The neurological complications of sleeping sickness in man caused by Trypanosoma brucei gambiense and T.b. rhodesiense are attributed to the penetration of the central nervous system by trypanosomes. Yet, how African trypanosomes cross the blood-brain barrier (BBB) remains an unresolved issue. Using our in vitro BBB model constructed of human brain micro vascular endothelial cells (BMEC), we find that bloodstream form (BSF) T. b. gambiense parasites bind at or near intercellular junctions and cross the in vitro BBB model paracellularly more efficiently than do animal infective T. b. brucei. Insect infective procyclics do not bind nor cross BMEC even in the presence of BSF parasites. Human infective BSF induce very rapid transient or oscillatory changes in intracellular calcium ([Ca2+]i) levels in BMEC. The early changes in monolayer integrity from real-time measurements of transendothelial electrical resistance (TEER), though rapid, occur after the observed changes in [Ca2+];. Pretreatment of human BMEC with the PLC inhibitor U73211 increases human BMEC transendothelial electrical resistance (TEER), inhibits the trypanosome-induced changes in BMEC [Ca2+]j levels, and inhibits trypanosome transmigration across the barrier. Because our [Ca2+]j studies also suggest a possible role for bradykinin, we plan to investigate this possibility with our Brazilian counterparts. The Specific Aim for this FIRCA proposal therefore will be to determine the role of African trypanosome-associated proteases and kinin-receptors in human BMEC activation. This research will be done primarily in Brazil at Universidade Federal do Rio de Janeiro with Julio Scharfstein as an extension of NIH grant # 1 RO1 AI51464-01. The results obtained from the proposed study will be an important step in our understanding of the initial events of African trypanosome invasion into the brain. Armed with this knowledge we will be better prepared to design therapies that will stop the parasites from entering the brain and prevent many of the agonizing neurological symptoms, which eventually lead to death.