Study of permissive/non-permissive T. gondii infections

描述（由申请人提供）：刚地弓形虫每年感染约150万人，是美国每年食源性死亡的第三大原因。感染通常是无症状的，但对未出生的胎儿、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]。虽然这种生物在体外侵入大多数有核宿主细胞，但在脑组织中发现的坏死斑块是与严重或致命脑炎相关的主要病理，这仍然是一个悖论[1,5-7]。速殖子侵入大脑的神经元、小胶质细胞和星形胶质细胞[8-11]，但我们在混合原代培养中观察到，与允许的小胶质细胞相比，星形胶质细胞限制了寄生虫的复制。这些不同的宿主细胞环境可能是通过激活明显不同的生化和代谢途径来响应感染而产生的。很明显，人成纤维细胞中的多种宿主细胞mrna在寄生虫感染后被调节[12,41 -42]。在这里，我们建议使用微阵列技术来表征宿主基因表达谱，并鉴定与复制耐受小胶质细胞相比，非允许星形胶质细胞在寄生虫感染反应中表达差异的基因。识别与免疫防御、代谢、调节和信号通路相关的宿主细胞特异性基因，以响应速殖子感染，将允许对单个或共同调节的mrna组进行集中的、假设驱动的分子遗传学实验，定义这些截然不同的宿主细胞环境的分子相关性。并可能为开发针对急性弓形虫病和慢性疾病的有效治疗方法提供基础。为此，我们将完成以下具体目的：(1)使用流式细胞术和高速细胞分选仪从原代培养的小鼠脑细胞中分离出速殖子感染的星形胶质细胞和小胶质细胞；(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.