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The efficacy and safety of rivipansel, a predominantly E-selectin antagonist, were studied in a phase 3, randomized, controlled trial for vaso-occlusive crisis (VOC) requiring hospitalization (RESET). A total of 345 subjects (204 adults and 141 children) were randomized and 320 were treated (162 with rivipansel, 158 with placebo) with an IV loading dose, followed by up to 14 additional 12-hourly maintenance doses of rivipansel or placebo, in addition to standard care. Rivipansel was similarly administered during subsequent VOCs in the Open-label Extension (OLE) study. In the full analysis population, the median time to readiness for discharge (TTRFD), the primary end point, was not different between rivipansel and placebo (−5.7 hours, P = .79; hazard ratio, 0.97), nor were differences seen in secondary end points of time to discharge (TTD), time to discontinuation of IV opioids (TTDIVO), and cumulative IV opioid use. Mean soluble E-selectin decreased 61% from baseline after the loading dose in the rivipansel group, while remaining unchanged in the placebo group. In a post hoc analysis, early rivipansel treatment within 26.4 hours of VOC pain onset (earliest quartile of time from VOC onset to treatment) reduced median TTRFD by 56.3 hours, reduced median TTD by 41.5 hours, and reduced median TTDIVO by 50.5 hours, compared with placebo (all P < .05). A similar subgroup analysis comparing OLE early-treatment with early-treatment RESET placebo showed a reduction in TTD of 23.1 hours (P = .062) and in TTDIVO of 30.1 hours (P = .087). Timing of rivipansel administration after pain onset may be critical to achieving accelerated resolution of acute VOC. Trial Registration: Clinicaltrials.gov, NCT02187003 (RESET), NCT02433158 (OLE).

Patients with sickle cell disease (SCD) have a lower risk for HIV-1 infection. We reported restriction of ex vivo HIV-1 infection in SCD peripheral blood mononuclear cells (PBMCs) that was due, in part, to the upregulation of antiviral, inflammatory, and hemolytic factors, including heme oxygenase-1 (HO-1). Here, we investigated whether individuals with sickle cell trait (SCT), who develop mild hemolysis, also restrict HIV-1 infection. Ex vivo infection of SCT PBMCs exhibited an approximately twofold reduction of HIV-1 replication and lower levels of HIV-1 reverse transcription products, 2-long terminal repeat circle, HIV-1 integration, and gag RNA expression. SCT PBMCs had higher HO-1 messenger RNA (mRNA) and protein levels and reduced ribonucleotide reductase 2 (RNR2) protein levels. HO-1 inhibition by tin porphyrin eliminated ex vivo HIV-1 restriction. Among Howard University clinic recruits, higher levels of HO-1 and RNR2 mRNA and lower HIV-1 env mRNA levels were found in SCT individuals living with HIV-1. To determine the population-level effect of SCT on HIV-1 prevalence, we assessed SCT among women living with HIV (WLH) in the WIHS (Women Interagency HIV-1 Study). Among WIHS African-American participants, the prevalence of SCT was lower among women with HIV compared with uninfected women (8.7% vs 14.2%; odds ratio, 0.57; 95% confidence interval, 0.36-0.92; P = .020). WIHS WLH with SCT had higher levels of CD4+/CD8+ ratios over 20 years of follow-up (P = .003) than matched WLH without SCT. Together, our findings suggest that HIV-1 restriction factors, including HO-1 and RNR2, might restrict HIV-1 infection among individuals with SCT and limit the pathogenicity of HIV.

