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Background: Red blood cell (RBC) exchange for sickle cell disease presents unique difficulties due to RBC phenotyping, complex antibody work-ups, large number of RBC units required, and vascular access considerations, any of which can delay the procedure. Multidisciplinary coordination and systemic processes ensure monthly appointments remain on schedule. Study Design and Methods: A high-volume chronic red cell exchange program is described, highlighting the importance of multidisciplinary coordination and process improvement strategies involving initial referral, vascular access, order sets, and allocation of antigen-negative or phenotypically matched RBCs. Results: Approximately fifty outpatient red cell exchanges are performed each month with an 82% kept-appointment rate. Specific factors for program success include open communication across services and improvements to referrals and standardized order sets. Conclusion: A combination of multidisciplinary coordination and process improvement can ensure the success of a high volume red cell exchange program Frequent communication of upcoming appointments between the referring hematologists, the hemapheresis clinic, transfusion service, and interventional radiology is critical. Advance notice to the immunohematology reference lab of upcoming appointments is needed to allow enough time for allocating antigen-negative RBCs. Order sets can be leveraged to standardize and streamline red blood cell exchanges. Lastly, numerous mechanisms help patients compensate for the cognitive sequelae of stroke.

Purpose To examine the relations between patient-reported outcomes (PROs) within a conceptual model for adults with sickle cell disease (SCD) ages 18 – 45 years enrolled in the multi-site Sickle Cell Disease Implementation Consortium (SCDIC) registry. We hypothesized that patient and SCD-related factors, particularly pain, and barriers to care would independently contribute to functioning as measured using PRO domains. Methods Participants (N = 2054) completed a 48-item survey including socio-demographics and PRO measures, e.g., social functioning, pain impact, emotional distress, and cognitive functioning. Participants reported on lifetime SCD complications, pain episode frequency and severity, and barriers to healthcare. Results Higher pain frequency was associated with higher odds of worse outcomes in all PRO domains, controlling for age, gender and site (OR range 1.02–1.10, 95% CI range [1.004–1.12]). Reported history of treatment for depression was associated with 5 of 7 PRO measures (OR range 1.58–3.28 95% CI range [1.18–4.32]). Fewer individual barriers to care and fewer SCD complications were associated with better outcomes in the emotion domain (OR range 0.46–0.64, 95% CI range [0.34–0.86]). Conclusions Study results highlight the importance of the biopsychosocial model to enhance understanding of the needs of this complex population, and to design multi-dimensional approaches for providing more effective interventions to improve outcomes.

Introduction: Sickle cell disease (SCD)-associated PH is associated with increased mortality when tricuspid regurgitant jet velocity (TRV) is ≥2.5m/s and mPAP is ≥25mmHg on right heart catheterization. RASA3, a ubiquitously expressed GTPase-activating protein, is integral to angiogenesis and maintenance of endothelial barrier function. Hypothesis: Loss of RASA3 leads to the development of PH in patients with SCD-associated PH and PAH. Methods: Cis-eQTLs were queried for RASA3 using whole genome genotype arrays and gene expression profiles from PBMCs of subjects with SCD from 3 cohorts. Genome-wide SNPs near or in the RASA3 gene that may associate with lung RASA3 expression were identified using data from the Genotype-Tissue Expression project, reduced to 9 tagging SNPs for RASA3 and associated with TRV≥2.5m/s and RHC hemodynamics. Associations between the top RASA3 SNP and RHC hemodynamics were tested using whole genome genotype data from subjects in the PAH Biobank. PAECs collected from explanted lungs from patients with IPAH or control lungs were cultured with RASA3 expression evaluated on Western blot. Results: PBMC-derived RASA3 expression was significantly lower in patients with SCD-associated PH using TRV≥2.5m/s and RHC hemodynamics, with significantly higher mortality (Figure 1). The risk allele (T) at rs9525228, an eQTL for RASA3, correlated with PH risk, higher TRV and higher PVR among PBMCs associated with decreased RASA3 expression in patients with SCD-associated PH. The T allele at rs9525228 was significantly associated with increased mPAP (p=0.014), PVR (p=0.041) and mean right atrial pressure (p=0.04) in patients from the PAH Biobank. RASA3 expression was significantly lower in PAECs from patients with IPAH compared to control. Conclusions: RASA3 is a novel candidate gene in SCD-associated PH and PAH, with RASA3 expression appearing to be protective. Further studies are ongoing to delineate the role of RASA3 in PH.

