Malaria carries a clinical burden of up to 200 million episodes in Africa alone and a severe complication is cerebral malaria (CM), often leading to coma and death. A hallmark of human cerebral malaria (CM) is the sequestration of P. falciparum infected red blood cells (Pf- IRBC) in brain venules. However, the molecular interactions of Pf-IRBC with the blood brain barrier (BBB) endothelial interface and the host responses leading to neurological damage are unclear. Therefore we investigated the response of human brain micro-vascular endothelial cells (HBMEC) to Pf-IRBC exposure. We found that Pf-IRBC but not non-infected RBC activate BBB endothelium: increase ICAM-1 expression on HBMEC, release cytokines &chemokines and decrease barrier integrity. Pf-IRBC induced chemokine CCL20 (or MIP31) mRNA was increased ~700x and GRO-1,-2 and 3 (or CXCL-1, -2, -3) were increased up to 500 fold. CCL20 and GRO proteins were released to both apical and baso-lateral side of the endothelium. Preliminary data show that baso-laterally (brain side) released Pf-IRBC medium directly activates the neuropil (astrocytes, neurons) by increasing GFAP expression. It is hypothesized that Pf-IRBC activated BBB endothelium releases compounds from its baso-lateral side into the brain parenchyma, where it can directly activate neurons and/or astrocytes and subsequently cause neuronal dysfunction. CCL20 and GRO 1,-2 and 3 chemokines have the potential to mediate these effects. Therefore, it is proposed to explore the contribution of the chemokines released from Pf-IRBC activated brain endothelium to neuronal dysfunction. To study this, Pf-IRBC will be added to the upper (blood) compartment of an in-vitro model of the human BBB and the kinetics of chemokine secretions to both upper and lower (brain side) compartment studied. The specificity of immune cells activation and attraction will be assessed and the consequences of this for BBB integrity. Effects of baso-lateral chemokine secretion on activation of astro-neuronal cells and intracellular signaling mechanisms leading to chemokine secretion will be analyzed. It is expected that these exploratory R21 studies will clarify the role of CCL20 versus that of GRO 1,-2 and 3 mediated release from BBB endothelium in the activation of the neuropil as occurs in CM. Future plans include obtaining more sustainable RO1 funding to study the here identified targets in CM pathogenesis more in detail, using both in-vitro and in-vivo CM models. It is hoped that this will lead to identification of targets for clinical intervention of CM. NARRATIVE Malaria is a disease resulting from infection with the Plasmodium parasite and carries a clinical burden of up to 200 million episodes in Africa alone. A hallmark of human cerebral malaria (CM) is that the P. falciparum infected red blood cells (Pf-IRBC) adhere to the walls of the brain venules and clog them up. When this happens in the brain, this is called cerebral malaria (CM). Especially in childen CM often leads to coma and death and in surviving children neurological sequelae, such as seizures and loss of developmental milestones may occur. However, it is not clear how a parasite that resides inside red blood cells can cause brain damage. Since the walls of the blood vessels of the brain for a barrier in between the blood and brain, called blood brain barrier (BBB), it important to study the molecular events at the BBB occurring after Plasmodium infected red blood cells stick to the vessel wall and clog up the vessel. We made a model of the BBB in vitro by isolating human BBB cells. CM was then modeled by adding Plasmodium infected red blood cells to the BBB cells. Subsequently the responses of the BBB cells were studied We found that the Plasmodium infected red blood cells increased expression of cell adhesion molecules (ICAM-1) on HBMEC, released cytokines &chemokines and decrease barrier integrity. The secretion of specific chemokines such as CCL20 (or MIP31) and GRO-1,- 2 and 3 was increased to both the blood and brain side of the BBB cells. It is hypothesized that Pf-IRBC activated BBB cells releases compounds from their bottom side towards the brain, where it can interfere with the normal functioning of the brain cells, e.g. neurons and astrocytes. It has been shown by others that CCL20 and GRO 1,-2 and 3 chemokines have the potential to mediate these effects. Therefore, here it is proposed to explore the contribution of these specific chemokines that are released from BBB cells that were exposed to Plasmodium infected red blood cells, to neuronal dysfunction. It is expected that these exploratory R21 studies will clarify the role of these chemokines in the activation of the brain cells as this occurs in CM. Future plans include obtaining more sustainable RO1 funding to study the here identified targets in CM pathogenesis more in detail, using both in-vitro and in-vivo CM models. It is hoped that this will lead to identification of targets to develop drugs that can treat CM.