In the US, mortality in sickle cell disease (SCD) increases after age 18–20 years. Biomarkers of mortality risk can identify patients who need intensive follow-up and early or novel interventions. We prospectively enrolled 510 SCD patients aged 3–20 years into an observational study in 2006–2010 and followed 497 patients for a median of 88 months (range 1–105). We hypothesized that elevated pulmonary artery systolic pressure as reflected in tricuspid regurgitation velocity (TRV) would be associated with mortality. Estimated survival to 18 years was 99% and to 25 years, 94%. Causes of death were known in seven of 10 patients: stroke in four (hemorrhagic two, infarctive one, unspecified one), multiorgan failure one, parvovirus B19 infection one, sudden death one. Baseline TRV ≥2.7 m/second (>2 SD above the mean in age-matched and gender-matched non-SCD controls) was observed in 20.0% of patients who died vs 4.6% of those who survived (P = .012 by the log rank test for equality of survival). The baseline variable most strongly associated with an elevated TRV was a high hemolytic rate. Additional biomarkers associated with mortality were ferritin ≥2000 μg/L (observed in 60% of patients who died vs 7.8% of survivors, P < .001), forced expiratory volume in 1 minute to forced vital capacity ratio (FEV1/FVC)

Genetic studies on telomere length are important for understanding age-related diseases. Prior GWAS for leukocyte TL have been limited to European and Asian populations. Here, we report the first sequencing-based association study for TL across ancestrally-diverse individuals (European, African, Asian and Hispanic/Latino) from the NHLBI Trans-Omics for Precision Medicine (TOPMed) program. We used whole genome sequencing (WGS) of whole blood for variant genotype calling and the bioinformatic estimation of telomere length in n=109,122 individuals. We identified 59 sentinel variants (p-value

Direct-acting antiviral inhibitors have revolutionized the treatment of hepatitis C virus (HCV) infected patients. Herein is described the discovery of velpatasvir (VEL, GS-5816), a potent pan-genotypic HCV NS5A inhibitor that is a component of the only approved pan-genotypic single-tablet regimens (STRs) for the cure of HCV infection. VEL combined with sofosbuvir (SOF) is Epclusa®, an STR with 98% cure-rates for genotype 1-6 HCV infected patients. Addition of the pan-genotypic HCV NS3/4A protease inhibitor voxilaprevir to SOF/VEL is the STR Vosevi®, which affords 97% cure-rates for genotype 1-6 HCV patients who have previously failed another treatment regimen.

Treatment of hepatitis C virus (HCV) infection has been historically challenging due the high viral genetic complexity wherein there are eight distinct genotypes and at least 86 viral subtypes. While HCV NS3/4A protease inhibitors are an established treatment option for genotype 1 infection, limited coverage of genotypes 2 and/or 3 combined with serum alanine transaminase (ALT) elevations for some compounds has limited the broad utility of this therapeutic class. Our discovery efforts were focused on identifying an NS3/4A protease inhibitor with pan-genotypic antiviral activity, improved coverage of resistance associated substitutions, and a decreased risk of hepatotoxicity. Towards this goal, distinct interactions with the conserved catalytic triad of the NS3/4A protease were identified that improved genotype 3 antiviral activity. We further discovered that protein adduct formation strongly correlated with clinical ALT elevation for this therapeutic class. Improving metabolic stability and decreasing protein adduct formation through structural modifications ultimately resulted in voxilaprevir. Voxilaprevir, in combination with sofosbuvir and velpatasvir, has demonstrated pan-genotypic antiviral clinical activity. Furthermore, hepatotoxicity was not observed in Phase 3 clinical trials with voxilaprevir, consistent with our design strategy. Vosevi® (sofosbuvir, velpatasvir, and voxilaprevir) is now an approved pan-genotypic treatment option for the most difficult-to-cure individuals who have previously failed direct acting antiviral therapy.

Introduction: Chronic kidney disease (CKD) is a prevalent complication of sickle cell anemia (SCA). Hyperfiltration that delayed detection of CKD is common in SCA patients. Identification of novel urinary biomarkers correlating with glomerular filtration rates may help to detect and predict progression of renal disease. Methods: Reanalysis of mass spectra of urinary samples obtained from University of Illinois at Chicago identified kringle domain-containing protein HGFL. Results: HGFL levels correlated with hyperfiltration, were significantly reduced at CKD stage 1 compared to stage 0, negatively correlated with progression of CKD and were suitable for differentiation of stage 1. Better prediction of CKD progression to stage 2 was observed for HGFL-based risk prediction compared to the estimated glomerular filtration rate (eGFR)-based prediction. Results from a Howard University patient cohort supported the utility of HGFL-based test for the differentiation of stage 1 of CKD. Conclusion: Urinary HGFL may contribute additional information beyond eGFR and improve diagnosis of early-stage CKD in SCA patients.

