APOPTOSIS:A MEANS OF IMMUNE REGULATION TO TREAT DIABETES

描述（由申请人提供）：本补助金的主要目标是 特异性诱导细胞凋亡， 自体和同种异体反应性淋巴细胞 激活内源性半胱天冬酶。这些淋巴细胞的物理消除， NOD小鼠有望消除糖尿病前期的自身免疫过程 动物和防止胰岛移植排斥在患病动物，导致 糖尿病的预防和治疗。 I型胰岛素依赖型糖尿病（IDDM）是一种慢性自身免疫性糖尿病， 这种疾病影响着全世界很多人。 进展 了解胰岛素依赖型糖尿病发病机制的分子和细胞基础， 医疗实践的改进导致了有效的治疗 预防胰岛素依赖型糖尿病的方法。尽管医学治疗取得了进步， 胰岛素依赖型糖尿病仍然是长期发病率和死亡率的主要原因。是 唯一最 肾衰竭的常见原因 需要移植的失败。 移植 的 整个 胰腺 或 纯化 胰岛 含有 产生胰岛素的β细胞有望成为实现 胰岛素依赖型糖尿病患者中的优泌肽。然而，免疫排斥限制了长期移植 生存诱导胰岛特异性耐受，无需 因此，慢性免疫抑制将提供一个重大的进步。 针对独特的胰腺β细胞抗原的T细胞发挥了最大的作用， 在胰岛细胞破坏和胰岛素依赖型糖尿病的发展中起关键作用。在这份赠款中， 应用中，我们建议诱导凋亡的自身反应和同种异体反应， 通过IL-2 R靶向递送半胱天冬酶激活分子， 治疗糖尿病。细胞凋亡是最有效的生理性细胞死亡， 调节免疫稳态和对自身抗原的耐受性。失调 凋亡过程导致自身免疫，包括糖尿病。半胱天冬酶是 调节凋亡机制的最重要的分子。Caspases 在身体的所有细胞中作为不活跃的酶原合成。的 一个半胱天冬酶分子的激活足以使细胞死亡 因为它可以通过转录激活其它半胱天冬酶（在哺乳动物中>13）。 一旦被激活，半胱天冬酶就会覆盖所有的调节过程，并将其提交给 细胞死亡。 由于激活的自身反应性和同种异体反应性T细胞 表达高亲和力IL-2 R，我们假设这些细胞可以靶向 使用IL-2 R嵌合半胱天冬酶激活蛋白进行细胞凋亡。物理 消除自身和同种异体反应性T细胞预计将导致 糖尿病前期疾病的预防和糖尿病动物的治疗 移植了同种异体胰岛。这种方法可能具有直接的临床意义。 作为嵌合蛋白的应用可以容易地大量产生 使用重组DNA技术。 本研究的具体目的包括：1）构建重组基因 编码与半胱天冬酶激活嵌合的IL-2 分子和评估 用于自身反应性和同种异体反应性的特异性细胞凋亡的嵌合蛋白 2）在体内测试嵌合蛋白以预防 糖尿病前期NOD小鼠的糖尿病; 3）研究嵌合体的能力， 蛋白质以诱导对自身抗原和同种异体抗原的耐受性， 预防胰岛移植排斥反应和自身免疫复发。

DESCRIPTION (provided by applicant): The main objective of this grant is to induce apoptosis specifically in auto and alloreactive lymphocytes by activating endogenous caspases. Physical elimination of these lymphocytes in NOD mouse is expected to abrogate the autoimmune process in prediabetic animals and to prevent islet allograft rejection in diseased animals, leading to the prevention and treatment of diabetes. Type I insulin-dependent diabetes mellitus (IDDM) is a chronic autoimmune disorder that affects a large number of people worldwide. Advances in understanding of the molecular and cellular basis of IDDM pathogenesis and improvements in medical practice have resulted in effective therapeutic approaches for the prevention of IDDM. Despite advances in medical therapies, IDDM remains a major cause of long-term morbidity and mortality. It is the single most common cause of renal failure requiring transplantation. Transplantation of whole pancreas or purified islets containing insulin-producing beta-cells promises an efficient approach to achieving euglycemia in IDDM patients. However, immune rejection limits long-term graft survival. Induction of islet-specific tolerance without the requirement for chronic immunosuppression would, therefore, offer a major advancement. T cells, directed at unique pancreatic beta-cell antigens, play the most critical role in islet cell destruction and development of IDDM. In this grant application, we propose to induce apoptosis in autoreactive and alloreactive lymphocytes by IL-2R-targeted delivery of caspase-activating molecules to treat diabetes. Apoptosis is the most potent physiological cell death that regulates immune homeostasis and tolerance to self-antigens. Dysregulation of the apoptotic process leads to autoimmunity, including diabetes. Caspases are the most important molecules that regulate the apoptotic machinery. Caspases are synthesized as inactive proenzymes in all the cells of the body. The activation of one caspase molecule is sufficient to commit the cell to death since it can activate the other caspases (>13 in mammals) by transcatalysis. Once activated, caspases override all the regulatory processes and commit the cell to death. Inasmuch as activated autoreactive and alloreactive T cells express high affinity IL-2R, we hypothesize that these cells can be targeted for apoptosis using IL-2R chimeric with caspase-activating proteins. Physical elimination of auto and alloreactive T cells is expected to result in the prevention of disease in prediabetic and treatment in diabetic animals transplanted with islet allografts. This approach may have direct clinical application as chimeric proteins can easily be generated in large quantities using recombinant DNA technology. The Specific Aims of this proposal include: 1) constructing recombinant genes encoding IL-2 chimeric with caspase-activating molecules and assessing chimeric proteins for specific apoptosis of autoreactive and alloreactive lymphocytes in vitro; 2) testing chimeric proteins in vivo for the prevention of diabetes in prediabetic NOD mice; 3) investigating the ability of chimeric proteins to induce tolerance to autoantigens and alloantigens for the prevention of islet allograft rejection and recurrence of autoimmunity.