ANIMAL MODELS OF HYPERTHYROIDISM

描述(申请人提供)：Graves病是一种常见的人类自身免疫性疾病，由刺激TSH受体(TSHR)的自身抗体引起。目前还没有这种疾病的自发动物模型。我们将使用两种诱导小鼠模型：“裸”TSHR-DNA疫苗接种和“Shimojo”方法(注射表达TSHR的成纤维细胞)。利用这些模型，我们将分析TSHR免疫反应中的以下问题： 1.微生物或缺乏自身耐受性的影响：我们将研究TSHR-DNA疫苗联合微生物制品诱导的TSHR抗体的效价和功能活性，以及对TSHR不能获得自身耐受性的TSHR基因敲除小鼠。 2.细胞因子和细胞相互作用：野生型小鼠和B细胞敲除小鼠将被用来确定(A)TSHR-蛋白攻击的脾细胞产生哪些细胞因子；(B)CD4+或CD8+T细胞是否参与反应；(C)如果需要B细胞来诱导TSHR特异的记忆T细胞， 3.TSHR特异性T细胞的识别表位：合成的TSHR多肽将用于确定T细胞识别的表位：(A)从TSHR-DNA疫苗接种的BALB/c小鼠克隆的；(B)对TSHR缺乏耐受性的TSHR基因敲除小鼠；(C)易患(DR3)或保护(DQ6)Graves病(DQ6)的人类白细胞抗原转基因小鼠。自然加工的TSHR多肽也将被研究， 4.共刺激信号：将探索共刺激分子的作用：(A)在体外，通过使用抗体来阻断CD40/CD40-1配体和CD28/B7-1/2的相互作用；(B)在体内使用CD28、CD40、B7-1或B7-2基因被敲除的小鼠；(C)在体内通过注射抗B7-1抗体(或对照)和TSHR-成纤维细胞(表达B7-1)。 5.TSHR裂解和脱落的作用：我们将检验TSHR裂解之后高度糖化的A亚基脱落在对TSHR的免疫反应中发挥作用的假设：-(A)使用编码非裂解或脱落TSHR的DNA；(B)检测高度糖基化的TSHR A亚单位与糖基化程度较低的抗原(如TPO)与甘露糖受体的结合，甘露糖受体捕捉糖基化生物并呈递给T细胞。

DESCRIPTION (provided by applicant): Graves' disease is a common human autoimmune disorder caused by autoantibodies that stimulate the TSH receptor (TSHR). There are no spontaneous animal models of the disease. We will use two induced mouse models; "naked" TSHR-DNA vaccination, and the "Shimojo" approach (injecting TSHR-expressing fibroblasts). With these models, we will analyze the following issues in the immune response to the TSHR:- 1. Influence of micro-organisms or lack of self tolerance: We will study the titers and functional activity of TSHR antibodies induced by TSHR-DNA vaccination in combination with microbial products, as well as in TSHR-knockout mice that cannot acquire self tolerance to the TSHR. 2. Cytokine and cellular interactions: Wild type mice and B-cell knockout mice will be used to determine (a) which cytokines are produced by splenocytes challenged with TSHR-protein; (b) whether CD4+ or CD8+ T cells are involved in the response; (c) if B cells are required to induce memory T cells specific for the TSHR, 3. Epitopes recognized by TSHR-specific T cells: Synthetic TSHR peptides will be used to determine the epitopes recognized by T cells (a) cloned from TSHR-DNA vaccinated BALB/c mice; (b) TSHR knockout mice that lack tolerance to the TSHR; (c) mice transgenic for HLA that predispose (DR3) or protect against (DQ6) Graves' disease. Naturally processed TSHR peptides will also be studied, 4. Co-stimulatory signals: The role of co-stimulatory molecules will be explored (a) in vitro by using antibodies to block CD40/CD40-1igand and CD28/B7-1/2 interactions; (b) in vivo using mice with disrupted genes ("knockouts") for CD28, CD40, B7-1 or B7-2; (c) in vivo by injecting mice with anti-B7-1 (or control) together with TSHR -fibroblasts (that express B7-1). 5. Role of TSHR cleavage and shedding: We will test the hypothesis that TSHR cleavage followed by shedding of the heavily glycosylated A subunit plays a role in the immune response to the TSHR:- (a) using DNA encoding a non-cleaving or shedding TSHR; (b) examining binding of the heavily glycosylated TSHR A-subunit vs less glycosylated antigens (such as TPO) to the mannose receptor that captures glycosylated organisms for presentation to T cells.