Background: Sickle cell nephropathy (SCN) is one of the most common complications of SCD, leading in most cases to chronic kidney disease (CKD) and end-stage renal disease (ESRD). Despite the high prevalence of CKD in sickle cell disease (SCD) patients, there remains a poor understanding of the pathophysiological mechanism of SCN and a lack of biomarkers for early detection of SCD-associated CKD. Soluble urokinase-type plasminogen activator receptor (suPAR) is an emerging biomarker of CKD. suPAR is a member of the fibrinolytic system, which is dysregulated in SCD patients. Objective: To evaluate suPAR as a biomarker of SCD-associated nephropathy and identify plasma proteases responsible for its increase in SCD. Methods: The study was approved by Howard University review board (IRB) and all subjects provided written inform consent prior to the sample collection. Whole blood and urine samples were collected from 77 SCD patients and 10 healthy individuals, and plasma was isolated. Levels of creatinine and cystatin C in plasma and albumin and creatinine in urine were measured by ELISA. eGFR was calculated using CKD-EPI creatinine-cystatin equation, and CKD stages were assigned. Plasma suPAR was measured by ELISA and was correlated with CKD stages. The activities of candidates uPAR proteases: Neutrophile elastase (NE), urokinase-type plasminogen activator (uPA) and plasmin in plasma samples from SCD patients were measured and compared to healthy participants. Results: The average age of SCD patients was 42.5 years (range 18-67 years). Most patients had HbSS genotype (67.5%),19.5% of patients were HbSC (hemoglobin C sickle cell compound heterozygous), and 13% had HbS β-thalassemia. More than half (53.2 %) were females. We observed an increased level of plasma suPAR (>3ng/ml) in more than 60% of SCA patients without renal disease, representing a risk factor for CKD progression. Plasma suPAR levels further increased in the patients with CKD and positively correlated with stages of CKD (r=0.419, R2=0.1696). Analysis of plasma proteases that cleaved uPAR producing soluble peptides (suPAR) demonstrated increased urokinase-type plasminogen activator (uPA) activity without significant changes in neutrophile elastase. Conclusion: This study validated plasma suPAR as a potential marker of CKD in SCD patients and identified plasma uPA as a uPAR protease that may increase circulating suPAR in SCD. Future longitudinal analysis of suPAR levels in patients with SCA is needed. Acknowledgments: We thank Drs. Namita Kumari and Xiaomei Niu for their help in samples identification. This work was supported by NIH Research Grants 1R01HL125005-06A1, 5U54MD007597, 1P30AI117970-06,1UM1AI26617, and 1SC1HL150685. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

BackgroundSickle Cell Disease (SCD) is an inherited blood disorder that leads to hemolytic anemia, pain, organ damage and early mortality. It is characterized by polymerized deoxygenated hemoglobin, rigid sickle red blood cells and vaso-occlusive crises (VOC). Recurrent hypoxia-reperfusion injury in the gut of SCD patients could increase tissue injury, permeability, and bacterial translocation. In this context, the gut microbiome, a major player in health and disease, might have significant impact. This study sought to characterize the gut microbiome in SCD.MethodsStool and saliva samples were collected from healthy controls (n = 14) and SCD subjects (n = 14). Stool samples were also collected from humanized SCD murine models including Berk, Townes and corresponding control mice. Amplified 16S rDNA was used for bacterial composition analysis using Next Generation Sequencing (NGS). Pairwise group analyses established differential bacterial groups at many taxonomy levels. Bacterial group abundance and differentials were established using DeSeq software.ResultsA major dysbiosis was observed in SCD patients. The Firmicutes/Bacteroidetes ratio was lower in these patients. The following bacterial families were more abundant in SCD patients: Acetobacteraceae, Acidaminococcaceae, Candidatus Saccharibacteria, Peptostreptococcaceae, Bifidobacteriaceae, Veillonellaceae, Actinomycetaceae, Clostridiales, Bacteroidacbactereae and Fusobacteriaceae. This dysbiosis translated into 420 different operational taxonomic units (OTUs). Townes SCD mice also displayed gut microbiome dysbiosis as seen in human SCD.ConclusionA major dysbiosis was observed in SCD patients for bacteria that are known strong pro-inflammatory triggers. The Townes mouse showed dysbiosis as well and might serve as a good model to study gut microbiome modulation and its impact on SCD pathophysiology.