Background Across complex traits, common variants explain only a modest amount of variance, with SNP-heritability consistently below heritability estimates from close relatives. Here, we examined the contribution of rare variant to tobacco use risk in up to 26,000 individuals of European ancestry in the Trans-Omics for Precision Medicine (TOPMed) program with whole genome sequence (WGS;~30X coverage).Method We grouped about 35 million genetic variants by their minor allele frequencies (MAF) and linkage disequilibrium (LD) and estimated SNP-heritability for age of smoking initiation (N = 14,747), cigarettes smoked per day (N = 15,425), smoking cessation (N = 17,871) and initiation (N = 26,340) using linear mixed model. Rare variant population structure is detected and adjusted for by permutation procedure. We estimated an upper bound for narrow-sense heritability for tobacco use using available pedigrees consisting of close relatives in TOPMed.Results Rare variants with MAF 0.1–0.01%, mostly from non-protein altering region, accounted for 26% of variation in age of initiation and 15% for cessation. Follow-up analysis indicated that about one-third of these rare variants contribtion is potentially confounded with rare variants structure even after adjusting for principal components. After further conservative adjustment of population structure, we estimated SNP-based heritability to be 0.21 (SE = 0.08) for age of initiation, 0.15 (0.06) for cigarettes per day, 0.21 (0.09) for cessation, and 0.24 (0.07) for initiation, 1.8–4.5 times higher than previous SNP-based estimates. Our pedigree-based upper-bound for SNP-based heritability ranged from 0.18–0.35.Conclusion The substantial contribution of rare variants for several smoking phenotypes sheds light on the missing heritability and genetic etiology of tobacco use. It also informs fine-mapping strategies since the majority of the rare variant contribution was located in non-coding regulatory regions.

Impaired soluble guanylyl cyclase (sGC)-dependent nitric oxide (NO) signaling has been linked to numerous cardiovascular diseases (CVD) such as hypertension, myocardial infarction and atherosclerosis. Despite emerging evidence indicating the importance of sGC function within the cardiovascular system, the basic mechanisms that regulate sGC activity remain incompletely understood. Herein, we provide in vitro and in vivo evidence that cytochrome b5 reductase 3 (Cyb5R3) is an sGC heme iron reductase and regulates downstream cGMP signaling. Of major significance, we also demonstrate that a Cyb5R3 T116S polymorphism with allele frequency of 0.23 in African Americans associates with increase blood pressure and is incapable of reducing sGC. Proximity ligation assay (PLA) experiments show that endogenous Cyb5R3 and oxidized sGC associate. Knockdown of Cyb5R3 results in reduced cGMP production and downstream signaling in rat aortic smooth muscle cells (SMC). Overexpression of Cyb5R3 not only rescues cGMP production but also increases baseline cGMP, whereas T116S mutant does not. Finally, inhibition of Cyb5R3 in mice significantly increases systemic blood pressure. Our studies are the first to identify an sGC heme iron reductase, provide evidence for Cyb5R3 as a key biological regulator of sGC activity and vascular tone in SMC, and link a human polymorphism of Cyb5R3 to increased blood pressure; all of which may lead to the development of novel therapeutics targeting Cyb5R3 for the treatment of CVD. Importantly, the co-expression of Cyb5R3 and sGC in multiple cells types suggests that this regulation of sGC activity may have broad applications for multiple physiological and pathophysiological processes. Results: Conclusions

