Project 1: Arginine and Methylarginine concentrations in Gambian children with severe malaria: associations with endothelial activation and tissue perfusion. To test the hypothesis that elevated ADMA contributes to severe malaria, we measured arginine and ADMA in plasma from children presenting to community health clinics for treatment of malaria in the Gambia, West Africa, in a study conducted by Michael Walther. We found that children clinically defined as severe malaria had elevated ratios of ADMA to arginine, the major biochemical determinant of nitric oxide synthase activity. To assess the impact of elevated ADMA on endothelial pathology and tissue perfusion, we performed correlation analysis between plasma ADMA concentration and soluble VCAM-1, a marker of endothelial activation that is normally downregulated by nitric oxide via NF-kappaB. We found a strong positive correlation between plasma ADMA concentration and plasma sVCAM-1 concentration (r = 0.57, p <0.001). This is consistent with in vitro models in which ADMA inhibits endothelial NOS and triggers sVCAM-1 secretion via NF-kappaB activation. Plasma ADMA concentration was also positively correlated with lactic acid concentration (r = 0.38, p <0.001), a product of anaerobic glycolysis that indicates impaired tissue perfusion and is a predictor of death from severe malaria. This is consistent with a model of impaired nitric oxide synthesis leading to adherence, coagulation/thrombosis, and dysfunctional vasoregulation, all culminating in decreased tissue perfusion and hypoxia. In summary, severe malaria in Gambian children is associated with an acute increase in ADMA relative to arginine, and the level of ADMA is associated with endothelial activation and impaired tissue perfusion. In the mouse model of malaria, Jessica Chertow analyzed tissue levels of DDAH to better understand how malaria infection alters DDAH activity and ADMA to Arginine ratios. While DDAH gene expression in mice was unchanged by infection with P berghei (a rodent malaria species), DDAH protein concentration and activity were decreased by about 50% in liver tissue by day 6 after infection. A concomitant rise in the ADMA:Arginine ratio was observed. The results from this animal model suggest that dysregulation of the ADMA:Arginine ratio may be caused by accelerated degradation of DDAH in liver endothelium, a major site for ADMA metabolism. Project 2: Mechanisms of Arginine depletion in experimental cerebral malaria. (Work by: Matthew Alkaitis) Previous studies suggest that impaired nitric oxide (NO) signaling contributes to severe malaria pathogenesis in human patients and in mice. The amino acid L-arginine is an essential substrate for NO production by nitric oxide synthase (NOS). L-arginine synthesis and degradation are regulated as part of the urea cycle, which consists of the cyclical interconversion of the amino acids L-arginine, L-ornithine and L-citrulline. In previous studies in vitro, blood stage malaria infection has been associated with activation/release of parasite- and host-derived arginase enzymes, which convert L-arginine to L-ornithine and urea. All other variables remaining constant, accelerated arginase activity should produce an increase in the ratio of L-ornithine to L-arginine. Using HPLC analysis, we sought to confirm these findings in vivo. In mice infected with P. berghei ANKA, we observed that plasma L-arginine, L-citrulline and L-ornithine are depleted to similar degrees, leaving the relative ratios among them unchanged. Genetic knock-out of parasite arginase did not prevent depletion of these amino acids in infected mice. To isolate the effect of reduced nutrient intake, we restricted the amount of feed available to a group of uninfected mice to match the gram amount consumed the previous day by a group of infected mice. Such nutrient restriction was not sufficient to produce significant depletion of L-arginine, L-citrulline or L-ornithine in uninfected mice. These findings suggest that neither dietary insufficiency nor parasite arginase activity provide a complete explanation of L-arginine depletion. We suspect that arginine is exiting the urea cycle via ornithine, and perhaps is used for energy production or polyamine synthesis by rapidly divided immune cells in the spleen. Heavy isotope-labeled tracer studies are ongoing to quantify changes in urea cycle metabolic flux in infected mice and may suggest alternate metabolic fates of L-arginine. Project 3: In vivo assessment of endothelial function in patients with sickle cell disease or malaria. In order to develop sensitive measures of endothelial function, we have performed studies of microvascular blood flow and perfusion in forearm skin after inducing a controlled period of ischemia via transient occlusion of the brachial artery. Under protocol 12-H-0101 "Blood Flow in Sickle Crisis" a multi-institute collaboration between NIAID, NHLBI and NIBIB (Principal Investigator Hans Ackerman, NIAID;Lead Associate investigator Alexander Gorbach, NIBIB;Accountable Investigator Gregory Kato, NHLBI). Allison Ikeda found that patients with sickle cell disease have prolonged time to reach maximum blood flow compared to healthy African Americans, and that the rate of thenar muscle re-oxygenation is slower in patients with sickle cell disease compared to healthy African Americans. These results suggest that sickle cell patients have delayed blood flow responses after transient brachial artery occlusion, possibly due to delayed vasodilation. The slower re-oxygenation rate seen in patients with sickle cell disease might reflect subclinical vascular dysfunction. Furthermore, Carol Rowley has analyzed oxygen consumption rates in sickle cell patients and have discovered that pain crisis is accompanied by an elevation of oxygen consumption rate, possibly due to inflammation, that may exacerbate sickle cell disease by increasing the demand for oxygen delivery. The mismatch between oxygen delivery and oxygen consumption would drive down the equilibrium oxygen saturation of hemoglobin and promote polymerization of hemoglobin and sickling of red blood cells. This identifies oxygen consumption as a potential therapeutic target for resolving pain crisis. Allison Ikeda applied the same approach to studying young children with cerebral malaria in a collaborative study with Karl Seydel and Terrie Taylor. She found that children with any malaria infection had lower baseline perfusion levels and that children with cerebral malaria in particular had prolonged (slower) reperfusion. This could be attributable to increased sequestration of parasitized red blood cells causing a fixed reperfusion limitation, or due to endothelial dysfunction and impaired vasodilation responses to hypoxia and shear stress.
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