The collection of human blood from both patients and healthy volunteers is necessary for the development of laboratory assays required for studies of the role of nitric oxide, inflammatory mediators and endothelial function in inflammatory diseases that involve the blood vessels. This protocol continues to be used for the purposes stated, to collect blood from patients and volunteers for the development of laboratory assays. To date we have enrolled 387 individuals from the NIH site and collected blood numerous times to be used in experimental assays. Some of these samples have been used for gene expression studies and nitrite bioavailability studies. To date we have performed the following sub-studies: 1. Red cell pellets were collected from 32 subjects with SCD and 17 healthy African-American control subjects, and were stored frozen until assayed. A novel microplateimmunoaffinity capture assay was used that measures both immunoreactive GPX-1 protein and enzyme activity. Chronic redox imbalance in erythrocytes of individuals with sickle cell disease (SCD) contributes to oxidative stress and likely underlie common etiologies of hemolysis. We measured the amounts of six antioxidant enzymesSOD1, catalase, glutathione peroxidase 1 (GPx1), as well as peroxiredoxins (Prxs) I, II, and VIinrbcs of SCD patients and control subjects. The amounts of SOD1 and Prx VI were reduced by 17 and 20%, respectively, in SCD RBCs compared with control cells. The amounts of Prx II and GPx1 did not differ between SCD and normal rbcs. However, 18 % of Prx II was inactivated in SCD RBCs as a result of hyperoxidation, whereas inactive Prx II was virtually undetectable in control cells. Furthermore, GPx1 activity was reduced by 33% in SCD RBCs and the loss of activity was correlated with hemolysis in SCD patients. RBCs from SCD patients on hydroxyurea demonstrated 90% higher GPx1 activity than those from untreated SCD patients, with no differences seen for the other catalytic antioxidants. Hydroxyurea induced GPx1 expression in multiple cultured cell lines in a manner dependent on both p53 and NO-cGMP signaling pathways. GPx1 expression represents a previously unrecognized potential benefit of hydroxyurea treatment in SCD patients. 2. We enrolled 15 subjects from Children's National Medical Center. We tested a number of compounds, present in BCAA metabolism, for their ability to up-regulate embryonic globin gene expression in mouse erythroleukemias cells, an adult murine erythroid cell. In 10 subjects, 3 to 24 years of age, with disorders of intermediary BCAA metabolism, there was a modest, but statistically significant, elevation in HbF in patients with IVA, PA, or MMA than was seen in patients with MSUD, at 0.9 % vs. 0.25% (p= 0.017, figure 1), when analyzed by HPLC. Further, we found that the predominant gamma globin chain in patients with BCCA-abnormalities was gamma-2, gG at 8-12% of total betaglobin chains (gamma + beta, figure 2), although less meaningful in a single patient with HbSb-thalassemia. However, no gamma-2 globin was detectable in patients with MSUD. 3. 29 subjects have given blood for the study of gene expression, peripheral blood mononuclear cells and platelets from subjects with pulmonary hypertension and healthy volunteers. 4. Blood was collected from SCD subjects to measure oxidized and modified proteins in plasma and red cells by mass spectrometry. 5. Blood was collected for the study of hemolysis. The mechanism of hemolysis or relative contribution of sickling due to hemoglobin polymerization vs. oxidative damage remains unknown. Earlier studies aimed at deciphering the relative importance of these two mechanisms have been complicated by the fact that sickle red cells (SS) have already been affected by multiple rounds of sickling and oxidative damage before they are collected. In our study, we examine the mechanical fragility of sickle cell trait cells, which do not sickle in vivo, but can be made to do so in vitro. Thus, our novel approach explores the effects of sickle hemoglobin polymerization on cells that have never been sickled before. We find that the mechanical fragility of these cells increases dramatically after a single sickling event, suggesting that a substantial amount of hemolysis in vivo probably occurs in polymer-containing cells. A manuscript has been published. 6. Blood specimens are being collected from SCD subject and health controls for measurement of plasma hemopexin and other markers of hemolysis. Samples collected from SCD subjects and health controls were tested in the research laboratory for plasma hemoglobin and other markers of hemolysis. Coded aliquots are being provided to Dr. James Casella (The Johns Hopkins University School of Medicine) for measurement of plasma hemopexin and other markers of hemolysis. 7. Another sub-study is to determine PBMC of microRNA and their potential perturbations in disease. MicroRNAs have been shown to be global regulators of gene and protein expression in health, development and disease (pulmonary fibrosis, cancer, heart failure). To that end we are going to evaluate, along with our collaborators, microRNA expression in PBMCs from subjects with SCD with and without pulmonary hypertension. An abstract has been written and submitted. 8. MicroRNAs have been shown to be global regulators of gene and protein expression in health, development and disease (pulmonary fibrosis, cancer, heart failure). Total RNA from platelets will be labeled and hybridized to Agilent MicroRNA arrays. Analysis of (a) a repertoire of platelet microRNA and (b) differential expression in diseased and healthy controls will be performed using traditional statistical methods. Prioritization will be performed based on predicted targets. Validation of expressed and differentially expressed microRNAs will be performed by RT PCR. Future studies regarding biological role of platelet expressed microRNAs will be performed. 9. We collected blood to compare SCD subjects with renal insufficiency with SCD subjects who do not have renal insufficiency. We intend to look for vasoactive substances that are differentially regulated in chronic kidney disease, compared to patients with more normal kidney function in SCD, to determine in renally cleared substances contribute to underlying pathogenesis in SCD. 10. The Role of Nitric Oxide Synthase Inhibitors in Severe Malaria. We have developed an HPLC-based assay to measure arginine and methylated arginine metabolites in blood plasma. As part of the assay development process, we drew blood from 4 healthy volunteers at the NIH Clinical Center. The plasma from these blood samples were analyzed using our HPLC assay. We later pooled these four samples and froze aliquots. We periodically thaw these aliquots and run them to assess the intra-day variability in the HPLC assay. The frozen aliquots also serve as an external reference that will alert us to any changes in the calibration of our HPLC assay. 11. G6PD Deficiency and Protection from Malaria. The role of G6PD deficiency in malaria infection appears complex, it may exacerbate anemia during malaria infection but may offer protection from cerebral malaria. One question is whether G6PD deficient erythrocytes can support parasite growth and development to the same extent as G6PD normal erythrocytes. We are developing techniques to distinguish G6PD deficient from G6PD normal erythrocytes in a mixed culture, using both microscopy and flow cytometry.
The aim i s to assess the relative growth of parasites in G6PD deficient vs. G6PD normal erythrocytes. We drew blood from 4 healthy controls at the NIH Clinical Center and used those freshly drawn erythrocytes to optimize the assays. In the future we hope to identify individuals with G6PD deficiency who can provide blood donations to support this project further.
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