CEREBRAL MALARIA-INDUCED APOPTOSIS AND BRAIN PATHOLOGY

脑疟疾引起的细胞凋亡和脑病理学

• 批准号: 7715262
• 负责人: Jonathan K. Stiles
• 金额: $ 16.57万
• 依托单位: MOREHOUSE SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7715262
• 关键词: Antibodies; Apoptosis; Apoptotic; Biological Assay; Blocking Antibodies; Brain; Brain Injuries; Brain Pathology; Cell Line; Cerebral Malaria; Child; Cognition; Computer Retrieval of Information on Scientific Projects Database; DNA; Data; Endothelial Cells; Funding; Gene Proteins; Gene Silencing; Genes; Genomics; Goals; Grant; Human; Imaging Techniques; In Situ Nick-End Labeling; In Vitro; Institution; Malaria; Mass Spectrum Analysis; Mediating; Methods; Modeling; Mus; Neuroglia; Neurologic; Neurons; Numbers; Parasites; Pathology; Patients; Plasmodium; Platelet Activating Factor; Procedures; Proteins; Proteomics; RNA Interference; Research; Research Personnel; Resources; Rodent; Role; Source; Survivors; Therapeutic; Two-Dimensional Gel Electrophoresis; United States National Institutes of Health; brain tissue; doxorubicin/fluorouracil/melphalan protocol; killings; knock-down; outcome forecast; platelet activating factor receptor; prevent; protein expression

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 具体目标 疟疾 (P. falciparum) 每年感染全球 200 至 3 亿人，并导致 200 万人死亡（主要是儿童）。 15% 的死亡病例是由脑型疟疾 (CM) 和其他严重形式的疟疾引起的。 无论推荐的治疗方法如何，很大一部分 CM 患者都会死亡。大量幸存者出现神经系统并发症和认知问题。 CM 引起的脑损伤和不良预后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 (PAF) 诱导神经元和微血管内皮细胞凋亡，选择性阻断 PAF 介导的细胞凋亡将消除或显着减少细胞凋亡和 CM 诱导的病理。 我们的目标是分别使用人脑内皮细胞 (HBVEC)、神经胶质细胞和神经元细胞系以及我们建立的啮齿动物 CM 模型来识别和表征 PAF 在 CM 诱导的脑病理学中的作用。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。 我们的长期目标是将 PAF 作为预防或治疗 CM 引起的脑部病理的治疗策略。研究细胞凋亡在 CM 诱导的脑病理学中的作用提出了三个具体目标。 具体目标 1 将使用死后脑组织对小鼠 CM 模型中 CM 诱导的细胞凋亡与恶性疟原虫诱导的人类 CM 中的细胞凋亡进行比较和对比。将通过基因和蛋白质表达分析、超微结构、免疫组织学和 DNA 切割 (TUNEL) 分析来比较 CM 诱导的小鼠和人类 CM 中细胞凋亡因子的表达变化。 具体目标 2 将鉴定和表征介导 CM 诱导细胞凋亡的关键细胞凋亡因子 (PAF)。基因和蛋白质表达数据（基因微阵列芯片、1D 和 2D 凝胶电泳）和蛋白质质谱 (MALDI-TOF) 将用于鉴定和表征 PAF 在体外 CM 诱导的 HBVEC、神经胶质细胞和神经元细胞系细胞凋亡中的潜在作用。 具体目标 3 将在功能上表征 CM 介导的细胞凋亡。抗体封闭程序以及必要时的 RNA 干扰 (RNAi) 策略将用于敲低由寄生虫感染的红细胞 (IRBC) 和未感染的对照诱导的 HBVEC、神经胶质细胞和神经元细胞系中宿主 PAF 受体的表达，并检测细胞凋亡。 将在存在或不存在 IRBC 的情况下在体外 BBB 模型中评估 BBB 完整性。我们还将在小鼠 CM 模型中用抗 PAF 抗体外源性阻断 PAF，并检查对 BBB 和 CM 病理学的影响。