Pain is the hallmark of sickle cell anemia (SCA), presenting as recurrent acute events or chronic pain. Central sensitization, or enhanced excitability of the central nervous system, alters pain processing and contributes to the maintenance of chronic pain. Individuals with SCA demonstrate enhanced sensitivity to painful stimuli however central mechanisms of pain have not been fully explored. We hypothesized that adults with SCA would show evidence of central sensitization as observed in other diseases of chronic pain. Methods We conducted a prospective study of static and dynamic quantitative sensory tests in 30 adults with SCA and 30 matched controls. Results Static thermal testing using cold stimuli showed lower pain thresholds (p = 0.04) and tolerance (p = 0.04) in sickle cell subjects, but not for heat. However, SCA subjects reported higher pain ratings with random heat pulses (p < 0.0001) and change in scores with temporal summation at the heat pain threshold (p = 0.002). Similarly, with the use of pressure pain stimuli, sickle cell subjects reported higher pain ratings (p = 0.04), but not higher pressure pain tolerance/thresholds or allodynia to light tactile stimuli. Temporal summation pain score changes using 2 pinprick probes (256 and 512 mN) were significantly greater (p = 0.004 and p = 0.008) with sickle cell, and delayed recovery was associated with lower fetal hemoglobin (p = 0.002 and 0.003). Conclusions Exaggerated temporal summation responses provide evidence of central sensitization in SCA. Implications The association with fetal hemoglobin suggests this known SCA modifier may have a therapeutic role in modulating central sensitization.

Pancreatic cancer has one of the worst survival rates amongst all forms of cancer because its symptoms manifest later into the progression of the disease. One of those symptoms is jaundice, the yellow discoloration of the skin and sclera due to the buildup of bilirubin in the blood. Jaundice is only recognizable to the naked eye in severe stages, but a ubiquitous test using computer vision and machine learning can detect milder forms of jaundice. We propose BiliScreen, a smartphone app that captures pictures of the eye and produces an estimate of a person's bilirubin level, even at levels normally undetectable by the human eye. We test two low-cost accessories that reduce the effects of external lighting: (1) a 3D-printed box that controls the eyes' exposure to light and (2) paper glasses with colored squares for calibration. In a 70-person clinical study, we found that BiliScreen with the box achieves a Pearson correlation coefficient of 0.89 and a mean error of -0.09 ± 2.76 mg/dl in predicting a person's bilirubin level. As a screening tool, BiliScreen identifies cases of concern with a sensitivity of 89.7% and a specificity of 96.8% with the box accessory.