What is the topic of this review? The topic of this review is how Tibetans have adapted genetically to high altitude, particularly with reference to altitude-induced changes in metabolism. What advances does it highlight? It highlights recent work on metabolic phenotyping in Tibetans and demonstrates that selected genetic haplotypes influence their metabolism of fats and glucose. Recent studies have identified genes involved in high-altitude adaptation in Tibetans. Three of these genes (EPAS1, EGLN1 and PPARA) are associated with decreased haemoglobin levels compared with non-Tibetans living at altitude. Consistent with the phenotype, EGLN1 in Tibetans has a gain-of-function mutation that confers a higher affinity for oxygen, hence less sensitivity to hypoxia. Considering the demands imposed upon metabolism in meeting energy demands despite limitations on fuel oxidation, we hypothesized that other selected genes might alter metabolism to allow adaptation to altitude despite the desensitization of the upstream hypoxia sensing caused by the EGLN1 mutation that results in the failure to sense hypoxia. A shift in fuel preference to glucose oxidation and glycolysis at the expense of fatty acid oxidation would provide adaptation to decreased oxygen availability. Measurements of serum metabolites from Tibetans living at high altitude are consistent with this hypothesis; the EPAS1 haplotype is significantly associated with increased lactate levels (suggesting increased anaerobic metabolism), and the PPARA haplotype and serum free fatty acids are positively related (suggesting decreased fat oxidation). These data suggest that the high-altitude adaptations may offer protection from diabetes at high altitude but increase the risk of diabetes at lower elevations and/or with adoption of a non-traditional diet. It should also be considered in future work in the field that because iron is a cofactor for EGLN1, there may be significant associations of phenotypes with the significant degrees of variation seen in tissue iron among human populations.

Introduction: Interactions between platelets, leukocytes and endothelial cells are important during microvascular occlusion. Although the major receptors and counter receptors have been well identified, it remains unclear how heterotypic cell-cell interactions are regulated under inflammatory conditions. Hypothesis: We assessed the hypothesis that AKT, a Ser/Thr protein kinase, plays a regulatory role in platelet-neutrophil-endothelial cell interactions during vascular inflammation. Methods and Results: Using real-time fluorescence intravital microscopy to evaluate mice lacking specific Akt isoform, we demonstrated that Akt2, but not Akt1 and Akt3, regulates neutrophil adhesion and crawling, and platelet-neutrophil interactions on the activated endothelium during TNF-α-induced cremaster venular inflammation. Further, using chimeric mice generated from bone marrow transplants on wild-type (WT) and Akt2 knockout (KO) mice, we found that hematopoietic cell-associated AKT2 is important for the regulatory effect in vivo. Studies with an AKT2-specific inhibitor and cells isolated from WT and Akt KO mice revealed that platelet- and neutrophil-associated AKT2 regulates heterotypic neutrophil-platelet aggregation under conditions of shear. In particular, neutrophil AKT2 was critical for intracellular calcium mobilization, β2-talin1 interaction, and membrane translocation of αMβ2 integrin - a crucial receptor for neutrophil-platelet interactions. We observed that the basal phosphorylation levels of AKT isoforms are markedly increased in neutrophils and platelets from patients with sickle cell disease (SCD), an inherited hematological disorder associated with vascular inflammation and occlusion. AKT2 inhibition significantly reduced heterotypic aggregation of neutrophils and platelets isolated from SCD patients and diminished neutrophil adhesion and neutrophil-platelet aggregation in venules of Berkeley (SCD) mice, thereby improving blood flow rates. Conclusion: Our results provide genetic and pharmacologic evidence that neutrophil AKT2 regulates αMβ2 integrin function and is important for neutrophil recruitment and neutrophil-platelet interactions under thrombo-inflammatory conditions such as SCD.

