Measurement of Beta Cell Death in Diabetes

描述(申请人提供)：所有形式的糖尿病都以产生胰岛素的细胞死亡为特征。在1型糖尿病(T1D)中，这导致依赖外源性胰岛素生存。此外，在T2D和胰岛或胰岛缺失的个体中存在细胞丢失 同种异体胰腺移植。已开发出可调节T1D病程的新疗法，但这些疗法的有效应用需要更清楚地了解病理过程，包括细胞死亡的时间和诱因，因为这些疗法并未导致疾病的永久缓解。然而，细胞的死亡是沉默的--在体内无法检测到它，直到它进展到代谢功能受损的程度。这项拟议工作的目标是通过开发一种能够在体内定量测量细胞死亡的方法来满足这一需求。我们提出的分析是基于这样一种理解，即表观遗传修饰控制着胰岛素基因的组织特异性表达。由于胰岛素的唯一重要来源是细胞，血清或血浆中去甲基化的胰岛素DNA反映了细胞的死亡和DNA的释放。我们开发了一种新的检测方法，通过在小鼠中用套式PCR反应来测量去甲基化的胰岛素DNA，并验证了它在两个T1D模型中检测细胞死亡的能力。在NOD小鼠中，在血糖水平没有升高的情况下，可以在血清中检测到脱甲基化胰岛素DNA水平的增加。我们现在建议开发这一方法，用于人类样本。在第一个目标中，我们将优化检测人血清中去甲基化胰岛素编码DNA的方法。我们将选择检测去甲基化胰岛素DNA的引物以及套式聚合酶链式反应的条件。然后，我们将对该分析的敏感性和重复性进行表征，并测试血清或血浆是否为最佳起始材料。我们还将确定DNA总量如何影响检测结果。在第三个目标中，我们将确定健康对照受试者、长期存在T1D但未检测到C肽的患者以及新发疾病的基线反应。我们将测试化验结果在儿童中是否有所不同，因为T1D最常在儿童中被诊断出来。一旦这一检测方法被优化，它可能会用于临床环境中研究细胞死亡，例如在T1D高危人群中(在TrialNet自然病史研究中)、免疫治疗试验、跟踪胰岛和胰腺移植等。此外，这项测试的未来应用将包括对T2D的研究和了解对人类细胞生长和再生的控制。 公共卫生相关性：所有形式的糖尿病都与细胞死亡有关，但我们没有测量体内细胞死亡的手段。我们建议开发一种新的方法，基于细胞特异性胰岛素DNA的鉴定来满足这一需求。该检测方法将广泛用于了解糖尿病患者的死亡情况，以及开发治疗和预防糖尿病的新疗法。

DESCRIPTION (provided by applicant): All forms of diabetes are characterized by the death of insulin producing ¿ cells. In Type 1 diabetes (T1D), this leads to the reliance on exogenous insulin for survival. In addition, there is ¿ cell loss in T2D and in individuals who lose islet or pancreas allografts. New therapies have been developed that can modulate the course of T1D but the effective application of these therapies requires a clearer understanding of the pathologic processes including the timing and precipitants of ¿ cell death because these therapies have not led to permanent remission of disease. However, death of ¿ cells is silent - it cannot be detected in vivo until it has progressed to such an extent that metabolic function is impaired. The goal of the proposed work is to address this need by developing an assay that can measure ¿ cell death in a quantitative manner in vivo. Our proposed assay is based on the understanding that epigenetic modifications control the tissue specific expression of the insulin gene. Since the only significant source of insulin is ¿ cells, demethylated insulin DNA in the serum or plasma reflects the death of ¿ cells and release of their DNA. We have developed a novel assay that measures demethylated insulin DNA with a nested PCR reaction in mice and have validated its ability to detect ¿ cell death in 2 models of T1D. In NOD mice, increased levels of demethylated insulin DNA were detectable in the serum at a time when the glucose level was not elevated. We now propose to develop this approach for use with human samples. In the first aim we will optimize the assay for detection of demethylated insulin-coding DNA in human serum. We will select primers for detection of demethylated insulin DNA as well as the conditions for the nested PCR reactions. We will then characterize the assay including the sensitivity and reproducibility, and test whether serum or plasma is optimal starting material. We will also determine how the total amount of DNA affects the assay results. In the 3rd aim, we will determine baseline responses in healthy control subjects, patients with long standing T1D without detectable C-peptide, and new onset disease. We will test whether the assay results vary in childhood since T1D is most frequently diagnosed in children. Once this assay is optimized, it may have use in clinical settings to study ¿ cell death such as in individuals at hig risk for development of T1D (in the TrialNet Natural History Study), immune therapy trials, tracking islet and pancreas transplants, and others. Moreover, future application of this assay will include studies of T2D and understanding control of human ¿ cell growth and regeneration. PUBLIC HEALTH RELEVANCE: All forms of diabetes involve death of ¿ cells but we do not have a means of measuring ¿ cell death in vivo. We propose to develop a new assay that will address this need based on identification of ¿ cell specific insulin DNA. The assay will have broad use for understanding ¿ death in diabetes and for developing new therapies to treat and prevent diabetes.