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-在遗传风险人群中减少胰岛素依赖型糖尿病的试验（TRIGR）。这项研究将产生重要的新信息的主要环境候选危险因素在1型糖尿病（T1 D）的发病机制，利用TRIGR（饮食干预）的设置。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以及胰岛自身免疫诱导之间的关联。胰岛自身免疫定义为在所测量的四种糖尿病相关自身抗体中至少两种抗体重复阳性。 目前的研究将基于TRIGR队列中收集的生物样本。TRIGR研究是一项国际双盲随机临床试验，2002-2007年在15个国家招募了2159名具有HLA赋予的疾病易感性和T1 D一级亲属的婴儿。在TRIGR中，将广泛水解的酪蛋白配方与常规牛奶配方进行了比较。对所有受试者进行随访，直至最小的孩子年满10岁。每隔3至12个月采集一次血样。 本研究采用队列或巢式病例对照设计，对血清中脂肪酸组成、25-OH-维生素D浓度、病毒抗体、牛乳抗体、牛胰岛素抗体、25-OH-维生素D浓度进行了测定，其中脂肪酸组成测定采用气相色谱法，25-OH-维生素D浓度测定采用荧光微粒免疫法，病毒抗体测定采用空斑中和法和酶免疫法，牛乳抗体测定采用酶联免疫吸附法，牛胰岛素抗体测定采用放射性和冷胰岛素竞争性放射免疫法，25-OH-维生素D浓度测定采用放射免疫法。以及细胞因子和趋化因子。当研究对柯萨奇病毒B、β-乳球蛋白和牛胰岛素的细胞介导的应答时，我们使用冷冻的外周血单核细胞（PBMC），我们使用RT-qPCR方法在牛乳蛋白刺激的PBMC中从其测量Treg Th 1、Th 2和Th 17标志物、FoxP 3、CTLA-4、IFN-g、IL-4、IL-5、IL-13、IL-17和IL-22。我们还将从冷冻的PBMC中分析负责调节性T细胞发育的两个基因（FOXP 3，IL-2）的表观遗传调节。这些方法在我们的实验室中非常成熟和运作良好。从TRIGR收到样品后，可以直接开始分析。 该应用涵盖了血清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等分。

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.