Non-Human Primate Model of Gluten-Sensitive Enteropathy

描述(申请人提供)：在人类中，面筋敏感型肠病(GSE)通常在遗传上易患乳糜泻(CD)的个体中发现，在恒河猴和人类中都可以由含面筋的饮食诱发。我们最近进行了一项实验，让对面筋敏感的猕猴和对照组的猕猴分别喂以含面筋的饮食和不含面筋的饮食。基于停止饮食中的面筋，GSE猕猴实现了完全恢复。此外，我们确定了2个DRB单倍型和/或4个DQ等位基因对作为候选MHC II基因与恒河猴面筋敏感性的免疫遗传相关。虽然已经建立了一些有用的模型来研究CD，包括表达HLA-DQ2等位基因的转基因小鼠，但还没有一个令人满意的动物模型来同时满足这种自身免疫性疾病的遗传和病理标准。我们已经部分地刻画了恒河猴GSE模型。我们相信这样的模型对于CD的免疫发病机制和治疗的研究将是非常有用的。为了进一步发展这一模型，我们计划：目标1：评估猕猴GSE的临床、组织病理学和免疫学替代物之间的关系。将评估临床或亚临床GSE猕猴的临床症状(腹泻、体重减轻、皮肤损害等)、AGA的存在、抗转谷氨酰胺酶2(TG2)抗体、绒毛萎缩、IEL的存在以及产生炎性细胞因子的肠道T淋巴细胞与对照组之间的关系。目的：确定恒河猴MHC II等位基因与面筋蛋白敏感性相关的候选免疫遗传基因。从至少100只对面筋敏感的印度猕猴和100只对照印度猕猴中提取的DNA将被用来检测与我们最近确定为MHC II候选等位基因的2个DRB单倍型和/或4个DQ等位基因对的关联。我们预测，类似于乳糜泻患者的DQ2/8关联也将在患有GSE的恒河猴中得到证实。目的3：评价面筋敏感猕猴和对照猕猴在α-醇溶蛋白消化方面的差异。在对面筋敏感猕猴和对照猕猴的初步研究中，发现GSE动物，而不是对照，也不是缓解的动物，通过肠道上皮吸收未消化的33-聚体。因此，推测GSE猕猴对食物面筋蛋白的体液免疫反应是由MHC II型正常的GSE猕猴未消化的α2-醇溶蛋白通过渗漏上皮吸收引起的。目的：评价口服酶制剂治疗GSE猕猴的效果。MHC II-预先选择的对面筋敏感的猕猴将首先接受无面筋饮食，以实现缓解。在后续的面筋挑战中，将通过增加面筋水平和每日固定剂量的鞘氨醇单胞菌Pro内肽酶、大麦半胱氨酸内蛋白酶EP-B2和这两种酶一起给面筋敏感型猕猴服用，以评估它们的治疗潜力。据估计，有100多万美国人也患有乳糜泻。 称为乳糜泻(CD)。CD是一种自身免疫性小肠疾病 由摄入谷类谷物中的面筋蛋白引起的。据报道，有 慢性胃肠道炎症的临床和免疫学相似性 在恒河猴(Macaca Mulatta)和人类中的束，暗示了其潜在的用途 非人灵长类动物作为这种NIDDK相关疾病的模型。面筋- 伴抗醇溶蛋白抗体(AGA)的过敏性肠病(GSE)，绒毛 萎缩，绒毛减少：隐窝比率和上皮内淋巴细胞数量增加 (IEL)可在相对较大比例(~25%)的圈养幼年恒河猴中发现 起源于印度的猕猴，有特发性慢性腹泻症状。在人类身上， GSE通常见于遗传易感性为CD的个体和恒河猴 在猴子和人类中，含面筋的饮食都可以诱发这种疾病。我们最近 在对面筋敏感的猕猴和对照组猕猴喂食面筋的实验中- 包含饮食，然后是无麸质饮食。结论是完全缓解 可以在GSE猕猴中实现，这是基于从它们的饮食中去除面筋。 此外，我们还确定了2个DRB单倍型和/或4个DQ等位基因对作为候选 MHC II基因与恒河猴面筋敏感性的免疫遗传关联。 肠粘膜相关淋巴组织可在非人类中取样 通过无创和重复的内窥镜(PIPCH)活检研究特定的灵长类动物 关于发病机制和免疫的问题。这是不可能在 对人类病人或小鼠进行的临床研究。尽管已经有几个有用的模型 为研究CD而建立的，包括表达人类白细胞抗原-DQ2等位基因的转基因小鼠，有 没有一种令人满意的动物模型能同时满足遗传和病理标准 自身免疫性疾病。我们已经部分地描述了非人类的GSE模型 灵长类动物。我们相信，这样的模型将对研究 CD的免疫发病机制及治疗。

DESCRIPTION (provided by applicant): In humans, gluten-sensitive enteropathy (GSE) is typically found in individuals genetically predisposed to celiac disease (CD), and in rhesus monkeys as well as in humans it can be induced by a gluten-containing diet. We recently performed experiments where gluten-sensitive and control macaques were fed gluten- containing diets followed by gluten-free diets. Complete recovery was achieved in GSE macaques - based on withdrawal of gluten from their diet. Furthermore, we identified 2 DRB haplotypes and/or 4 DQ allelic pairs as candidate MHC II genes for immunogenetic association with gluten sensitivity in rhesus macaques. Although several useful models have been established to study CD, including transgenic mice expressing HLA-DQ2 allele, there is no satisfactory animal model that would fulfill both genetic and pathologic criteria of this autoimmune disease. We have partially characterized a rhesus GSE model. We believe that such a model will be extremely useful for studies of the immunopathogenesis and treatment of CD. To develop this model further, we plan to: Aim 1: Evaluate an association between clinical, histopathological and immunological surrogates of GSE in rhesus macaques. An association between clinical symptoms (diarrhea, weight loss, skin lesions, etc.), presence of AGA, anti-transglutaminase 2 (TG2) antibodies, villous atrophy, increased presence of IELs and inflammatory-cytokine producing intestinal T lymphocytes in macaques with clinical or subclinical GSE vs. controls will be evaluated. Aim 2: Confirm MHC II alleles that were identified in rhesus macaques as candidates for immunogenetic association with gluten sensitivity. DNA extracted from at least 100 gluten-sensitive and 100 