This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.
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