Vaso-Occlusive Crises (VOCs) in patients with SCD cause acute and chronic morbidity including disabling pain, hospitalizations, missed school and work, end-organ damage, and early mortality. VOC prophylaxis, while beneficial, does not address the disabling pain and morbidity of breakthrough VOC events that still occur. E-selectin upregulation on vascular endothelium leads to leukocyte trapping, activation and aggregation and is a critical driver of acute VOC (Morikis et al, Blood 2017). Rivipansel, a pan-selectin inhibitor with potent activity against E-selectin, prevents interaction between leukocytes and vascular endothelium (Morikis et al, Blood 2017) and increases blood flow by reducing cell-cell aggregates and vascular occlusion in a SCD mouse model (Chang et al, Blood 2010). A phase 2 study of rivipansel in VOC demonstrated shorter hospital stays and reduced opioid use (Telen et al Blood, 2015). The RESET trial (NCT02187003) was a phase 3, randomized, double-blind, placebo-controlled, study of the efficacy and safety of rivipansel for VOC requiring hospitalization. Three hundred forty-five patients (204 patients ≥18 and 141 patients 6-17 years of age) were randomized to an intravenous rivipansel loading dose, followed by up to 14 additional doses Q 12 hr, or placebo, in addition to standard care. Overall, 320 were treated: 162 with rivipansel, 158 with placebo. Pre-treatment variables were well balanced, including concomitant hydroxyurea use, genotype, chronic opioid use, gender, and country. The primary endpoint was time to readiness for discharge (TTRFD), and key secondary endpoints were time to discharge (TTD), time to discontinuation of IV opioids (TTDIVO), and cumulative IV opioid use (CIVO). Hazard ratios or ratios of medians, and confidence intervals were calculated for each. Both study arms had comparable baseline soluble E-selectin (sE-sel) levels and inflammatory/coagulation biomarkers. Median sE-sel in the rivipansel group decreased by 59% from baseline after a loading dose while increasing by 9% in the placebo group. Although the RESET study did not show statistically significant improvements in outcomes for the total population, additional analysis demonstrated that rivipansel treatment within 26.4 hr of pain onset (earliest quartile of duration of VOC until treatment) reduced median TTRFD by 56.3 hrs (from 122.0 to 65.7 hrs), reduced median TTD by 41.5 hrs (from 112.8 to 71.3 hrs), and reduced median TTDIVO by 50.5 hrs (from 104.0 to 53.5 hrs), compared to placebo. There was also a consistent trend for lower hazard ratios for rivipansel treatment earlier during VOC (up to ~36 hr from onset) for all endpoints (Figure 1). Pediatric subjects (6-17 yrs, n = 141) were 41% of patients treated in the RESET trial (71 in rivipansel arm, 70 in placebo arm). As in the overall population the observed benefit with rivipansel in pediatric subjects depended on duration of VOC prior to treatment. Children 6-17 yrs of age treated with rivipansel within 30 hrs of onset of VOC experienced reduction in median TTRFD by 29.3 hrs (from 94.1 to 64.8 hrs), reduction in median TTD by 23.2 hrs (from 92.8 to 69.6 hrs), and reduction in median TTDIVO by 15.4 hrs (from 68.9 to 53.5 hrs). Early treatment with rivipansel decreased median TTRFD by more than one day and led to more children ready for discharge by 24, 48, and 72 hrs, compared to patients receiving placebo (Table 1). Additionally, pain scale assessments (VAS and Faces scale) showed a substantial reduction in the time to first clinically meaningful reduction in pain (data not shown). Thus, the hazard ratios for the efficacy endpoints favored early rivipansel treatment over placebo in the overall population and the pediatric population (Table 2). Summary: Rivipansel administered early in VOC results in clinically meaningful benefit for adults and children with SCD, shortening IV opioid use and hospital stay. Biomarker data confirm on-target effect, suggesting that the diminishing effect of later rivipansel treatment results from downstream pathophysiology. These findings suggest the utility of early treatment to shorten or interrupt acute VOCs, analogous to thrombolysis for heart attack or stroke. This could change the VOC treatment paradigm from deferral of hospitalization to one of early intervention to reduce length of hospitalization and IV opioid requirement, relieve VOC symptoms, and possibly mitigate end-organ damage from tissue ischemia. Disclosures Dampier: Merck: Research Funding; Hudson Publishing Company: Consultancy, Research Funding; CLS Behring: Consultancy, Other: DSMB Chair; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Micelle Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cyclerion: Consultancy, Other: DSMB Chair; Novartis: Research Funding. Telen:GlycoMimetics Inc.: Consultancy; Forma Therapeutics: Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Wun:GlycoMimetics, Inc.: Consultancy; Pfizer, Inc.: Other: Steering Committee for clinical study, Research Funding. Smith:Incyte: Other: Investigator; Pfizer: Consultancy; Ironwood: Consultancy; Novo Nordisk: Consultancy; Imara: Research Funding; Shire: Research Funding; NHLBI: Research Funding; Patient-Centered Outcomes Research Institute: Other: Investigator, Research Funding; Health Resources and Services Administration: Other: Investigator, Research Funding; Shire, Inc.: Other: Investigator, Research Funding; Global Blood Therapeutics, Inc.: Consultancy, Research Funding; Novartis, Inc.: Consultancy, Other: Investigator, Research Funding; Emmaeus Pharmaceuticals, Inc.: Consultancy; GlycoMimetics, Inc.: Consultancy. Brown:Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Forma Therapeutics, Inc,: Research Funding; Imara, Inc.: Consultancy, Research Funding; Novartis, Inc.: Consultancy, Research Funding. Desai:Pfizer, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; GBT, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ironwood Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Rockpointe Continuing Medical Education Company: Consultancy. El Rassi:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cyclerion: Research Funding; Pfizer: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bluebird bio: Membership on an entity's Board of Directors or advisory committees. Kanter:Wells Fargo: Honoraria; Medscape: Honoraria; Guidepoint Global: Honoraria; NHLBI Sickle Cell Advisory Board: Membership on an entity's Board of Directors or advisory committees; SCDAA Medical and Research Advisory Board: Membership on an entity's Board of Directors or advisory committees; AGIOS: Membership on an entity's Board of Directors or advisory committees; BEAM: Membership on an entity's Board of Directors or advisory committees; Jeffries: Honoraria; GLG: Honoraria; Sanofi: Consultancy; bluebird bio, inc: Consultancy, Honoraria; Novartis: Consultancy; Cowen: Honoraria. Pastore:Pfizer: Honoraria. Rothman:Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Agios Pharmaceuticals: Honoraria, Research Funding. Readett:Pfizer, Inc.: Current Employment. Lozier:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Thackray:GlycoMimetics, Inc.: Current Employment. Hassell:Pfizer, Inc.: Other: RESET Study Steering Committee; Pfizer, Inc.: Research Funding.

