CEREBRAL MALARIA-INDUCED APOPTOSIS AND BRAIN PATHOLOGY

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。 具体目标 疟疾(恶性疟原虫)感染全球2亿至3亿人，每年导致200万人死亡(主要是儿童)。15%的死亡病例是由脑型疟疾和其他严重形式的疟疾引起的。不管推荐的治疗方法如何，很大一部分CM患者都会死亡。相当数量的幸存者会出现神经系统并发症和认知问题。CM引起的脑损伤和预后不良的确切机制尚不清楚。该项目的主要假设是疟原虫凋亡因子S(PAF)诱导神经元和微血管内皮细胞凋亡，选择性阻断PAF介导的凋亡将否定或显着减少细胞凋亡和CM诱导的病理改变。 我们的目标是分别利用人脑内皮细胞(HBVEC)、神经胶质细胞和神经细胞系以及我们建立的啮齿动物CM模型来鉴定和表征PAF在CM诱导的脑病理中的作用。我们计划利用基因组学、蛋白质组学、免疫学方法、成像技术、超微结构分析和靶向基因失活(RNA干扰，RNAi)来实现这些目标。 我们的长期目标是以PAF为靶点，作为预防或治疗CM引起的脑病理的治疗策略。已经提出了三个特定的目标来研究细胞凋亡在CM诱导的脑病理中的作用。 具体目的1利用死后脑组织对CM诱导的小鼠CM模型和恶性疟原虫诱导的人CM模型的细胞凋亡进行比较和对比。通过基因和蛋白质表达分析、超微结构、免疫组织学和DNA裂解(TUNEL)分析，比较CM诱导小鼠和人CM中凋亡因子表达的变化。 特定目的2将鉴定和表征介导CM诱导细胞凋亡的关键凋亡因子S(PAF)。将利用基因和蛋白质表达数据(基因芯片、一维和二维凝胶电泳)和蛋白质质谱学(MALDI-TOF)来鉴定和表征PAF在CM诱导体外培养的HBVEC、胶质细胞和神经元细胞系凋亡中的潜在作用。 具体目标3将从功能上描述CM介导的CM中的细胞凋亡。抗体阻断程序和必要的核糖核酸干扰策略将被用来抑制寄生虫感染的红细胞(IRBC)诱导的HBVEC、神经胶质细胞和神经元细胞系中宿主PAF受体(S)的表达，并检测其凋亡情况。血脑屏障完整性将在体外血脑屏障模型中进行评估，无论是否存在红血球。我们还将用抗PAF抗体在小鼠CM模型中外源性阻断PAF，并观察其对血脑屏障和CM病理的影响。

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. Specific Aims Malaria (P. falciparum) infects 200 to 300 million people globally and kills 2 million (mostly children) every year. 15% of fatal cases are due to cerebral malaria (CM) and other severe forms of malaria. A significant segment of CM patients die regardless of recommended treatment. A significant number of survivors develop neurological complications and cognition problems. The precise mechanisms responsible for CM induced brain damage and poor prognosis is unclear. The main hypothesis of this project is that Plasmodium apoptotic factor(s) (PAF) induce neuronal and microvascular endothelial cell apoptosis and that selectively blocking PAFmediated apoptosis will negate or significantly reduce apoptosis and CM-induced pathology. Our objective is to identify and characterize the role of PAF in CM-induced brain pathology using human brain endothelial (HBVEC), glial, and neuronal cell lines, as well as our established rodent CM model respectively. We plan to utilize genomics, proteomics, immunological methods, imaging techniques, ultrastructural analysis, and targeted gene inactivation (RNA interference, RNAi) to pursue these goals. Our long-term goal is to target PAF as a therapeutic strategy for preventing or treating CM-induced brain pathology. Three Specific Aims have been proposed to study the role of apoptosis in CM-induced brain pathology. Specific aim 1 will compare and contrast CM induced apoptosis in murine CM models with that in P. falciparum-induced human CM using post mortem brain tissues. CM-induced expression changes in apoptotic factors in murine and human CM will be compared by gene and protein expression analysis, ultrastructure, immunohistology, and DNA cleavage (TUNEL) analysis. Specific aim 2 will identify and characterize the key apoptotic factor(s) (PAF) mediating CM-induced apoptosis. Gene and protein expression data (gene microarray chips, 1D & 2D gel electrophoresis) and protein Mass spectroscopy (MALDI-TOF) will be utilized to identify and characterize the potential role of PAF in CM-induced apoptosis in HBVEC, glia, and neuronal cell lines in vitro. Specific aim 3 will functionally characterize CM-mediated apoptosis in CM. Antibody blocking procedures and, if necessary, RNA interference (RNAi) strategies will be used to knock down expression of host PAF receptor(s) in HBVEC, glial, and neuronal cell lines induced by parasite infected RBC (IRBC) and uninfected controls and assayed for apoptosis. BBB integrity will be evaluated in an in vitro BBB model in the presence or absence of IRBC. We will also exogenously block PAF with anti-PAF antibody in murine CM model and examine effects on BBB and CM pathology.