SCREENING AND RECRUITMENT OF SUBJECTS WITH ALPHA GLOBIN DELETIONS To begin studying the function of endothelial alpha globin in humans, we need to identify individuals who have genetic deletions of alpha globin. Deletional mutations in the alpha globin gene are common in many parts of the world. We have chosen to focus on the alpha -3.7 kb deletion because it affects Africans and African-Americans and has an established protective effect against many complications of sickle cell disease. About 27.5% of African Americans have a deletion of one of four alpha globin genes; 1.9% have two of four genes deleted. We wish to identify men and women who have two of hour alpha globin genes deleted, but are otherwise healthy. To facilitate the screening of healthy volunteers for the presence of two alpha globin -3.7 kb deletions, we are developing digital PCR based molecular diagnostics that we can perform in our CLIA-compliant laboratory. We plan to screen 1,000 - 2,000 young healthy African Americans at mobile fields sites (health fairs, college campuses, etc) as well as by mail. We have an active clinical protocol and are ready to screen participants for alpha globin deletions. VASCULAR FUNCTION IN HEALTHY PEOPLE WITH ALPHA GLOBIN DELETIONS Alpha globin appears to restrict the diffusion of the vasodilator nitric oxide from endothelium to smooth muscle in cell and animal models. We wish to test whether alpha globin regulates blood pressure and blood flow in healthy humans. We have designed a study in which we will compare healthy young African American individuals with no alpha globin deletions against those with two alpha globin deletions. We will examine several endpoints that we hypothesize will be affected by loss of endothelial alpha globin: 1) blood pressure, 2) orthostatic blood pressure, 3) shear stress-induced vasodilation, and 4) response to vasoactive infusions. We have written the protocol for these studies which is through the first level of review. We plan to enroll people who are identified through our screening and recruitment protocol described above. EFFECT OF ENDOTHELIAL ALPHA GLOBIN DELETION ON SICKLE CELL DISEASE SEVERITY IN THE MOUSE We have developed techniques to quantify the expression of alpha globin in tissue and to control for the potential confounding effects of alpha globin transcripts present in circulating reticulocytes (immature red blood cells). We hypothesize that deletion of alpha globin from endothelium will affect nitric oxide signaling in endothelial cells and alter hemodynamics and local NO production. We will examine how alpha globin deletion can mitigate the vascular complications associated with sickle cell disease. HIGH THROUGHPUT SCREENING OF COMPOUNDS THAT INTERRUPT ENOS/ALPHA GLOBIN INTERACTIONS The ability of alpha globin to limit the diffusion of nitric oxide appears to be dependent on formation of a macro molecular complex with endothelial nitric oxide synthase. Disruption of this complex with a mimetic peptide greatly increases the amount of NO that leaves the endothelium. This presents a druggable target: displacing alpha globin from eNOS with a small molecule should increase NO signaling in the vasculature. To identify these molecules, we are developing a fluorescence polarization assay to rapidly screen compounds that displace alpha globin from eNOS. We hope to identify existing or novel drugs that disrupt this interaction, test them in model systems, and then bring promising leads to safety testing in humans. Our goal is to develop a new class of drug that increases endothelial nitric oxide signaling. This could potentially benefit patients with blood and vascular diseases such as sickle cell disease or malaria. RED CELL BIOMECHANICS The conventional hypothesis is that alpha thalassemia protects against the complications of sickle cell disease by changing the red blood cell. Specifically, in sickle cell disease, alpha thalassemia is associated with smaller red blood cells, fewer irreversibly sickled cells, and fewer reticulocytes. We have developed a microfluidic device to evaluate red blood cell biomechanic properties quantitatively. We can apply this device and analytical approach to answer several questions: 1) does alpha thalassemia improve the ability of red blood cells to flow through narrow channels; 2) do red cell biomechanical properties changes during the course of sickle cell disease pain crisis; 3) what impact do white blood cells and reticulocytes have on capillary transit. BUILDING CLINICAL RESEARCH CAPACITY AT THE CENTRE RECHERCHE ET DE LUTTE CONTRE LA DREPANOCYTOSE (CRLD) IN BAMAKO, MALI The NHLBI Sickle Cell Branch is a leading center for clinical studies of sickle cell disease in the United States. However, sickle cell disease is a global disease, and there is an urgent need to improve the diagnosis and care of children with sickle cell disease who are living in sub-Saharan Africa. To this end, we have identified a clinical research partner organization in Bamako, Mali, the CRLD. The CRLD sees approximately 5,000 patients with sickle cell disease on a regular basis. They provide comprehensive specialty care for patients with sickle cell disease, including molecular diagnosis, education, preventive care, and treatment. We are working with the CRLD to establish IRB-approved natural history protocols to enable the collection of data and specimens from patients, implement electronic capture of clinical data, and provide research training for their staff and fellows. Together we hope to build the clinical research capacity of the CRLD so that we can address questions of public health concern for Malian children with sickle cell disease. For example, how iron deficiency effects the severity of sickle cell disease. We have developed a proposal to study the effects of iron availability on sickling and red cell survival at the population level in Bamako, Mali. This proposal has been approved scientifically.

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3
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2017
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U.S. National Heart Lung and Blood Inst
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