There has been remarkable progress in the treatment of hepatitis C virus (HCV) infection with the approval of multiple direct acting antiviral (DAA) regimens. Although overall high HCV cure rates are now generally obtained, there are patients who fail the currently approved therapeutics and require a retreatment option. While HCV NS3/4A protease inhibitors (PIs) have proven highly effective for treating genotype (GT) 1 infection, decreased potency against GT2 and/or GT3 along with observed ALT elevation for some PIs has limited the broad utility of this treatment class. Described herein are the discovery efforts resulting in the HCV NS3/4A PI voxilaprevir. Distinct interactions with the conserved NS3 protease catalytic triad were identified that improve GT3 potency and activity against some common resistance variants. Furthermore, multiple parameters were analyzed to determine potential drivers of hepatotoxicity for HCV PIs in the clinic and it was determined that protein adduct formation had a high correlation with clinical ALT elevation. Therefore, structural modifications were made that both improved metabolic stability and reduced protein adduct formation, ultimately resulting in voxilaprevir. Consistent with our optimization strategy, the combination of voxilaprevir with sofosbuvir (NS5B inhibitor) and velpatasvir (NS5A inhibitor) has proven highly effective across genotypes in Phase 3 clinical trials and hepatoxicity has not been observed. Vosevi® (sofosbuvir, velpatasvir, and voxilaprevir) was approved in 2017 as a pan-genotypic treatment option for individuals who have previously failed DAA regimens and, for some DAA-naive patients, represents a treatment regimen of shortened duration (8 weeks).

