Study of permissive/non-permissive T. gondii infections

描述（由申请方提供）：弓形虫每年感染约150万人，是美国每年食源性死亡的第三大原因[1]。感染通常是无症状的，但对胎儿和接受化疗或器官移植的患者以及艾滋病患者可能是危险的[2-4]。虽然这种微生物在体外侵入大多数有核宿主细胞，但仍然存在一个矛盾，即在脑组织中发现的坏死斑是与严重或致命性脑炎相关的主要病理[1，5 -7]。速殖子侵入脑的神经元、小胶质细胞和星形胶质细胞[8-11]，但我们在混合原代培养物中观察到，与允许的小胶质细胞相比，星形胶质细胞限制寄生虫复制。这些不同的宿主细胞环境可能通过响应感染而激活明显不同的生化和代谢途径而产生。显然，在寄生虫感染后，人成纤维细胞中的多种宿主细胞mRNA被调节[12，41-42]。在这里，我们建议使用微阵列技术来表征全球宿主基因表达谱，并确定与复制耐受性小胶质细胞相比，在寄生虫感染的非允许性星形胶质细胞中差异表达的基因。鉴定与免疫防御、代谢、调节和响应于速殖子感染而调节的信号传导途径相关的宿主细胞特异性基因将允许对定义这些明显不同的宿主细胞环境的分子相关物的mRNA的单个或共调节组进行集中的、假设驱动的分子遗传学实验，并且可以为潜在开发针对急性弓形体病和慢性疾病的有效治疗提供基础。为此，我们将完成以下特定目标：（1）使用流式细胞术和高速细胞分选仪从原代小鼠脑细胞培养物中分离速殖子感染的星形胶质细胞和小胶质细胞，以及2）使用DNA微阵列比较速殖子感染和未感染的小鼠星形胶质细胞和小胶质细胞中的基因表达。

DESCRIPTION (provided by applicant): Toxoplasma gondii infects approximately 1.5 million human subjects annually, and is the third-leading cause of foodborne mortality in the US each year [1]. Infections are most often asymptomatic, yet can be dangerous to the unborn and to patients undergoing chemotherapy or organ transplant, and also to people with AIDS [2-4]. While this organism invades most nucleated host cells in vitro, it remains a paradox that necrotic plaques found in tissues of the brain are the primary pathology associated with severe or fatal encephalitis [1,5-7]. Tachyzoites invade neurons, microglia and astrocytes of the brain [8-11], but we have observed in mixed primary cultures that astrocytes limit parasite replication, when compared with the permissive microglia. These distinct host cell environments likely arise via activation of distinctly different biochemical and metabolic pathways in response to infection. It is evident that multiple host cell mRNAs in human fibroblasts are modulated following parasite infection [12, 41-42]. Here, we propose to characterize global host gene expression profiles using microarray technology, and to identify genes that are differentially expressed in response to parasite infection of the non-permissive astrocytes when compared with the replication-tolerant microglia. The identification of host cell-specific genes relevant to immune defense, metabolic, regulatory and signaling pathways modulated in response to tachyzoite infection will allow for focused, hypotheses driven molecular genetic experiments on individual or co-regulated groups of mRNAs defining the molecular correlates of these distinctly different host cell environments, and may provide the basis for potential development of an effective treatment against both acute toxoplasmosis and chronic disease. To this end, we will accomplish the following Specific AIMS: (1) isolate tachyzoite-infected astrocytes and microglia from primary murine brain cell cultures using flow cytometry and a high-speed cell sorter, and 2) compare gene expression in tachyzoite-infected and uninfected murine astrocytes and microglia using DNA microarrays.