VERMONT COBRE: PROJECT 2: INNATE IMMUNE RESPONSES TO CRYPTOSPORIDIUM PARVUM

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 微小隐孢子虫是引起持续性和慢性腹泻的一个重要的全球性原因。在幼儿中，特别是在不发达国家，隐孢子虫病临床上更为严重，与成人感染相比，与肠道炎症有关。对于粘膜对微小隐孢子虫的“先天”免疫反应，人们知之甚少。γ-增量(Y6)T细胞是幼儿和肠道中重要的T细胞群。作为对肠道感染的反应，T细胞似乎在外围和 通过肠道特异的归巢受体(a4p7，aEP7)，选择性地“回家”回到肠道粘膜。肠道yd T细胞似乎调节上皮细胞的恢复，对肠道“应激”作出反应，并可能具有重要的细胞溶解功能，特别是在杀死受感染的肠道上皮细胞(可能还有CD4+AP T)方面。 单元格)。这项建议旨在对研究人员进行经典和分子/遗传免疫学技术方面的培训，并应用这一基础科学知识来研究儿童，特别是发展中国家儿童对隐孢子虫病的粘膜免疫反应。该假说认为T细胞在隐孢子虫病的先天免疫反应中起重要作用，T细胞通过T细胞受体在肠道特异性MHC-I分子(MIC-A)的共同刺激下被激活，并通过细胞溶解和凋亡杀死受感染的上皮细胞。然后，这些信息将被应用于对来自 小群孟加拉儿童急性隐孢子虫病与活体相关性。这项建议的主要导师是一位在Y6 T细胞方面具有专业知识的全职免疫学教授，并拥有在研究建议中熟练进行拟议研究分析的实验室，目标1确定Y5细胞的激活是否通过TCR以及是否需要与MHC-I类分子MIC-A共同刺激。在这个目的中，克隆了T细胞受体的y链和y链 被转染到T细胞受体阴性的细胞系，并对其功能进行评估。目的2通过Fas-FasL和穿孔素来评价Y5的细胞毒作用，并证实其细胞毒作用是否具有抗原依赖性。最后，为了建立体内相关性，Aim 3检查了患有隐孢子虫病儿童的Y5 T细胞，确定这些Y6细胞(携带肠道归巢受体)是否可以从外周血中提取 淋巴细胞以及这些克隆是否具有类似体外数据的激活和细胞溶解功能。这些数据将促进我们对隐孢子虫病粘膜免疫应答的了解，从而进一步促进预防和治疗的进展。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cryptosporidium parvum is an important global cause of persistent and chronic diarrhesa. In young children, particularly in underdeveloped countries, cryptosporidiosis is clinically more severe and is associated with more intestinal inflammation than infection in adults. Little is understood about the mucosal "innate" immune respone to C. parvum. Gamma delta (y6) T cells are an important T cell population in young children and in the intestinal tract. In response to infection at the intestine, T cells appear to expand in the periphery and selectively "home" back to the intestinal mucosa, via gut-specific homing receptors (a4p7, aEp7). Intestinal yd T cells appear to modulate recovery of the epithelium, respond to intestinal "stress" and may have important cytolytic function, especially in killing of infected intestinal epithelial cells (and possibly CD4+ ap T cells). This proposal seeks to train the investigator in classic and molecular/genetic immunology techniques and to apply this basic science knowledge to study mucosal immune responses to cryptosporidiosis in children, especially those in the developing world. The proposal hypotheses that \o T cells are important in the innate immune response to cryptosporidiosis, are activated in response to C. parvum via the T cell receptor with necessary co-stimulation by the gut-specific MHC-I molecule (MIC-A), and kill infected epithelial cells by cytolysis and apoptosis. This information will then be applied to the study of gamma delta T cells from a small population of Bangladeshi children with acute cryptosporidiosis to demonstrate in vivo relevance. The primary mentor of this proposal is a full professor of Immunology with expertise in y6 T cells and with a laboratory skilled in the proposed research assays In the research proposal, Aim 1 establishes whether the activation of the y5 cells is via the TCR and requires co-stimulation with the MHC-I like molecule MIC-A. In this aim, cloned y and y chains of the T cell receptor are transfected to a T cell receptor negative cell line and evaluated for function. Aim 2 evaluates the cytotoxicity of y5 via Fas-FasL and perforin and confirms whether cytotoxicity is antigen-dependent. Finally, to establish in vivo relevance, Aim 3 examines the y5 T cells from children with cryptosporidiosis, establishes whether these y6 cells (carrying gut homing receptors) can be extracted from peripheral lymphocytes and if these clones have activation and cytolytic functions similar to the in vitro data. These data will advance our understanding of the mucosal immune response to cryptosporidiosis, which will lead to further advances in prevention and therapy.