The Republic of Congo (RC) ([Figure 1][1]) has one of highest prevalence rates of sickle cell trait (SCT) in the world. The prevalence of sickle cell disease (SCD) is estimated to be at least 1.5% with an expected nationwide SCD patient population of 70 000. RC has no established national newborn

Memory stem T cells (TSCMs) constitute a long-lived, self-renewing lymphocyte population essential for the maintenance of functional immunity. Hallmarks of autoimmune disease pathogenesis are abnormal CD4+ and CD8+ T cell activation. We investigated the TSCM subset in 55, 34, 43, and 5 patients with acquired aplastic anemia (AA), autoimmune uveitis, systemic lupus erythematosus, and sickle cell disease, respectively, as well as in 41 age-matched healthy controls. CD8+ TSCM frequency was significantly increased in AA compared with healthy controls. An increased CD8+ TSCM frequency at diagnosis was associated with responsiveness to immunosuppressive therapy, and an elevated CD8+ TSCM population after immunosuppressive therapy correlated with treatment failure or relapse in AA patients. IFN-γ and IL-2 production was significantly increased in various CD8+ and CD4+ T cell subsets in AA patients, including CD8+ and CD4+ TSCMs. CD8+ TSCM frequency was also increased in patients with autoimmune uveitis or sickle cell disease. A positive correlation between CD4+ and CD8+ TSCM frequencies was found in AA, autoimmune uveitis, and systemic lupus erythematosus. Evaluation of PD-1, CD160, and CD244 expression revealed that TSCMs were less exhausted compared with other types of memory T cells. Our results suggest that the CD8+ TSCM subset is a novel biomarker and a potential therapeutic target for AA.

Background: Blood transfusion is a common therapy for sickle cell disease (SCD). Although, highly effective, a major limitation is development of alloantibodies to minor blood group antigens on donor red cells. Alloimmunization has a prevalence of 2-5% for transfusions in the general population, but it is significantly higher in SCD. Risk factors for alloimmunization have been poorly characterized, although number of lifetime transfusions is an important risk factor. Alloimmunization has been clinically observed in children with a prevalence of about 7%. With development of each antibody, blood donor matching becomes increasingly difficult and expensive with an increased risk for transfusion reactions and diminished availability of compatible red cell units for treatment of SCD. The ability to identify risk factors for developing alloantibodies would be beneficial for clinicians. To identify markers for alloimmunization in SCD, we have analyzed children and adults who developed this complication. Methods: We analyzed The Pulmonary Hypertension and Hypoxic Response in Sickle Cell Disease (PUSH) study, which enrolled n=468 pediatric and n=59 adult SCD subjects. In both children and adults, alloimmunization cases were defined as a history of at least 1 alloantibody. Controls in both cohorts were defined as subjects with no history of alloantibodies and receipt of more than 10 lifetime red cell transfusions. All others within the study who did not meet these criteria were assigned to a third comparison group. To identify differences between cases, controls and all others, we performed univariate analyses (using ANOVA or Kruskal Wallace where appropriate) for clinical parameters and laboratories. Case control comparisons were also performed for selected variables and plasma levels for 11 cytokines. Results were further analyzed using regression modeling. Results: The overall prevalence of alloimmunization was 7.3% among children (34/468 subjects; median age 12, range 3-20 years) compared to 28.8% in adults (17/59 subjects; median age 37, range 18-73 years). When only considering those with >10 lifetime transfusions, the prevalence was considerably higher at 29.3% and 54.8% in children and adults, respectively. At the same time, 8 pediatric (23.5%) and 5 adult (29.4%) alloimmunization cases had received fewer than 10 transfusions. In a 3-way pediatric cohort comparison (cases, controls and all others), risk factors associated with alloimmunization included SS genotype, older age and markers of more severe disease (higher ferritin, WBCs, platelets and total bilirubin). Comparison of cases to controls showed alkaline phosphatase (P=0.05) was significantly lower in cases, whereas AST (P=0.02) was significantly higher even with adjustment for age. Levels of plasma cytokines MCP-1 (P=0.01) and IFNgamma (P=0.08) were lower in cases from a subset of the pediatric cohort. In adults, only 4/59 (6.8%) subjects had never received a lifetime transfusion (all non-SS). In the adult 3-way comparisons, only SS genotype and higher ferritin were associated with alloimmunization. The adult case control analysis showed higher absolute monocyte count (P=0.02), absolute eosinophil count (P=0.04) and absolute basophil count (P=0.008) in association with alloimmunization cases. In addition, alkaline phosphatase was again significantly lower among cases (P=0.02) as seen in the pediatric cohort. There were no significant differences in cytokine levels among adults. Conclusions: When considering only transfused SCD patients, the prevalence of alloimmunization is higher than 30%. As seen in prior studies, higher lifetime red cell transfusions are an important risk factor especially among adults where most patients have received transfusions. Children who develop alloantibodies appear to have laboratory markers of more severe disease, but this is not observed in adults. A novel association observed across both pediatric and adult subjects is a significantly lower serum alkaline phosphatase in those with alloantibodies. The results of this study suggest a need for improved tracking of red cell transfusion therapy in the US for SCD patients due to a high prevalence of alloimmunization. Further study is also needed to elucidate the significance of the alkaline phosphatase association. Disclosures Gordeuk: CSL Behring: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Novartis: Consultancy; Ironwood: Research Funding; Imara: Research Funding.

