DNA Immunizations with GAD65 to Induce Tolerance

1型糖尿病是一种自身免疫性疾病，其特征是T细胞介导的朗格汉斯胰岛胰岛素产生细胞的破坏。作为胰岛素依赖型糖尿病(IDDM)的小鼠模型，非肥胖糖尿病(NOD)小鼠已成为人类疾病的有用模型，因为该疾病的许多特征都是共同的。在小鼠和人类中，糖尿病的发展之前都有大量的胰岛淋巴细胞浸润，包括CD4+，CD8+T细胞，树突状细胞和单核细胞。最近发现在幼年NOD小鼠的脾中最先检测到T细胞对神经酶谷氨酸脱羧酶65(GAD65)的反应，最早在4周就检测到反应。此外，研究还发现，如果通过胸腺内或静脉注射使NOD对GAD65耐受，则可预防糖尿病。我们最近证明，用单一GAD65多肽冲击的树突状细胞治疗NOD小鼠可以预防糖尿病，这是通过启动GAD65特异性Th2反应来实现的。使用任何一种质粒DNA的基因免疫，取决于免疫途径，可以导致Th1或Th2反应。在非糖尿病模型中，我们有初步证据表明，转基因蛋白的细胞定位也深刻影响免疫反应的性质。当该蛋白是细胞质时，可获得较强的Th1反应和CTL反应，而当卵蛋白(OVA)基因枪免疫后，该蛋白的分泌则以Th2为主。因此，可以对基因免疫进行调整，以诱导所需的反应。在NOD小鼠患糖尿病的情况下，已经证明，对特定GAD65肽的Th2反应的诱导可以导致破坏性胰岛素炎的停止和糖尿病的预防。本研究的目的是：1)构建允许GAD65作为分泌蛋白和跨膜蛋白在细胞质中表达的GAD65 cDNA质粒；2)鉴定这三种GAD65构建体免疫NOD和非糖尿病易感菌株后所引发的免疫反应；3)确定DNA免疫GAD65构建体是否可以预防糖尿病和胰腺炎的发生。

Type 1 diabetes is an autoimmune disease characterized by T cell- mediated destruction of the insulin-producing cells of the islets of Langerhans. The murine model of insulin dependent diabetes mellitus (IDDM), the non-obese diabetic (NOD) mouse has served as a useful model of the human disease since many of the features of the disease are shared. In both mouse and human the development of diabetes is preceded by heavy islet infiltration with lymphoid cells, including CD4+, CD8+ T cells, dendritic cells and monocytes. Recently it was found that T cell responses to the neuronal enzyme glutamic acid decarboxylase 65 (GAD65) were among the first to be detected in the spleen of young NOD mice, with responses being detected as early as 4 weeks. In addition it was found that if NOD were made tolerant to GAD65, by intra-thymic or intravenous injection, diabetes was prevented. We have recently shown that treatment of NOD mice with dendritic cells pulsed with a single GAD65 peptide can prevent diabetes and that this occurs through the initiation of a GAD65-specific Th2 response. Genetic immunization using either plasmid DNA, depending on the route of immunization, can lead to either a Th1 or Th2 response. We have preliminary evidence in a non-diabetes model that the cellular localization of the transfected protein also profoundly influences the nature of the immune response. Strong Th1 responses, with CTL, are obtained when the protein is cytoplasmic, whereas a Th2 dominated response is seen when the protein is secreted following gene gun immunization of ovalbumin (OVA) cDNA. Genetic immunization can thus be tailored to induce the desired response. In the case of diabetes in the NOD mouse, it has been shown that the induction of Th2 responses to specific GAD65 peptides can lead to a halt in the destructive insulitis and the prevention of diabetes. In this proposal are: 1) To construct GAD65 cDNA plasmids which will allow expression of GAD65 in the cytoplasm, as a secreted protein and as a transmembrane protein; 2) To characterize the immune response initiated following immunization of NOD and non diabetes-prone strains with the three GAD65 constructs; 3) To determine whether DNA immunization GAD65 constructs prevent the initiation of diabetes and insulitis.