control macaques of Indian origin will be examined for the association with 2 DRB haplotypes and/or 4 DQ allelic pairs that we recently identified as MHC II candidate alleles. We predict that analogous to celiac patients DQ2/8 association will also be confirmed in rhesus macaques with GSE. Aim 3: Evaluate the differences in a2-gliadin digestion between gluten sensitive and control macaques. In pilot study with gluten sensitive and control macaques, it was found that GSE animals but not controls, nor remitted animals, absorb undigested 33-mer across intestinal epithelium. Thus, it was proposed that systemic humoral immune response to dietary gluten is caused by absorption of undigested a2-gliadin across leaky epithelium in GSE macaques with proper MHC II type. Aim 4: Evaluate the oral enzyme treatments in rhesus macaques with GSE. MHC II-pre- selected macaques with gluten sensitivity will be first placed on a gluten-free diet to accomplish remission. In a follow-up gluten challenge, gluten sensitive macaques will be dosed with increasing levels of gluten and a fixed daily oral dose of prolyl endopeptidase from Sphingomonas capsulata, cysteine endoprotease EP-B2 from barley, and the two-enzymes together to evaluate their therapeutic potential. It is estimated that more than one million Americans suffer from celiac sprue also known as celiac disease (CD). CD is an autoimmune disease of the small intestine caused by ingestion of gluten proteins from cereal grains. It was reported that there are clinical and immunological similarities between chronic inflammation of gastrointestinal tract in rhesus macaques (Macaca mulatta) and humans, suggesting the potential use of the non-human primates as a model for this NIDDK-relevant disease. The Gluten- Sensitive Enteropathy (GSE), accompanied with anti-gliadin antibodies (AGA), villous atrophy, reduced villus:crypt ratio and increased number of intraepithelial lymphocytes (IEL) can be identified in a relatively large proportion (~25%) of captive juvenile rhesus macaques of Indian origin with symptoms of idiopathic chronic diarrhea. In humans, GSE is typically found in individuals genetically predisposed to CD, and in rhesus monkeys as well as in humans it can be induced by a gluten-containing diet. We recently performed experiments where gluten-sensitive and control macaques were fed gluten- containing diets followed by gluten-free diets. It was concluded that complete remission could be achieved in GSE macaques ¿ based on withdrawal of gluten from their diet. Furthermore, we identified 2 DRB haplotypes and/or 4 DQ allelic pairs as candidate MHC II genes for immunogenetic association with gluten sensitivity in rhesus macaques. The intestinal mucosa-associated lymphoid tissues can be sampled in non-human primates by non-invasive and repetitive endoscopic (pinch) biopsies to study specific questions with respect to pathogenesis and immunity. This cannot be accomplished in clinical studies with human patients or mice. Although several useful models have been established to study CD, including transgenic mice expressing HLA-DQ2 allele, there is no satisfactory animal model that would fulfill both genetic and pathologic criteria of this autoimmune disease. We have partially characterized a model of GSE in non-human primates. We believe that such a model will be extremely useful for studies of the immunopathogenesis and treatment of CD.