Malaria is among the leading infectious causes of death in many of the worlds poorest countries. This parasitic mosquito-borne illness produces massive hemolysis in many infected human hosts. While much is known about parasite replication and cytoadherence, very little is known about the impact of hemolysis per se on vascular tone and endothelial function. Crossing a number of medical disciplines beyond the scope of malaria, intriguing new research on inherited hemolytic disorders such as sickle cell disease provides clues to pathogenic mechanisms that may be relevant to malaria. We have characterized a mechanism of disease, hemolysis-associated endothelial dysfunction, in which red blood cell hemoglobin spills into plasma and reacts with and oxidatively destroys nitric oxide. Additionally, erythrocyte arginase I is released into plasma and catabolizes arginine, the substrate for endothelial nitric oxide synthesis. As a result, the profound reduction in nitric oxide bioavailability produces vasomotor instability, oxidant stress, inflammation, endothelial adhesion molecule expression, activation of tissue factor, and platelet aggregation. Consistent with shared mechanisms, these same pathways are found to be activated during malarial infection. Chronic hemolysis in hemoglobinopathies also leads to a disease syndrome, hemolysis-associated pulmonary hypertension, which develops in all chronic hereditary and acquired hemolytic conditions and is associated with excessive morbidity and mortality. Despite the recent appreciation of these mechanisms, not one study can be found in the literature evaluating pulmonary hypertension in human malaria.? ? This protocol therefore aims to evaluate mechanisms governing interrelationships among malaria, intravascular hemolysis, nitric oxide bioavailability, endothelial function, pulmonary hypertension, and evolutionarily-selected host polymorphisms that regulate the host response to hemolysis. We will correlate our clinical observations in the field with laboratory assays of hemolysis and nitric oxide bioavailability related to scavenging by cell-free hemoglobin and arginine catabolism. Using a candidate gene approach, we will identify and selectively characterize polymorphisms in genes important for endothelial function, vascular inflammation and disease phenotype. Finally, the characterization of this mechanism in malaria may catalyze the development of novel therapies targeting this pathway, such as sodium nitrite, inhaled nitric oxide gas, and/or recombinant haptoglobin infusions.? ? This study will take place in a malaria-endemic region of Mali in West Africa. Both cases and controls will be recruited from the outpatient clinic of the Hospital Gabriel Tour in Bamako, which is the major pediatric referral hospital in Mali. Controls will be recruited from among healthy children attending the Immunization Clinic at the Hpital Gabriel Tour, and from among siblings of cases. All patients presenting with malaria symptoms are routinely tested via a simple finger prick and blood smear. Per our proposal, patients age 1-5 years with blood smears positive for P. falciparum infection will be considered for case study enrollment. Siblings may be enrolled as healthy or asymptomatic controls or uncomplicated cases. Using blood from the same fingerstick, a portable hemoglobin meter will determine if parasitemic patients meet criteria for severe malarial anemia (SMA). If consent is granted, a clinician investigator will perform a detailed history and physical examination to categorize malaria severity accordingto modified World Health Organization definitions. We will enroll up to 650 volunteers total, to compile complete studies on 125 persons per group. Due to expected limitations with echocardiograms, 125 studies should yield 100 readable tricuspid regurgitate jet velocity data points per subject group. ? ? SUBJECT GROUPS:? A. Healthy uninfected controls? B. Asymptomatic parasitemia controls? C. Uncomplicated malaria cases? D. Severe malarial anemia cases? ? These diagnostic assessments and clinical classifications represent current standard practice. We will initially screen volunteers for the Healthy control and SMA groups; we expect these groups to be most different in our targeted clinical and laboratory measurements. ? ? The NHLBI IRB approved the protocol on 8/7/07. The study was approved in Mali on 10/6/08 and at Tulane University on 10/8/08. The first subject was enrolled on 10/20/08. As of 6/25/08, 102 participants (50 males, 52 females) in Mali had been enrolled into the study. The mean age was 32 months. A standardized history and physical examination was performed. Each participant also underwent echocardiography (165 completed echocardiograms including follow-ups). Obtaining a blood sample for standard hematology and chemistry panels, research bloods, as well as DNA collection for isolation and plasma storage followed the echocardiogram.? ? Based on these studies the prevalence of PAH in the severe malarial anemia subjects was 64% versus 19% I the health controls. We also have echocardiographs on the asymptomatic parasitemia and uncomplicated malaria arms, but this data has not yet been analyzed.
