Non-Human Primate Model of Gluten-Sensitive Enteropathy

描述（由申请方提供）：在人类中，麸质敏感性肠病（GSE）通常见于遗传上易患乳糜泻（CD）的个体，在恒河猴和人类中，含麸质饮食可诱导GSE。我们最近进行了实验，其中谷蛋白敏感的猕猴和对照猕猴被喂食含谷蛋白的饮食，然后是无谷蛋白的饮食。在GSE猕猴中实现了完全恢复-基于从其饮食中去除麸质。此外，我们确定了2 DRB单倍型和/或4 DQ等位基因对作为候选MHC II基因的免疫遗传学关联与恒河猴面筋敏感性。虽然已经建立了几种有用的模型来研究CD，包括表达HLA-DQ 2等位基因的转基因小鼠，但还没有令人满意的动物模型能够满足这种自身免疫性疾病的遗传和病理标准。我们已经部分地描述了恒河猴GSE模型。我们相信，这样的模型将是非常有用的CD的免疫发病机制和治疗的研究。为了进一步开发这个模型，我们计划：目的1：评价恒河猴GSE的临床，组织病理学和免疫学替代品之间的关联。临床症状（腹泻、体重减轻、皮肤病变等）之间的关联，将评价临床或亚临床GSE猕猴与对照相比是否存在阿加、抗转氨酶2（TG 2）抗体、绒毛萎缩、IEL和产生炎性细胞因子的肠T淋巴细胞的存在增加。目的2：确认在恒河猴中鉴定的MHC II等位基因作为与谷蛋白敏感性免疫遗传学关联的候选者。从至少100个面筋敏感的和100个控制猕猴的印度起源的DNA提取将被检查与2 DRB单倍型和/或4 DQ等位基因对，我们最近确定为MHC II候选等位基因的关联。我们预测，类似于乳糜泻患者DQ 2/8的关联也将在患有GSE的恒河猴中得到证实。目的3：评估谷蛋白敏感猕猴和对照猕猴之间α 2-麦醇溶蛋白消化的差异。在谷蛋白敏感猕猴和对照猕猴的初步研究中，发现GSE动物而不是对照动物，也不是缓解动物，吸收未消化的33-mer穿过肠上皮。因此，提出对膳食麸质的全身体液免疫应答是由具有适当MHC II型的GSE猕猴中未消化的α 2-麦醇溶蛋白穿过渗漏上皮的吸收引起的。目的4：评价口服酶治疗恒河猴GSE。MHC II预选猕猴与面筋敏感性将首先放在一个面筋免费的饮食，以实现缓解。在后续的谷蛋白激发中，谷蛋白敏感性猕猴将被给予增加水平的谷蛋白和固定的每日口服剂量的来自荚膜鞘氨醇单胞菌的脯氨酰内肽酶、来自大麦的半胱氨酸内切蛋白酶EP-B2以及这两种酶，以评估它们的治疗潜力。据估计，超过一百万美国人患有乳糜泻也 称为乳糜泻（CD）。CD是一种小肠自身免疫性疾病 是由于摄入谷物中的谷蛋白引起的。据报道， 胃肠道慢性炎症的临床和免疫学相似性 道在恒河猴（Macaca mulatta）和人类，表明潜在的使用 非人灵长类动物作为这种NIDDK相关疾病的模型。谷蛋白- 敏感性肠病（GSE），伴有抗麦胶蛋白抗体（阿加），绒毛状 萎缩，绒毛：隐窝比例降低，上皮内淋巴细胞数量增加 (IEL)可以在相对较大比例（约25%）的圈养幼年恒河猴中鉴定出 具有特发性慢性腹泻症状的印度猕猴。在人类中， GSE通常存在于遗传上易患CD的个体和恒河猴中， 猴子以及人类的这种疾病都可以通过含麸质的饮食来诱导。我们最近 进行了实验，其中谷蛋白敏感的猕猴和对照猕猴被喂食谷蛋白， 含麸质的饮食，然后是无麸质的饮食。结论是完全缓解 GSE猕猴可以实现基于从他们的饮食中提取面筋。 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基因， 恒河猴中与麸质敏感性相关的免疫遗传学MHCII基因。 肠粘膜相关淋巴组织可在非人类中取样。 通过非侵入性和重复性内窥镜（捏）活检对灵长类动物进行研究， 关于发病机制和免疫力的问题。这是无法实现的， 人类患者或小鼠的临床研究。虽然有几个有用的模型， 建立了研究CD，包括表达HLA-DQ 2等位基因的转基因小鼠， 没有令人满意的动物模型，将满足遗传和病理标准，这是 自身免疫性疾病我们已经部分表征了非人类GSE模型， 灵长类动物我们相信，这样一个模型将是非常有用的研究， CD免疫发病机制和治疗。

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.