Host cell influences on the development of toxoplasmic encephalitis

描述（由申请人提供）： 项目概要：弓形虫是一种细胞内寄生虫，对中枢神经系统（CNS）具有特殊的嗜性，在中枢神经系统中建立潜伏的包囊感染。这种寄生虫可导致严重的神经系统后果和先天性感染或免疫功能低下患者的死亡。虽然中枢神经系统长期以来被认为是弓形虫包囊形式的主要器官，但中枢神经系统-寄生虫的相互作用知之甚少。我已经制定了一个建议，将帮助我获得我需要成为一个独立的，学术神经学家谁研究这种中枢神经系统感染的分子机制的科学工具。 研究计划的目的是使用弓形虫病小鼠模型结合位点特异性重组，以了解CNS细胞和不同株弓形虫如何特异性共同进化，以产生可变的免疫应答，导致慢性CNS感染伴或不伴脑炎。我所做的初步工作表明，通过给弓形虫配备一种分泌型Cre重组酶融合蛋白，位点特异性重组将仅发生在受感染的宿主细胞中。具体目标1是将该技术转移到更具生物学相关性的、形成包囊的弓形虫菌株中。具体目标2的目的是验证CNS中潜伏性囊肿的细胞内性质，并建立含囊肿宿主细胞的谱系。我将通过使用免疫组织化学和荧光共聚焦显微镜来评估慢性感染弓形虫Cre株的Cre报告小鼠的脑切片来实现这一目标。然后，具体目标3采用激光捕获显微切割来分离目标2中确定的感染的宿主细胞，以便在小鼠基因组表达阵列上对这些宿主细胞进行遗传分析，并最终确定宿主细胞修饰如何影响疾病结果。 了解最终导致CNS慢性感染的分子机制将指导实际治愈弓形虫病所需药物的开发，以及确定用于调节CNS自身免疫性疾病免疫反应的新基因靶点。 公共卫生相关性： 相关性：刚地弓形虫是一种细胞内病原体，偏好于大脑，慢性感染世界上大部分人口[Montoya JG 2005]。这种寄生虫在子宫内获得或免疫功能低下者（如艾滋病患者）重新激活时，会产生悲惨的神经系统后果。该项目的目标是了解这种慢性脑部感染是如何发生的，以便开发治疗这种疾病的方法。

DESCRIPTION (provided by applicant): PROJECT SUMMARY: Toxoplasma gondii is an intracellular parasite that has a particular tropism for the central nervous system (CNS) where it establishes a latent, encysted infection. This parasite can cause severe neurologic consequences and death in congenital infections or when reactivated in immunocompromised patients. Although the CNS has long been known to be the primary organ that harbors the encysted form of Toxoplasma, the CNS- parasite interaction is poorly understood. I have developed a proposal that will help me gain the scientific tools I need to become an independent, academic neurologist who studies the molecular mechanisms underlying this CNS infection. The goal of the research plan is to use the mouse model of toxoplasmosis in combination with site- specific recombination to understand how the cells of the CNS and different strains of Toxoplasma have specifically co-evolved to produce a variable immune response that results in a chronic CNS infection with or without concomintant encephalitis. Preliminary work I have done shows that by equipping Toxoplasma with a secretable Cre recombinase fusion protein, site-specific recombination will occur only in infected host cells. Specific aim 1 is to transfer this technology into more biologically relevant, cyst-forming Toxoplasma strains. The goal of specific aim 2 is to verify the intracellular nature of the latent cyst in the CNS and establish the lineage of the cyst- containing host cells. I will accomplish this goal by using immunohistochemistry and fluorescent confocal microscopy to evaluate brain sections of Cre-reporter mice chronically infected with Toxoplasma-Cre strains. Specific aim 3 then employs laser- capture microdissection to isolate the infected host cells determined in aim 2 in order to genetically profile these host cells on mouse genome expression arrays and ultimately determine how host cell modifications impact disease outcome. Understanding the molecular mechanisms that culminate in a chronic infection of the CNS will guide the development of drugs needed to actually cure toxoplasmosis, as well as identify novel gene targets for modulating the immune response in autoimmune diseases of the CNS. PUBLIC HEALTH RELEVANCE: RELEVANCE: Toxoplasma gondii is an intracellular pathogen with a predilection for the brain and that chronically infects much of the world's population [Montoya JG 2005]. The parasite produces tragic neurologic consequences when acquired in utero or when reactivated the immunocompromised, such as those with AIDS. The goal of this project is to understand how this chronic brain infection occurs so that treatments to cure the disease can be developed.