Pulmonary hypertension (PH) is associated with adverse outcomes in adults with sickle-cell disease (SCD), but its importance in children is less clear. The aim of this study was to define the incidence and causes of PH in pediatric patients with SCD. Children with SCD (n = 310) and matched controls (n = 54) were prospectively enrolled under basal conditions. Participants underwent echocardiography, pulse oximetry, 6-minute walk tests, and hematologic testing. Echocardiographic measures were compared between patients with SCD and control subjects before and after adjusting for hemoglobin. Correlations of echocardiographic and clinical parameters were determined. Tricuspid regurgitation velocity (TRV) was elevated compared to controls (2.28 vs 2.10 m/s, p0.0001). Increased TRV was associated with left ventricular diastolic diameter, hemoglobin, and estimated left atrial pressure. TRV remained elevated when controlling for left ventricular diameter and left atrial pressure. Echocardiographically derived pulmonary resistance was not significantly different between patients with SCD and controls, although it was elevated in the SCD subgroup with elevated TRV. When controlling for hemoglobin, TRV was no longer statistically different, but pulmonary insufficiency velocity, septal wall thickness, and estimated pulmonary resistance were statistically higher. TRV, pulmonary insufficiency end-diastolic velocity, and markers of increased cardiac output were correlated with indicators of adverse functional status, including history of acute chest syndrome, stroke, transfusions, and 6-minute walk distance. In conclusion, children with SCD had mildly increased TRV that was correlated with increased cardiac output and left ventricular filling pressures. Hemoglobin-adjusted analysis also suggested a contribution of primary vascular changes.

Hemoglobin, the sole carrier of oxygen to tissues, accounts for most cytoplasmic protein of the erythrocyte, an enucleate cell lacking protein synthesizing machinery and with limited energy metabolism. While a number of genetic mechanisms can result in decreased hemoglobin concentration in the blood, this review concentrates on those that lead to increased hemoglobin mass, i.e. polycythemia or erythrocytosis. Polycythemia may be due to (a) mutations of the enzyme synthesizing 2, 3 BPG, a metabolic intermediate which regulates hemoglobin-oxygen affinity and thus oxygen delivery, (b) mutation of the alpha or beta globin genes that increase hemoglobin-oxygen affinity and thus decrease oxygen delivery, and (c) mutations of the erythropoietin receptor gene or genes regulating erythropoietin production that lead to increased production of erythrocytes. Primary polycythemias are caused by inherited or acquired somatic mutations affecting the hematopoietic progenitors. In contrast, in secondary polycythemia normal progenitors are activated by external factors present in increased concentration, most commonly erythropoietin. Some hypoxia sensing disorders blur the distinction between primary and secondary polycythemias and may deserve their own category. Most polycythemias are acquired, but both primary and secondary polycythemias may be inherited. In this review we will discuss the genetic heterogeneity of individual responses to hypoxia, and the current understanding of inherited primary and secondary polycythemias.

Death from malaria occurs from the complications of the infection: cerebral manifestations leading to coma and a severe and refractory anemia leading to hypoxia and cardiac decompensation. Several mechanisms have been identified to play a role in the pathogenesis of malarial anemia, such as erythrocyte lysis and phagocytosis, and sequestration of parasitized red blood cells, but recent data indicate that these mechanisms (singly or in combination) do not adequately explain the severity of this anemia. By contrast, hematologic studies have shown that bone marrow suppression and ineffective erythropoiesis contribute importantly to the severe anemia of malaria infection. The host mechanisms responsible for suppression of erythropoiesis may involve an excessive or sustained innate immune response or a pathologic skewing of the T-cell differentiation response with the attendant production of certain proinflammatory cytokines. Experimental data also indicate that severe malarial anemia is associated with the immunologic expression of a circulating inhibitor of erythropoiesis that functionally antagonizes the action of erythropoietin. We review the clinical and experimental basis for these concepts and discuss ongoing experimental and genetic studies aimed at unraveling the molecular basis of this malaria-induced bone marrow suppression.