样本收集和计算。

DESCRIPTION (provided by applicant): Effect of early diet and virus infections on immune regulation and the development islet autoimmunity -Trial to Reduce IDDM in the Genetically at Risk (TRIGR). The study will yield important new information about the main environmental candidate risk factors in the pathogenesis of type 1 diabetes (T1D) utilizing the setup of TRIGR (dietary intervention). The aim is to evaluate associations between n-3 fatty acids, vitamin D, cow's milk exposure and viral infections with indicators of immune regulation and inflammation as well as induction of islet autoimmunity. Islet autoimmunity is defined as repeated positivity fo at least two diabetes-associated autoantibodies out of four ones measured. The current study will be based on biosamples collected in the TRIGR cohort. TRIGR study is an international double-blind randomized clinical trial of 2159 infants with HLA-conferred disease susceptibility and a first-degree relative with T1D recruited between 2002-2007 in 15 countries. In TRIGR an extensively hydrolyzed casein formula is compared to regular cow's milk based one. All subjects are followed until the youngest child will be 10-year-old. Blood samples are collected at 3 to 12 months' intervals. We measure from serum in cohort or nested case-control design: fatty acid composition with gas chromatography; 25-OH-vitamin D concentration with chemiluminescent mircoparticle immunoassay; virus antibodies with plaque neutralization and enzyme immune assay; cow's milk antibodies with enzyme-linked immunosorbent assay; antibodies to dietary bovine insulin by using the competitive radio-immunoassay with radioactive and cold insulin; and cytokines and chemokines with the Milliplex MAP kit. When studying cell-mediated responses to Coxsackievirus B, ß-lactoglobulin and bovine insulin, we use frozen peripheral blood mononuclear cells (PBMC), from which we measure Treg Th1, Th2 and Th17 markers, FoxP3, CTLA-4, IFN-g, IL-4, IL-5, IL-13, IL-17 and IL-22, using RT-qPCR methodology in cow's milk protein stimulated PBMCs. We will also analyze from frozen PBMCs the epigenetic modulation of two genes (FOXP3, IL-2) responsible for the development of regulatory T-cells. The methods are very well established and well-functioning in our laboratories. The analysis can start straight after the samples are received from TRIGR. This application covers the costs for the analysis of serum 25-OH-vitamin D concentration, serum fatty acids, immune regulation, inflammatory markers, and virus infections. It also covers coordination, data collection, management and statistical analysis as well as sample collection and aliquation for the current study.