Introduction: Transfusional iron (Fe) overload is a significant problem among patients with chronic, transfusion-dependent anemias. Iron overload is an important problem in pediatric sickle cell disease (SCD) patients on chronic transfusion regimens predominantly for primary and secondary prevention of stroke. Coexistent hereditary iron overload conditions contribute to the iron overload phenotype in SCD. For example, the Q248H mutation (rs11568350) in SLC40A1, which encodes ferroportin (FPN), is associated with a mild tendency to increase serum ferritin in the general population and with increased ferritin levels in SCD patients. Nevertheless, the molecular mechanisms underlying the progression to iron overload in SCD patients are poorly understood and more sensitive markers for outcome prediction that can be applied at early clinical stages are lacking. We hypothesize that genetic variation modifies the risk for iron overload in SCD patients and seek to validate previously identified mutation and identify novel genetic markers of iron overload among participants from TOPMed SCD cohorts by performing whole genome sequencing (WGS) association analyses. Methods: The WGS was performed by several national sequencing centers sponsored by NHLBI's TOPMed program at an average depth of 30× using DNA from SCD patient blood samples. Variant calling was performed jointly across TOPMed studies for all samples using the GotCloud pipeline by the TOPMed Informatics Research Center. The TOPMed data Coordinating Center performed quality control for sample identity. The data across the following studies were shared through the database of Genotype and Phenotype (dbGaP) exchange area: Howard PUSH SCD (N=370), OMG SCD (N=636), Walk PHaSST SCD (N=381) and REDS-III Brazil SCD (N=2620) with a total sample size of 4007. The study was approved by the appropriate institutional review boards (IRB) and informed consent was obtained from all participants. Genome Wide Association Analysis of iron overload was carried out using the University of Michigan ENCORE server. We performed single variant tests to test the association of log-transformed serum ferritin levels with single nucleotide variants (SNVs) while adjusting for sex, age, self-reported race, numbers of lifetime red blood transfusions and genetic substructure (PC's 1-10). We used a significance threshold of p Results: We first included PUSH SCD, OMG SCD and Walk PHaSST SCD cohorts with 840 serum ferritin samples in the WGS association analyses, which revealed at the genome-wide level a new rare variant rs137929759 (chr7:49538810 (GRCh38.p12), MAF=0.0043532, p=2.25×10−8). A few variants such as rs80097634 in gene AL163195.3 and RNASE11 (Chr14:20579417. MAF=0.050373, p=3.1×10-7) were close to the genome-wide significance level. We confirmed previously identified associations in SLC40A1 for ferritin (rs11568350, Chr2:189565370, MAF = 0.16853, p = 5.2×10−4). We also found several variants in AC105411.1, TJP1 and DCC that were close to genome-wide significance level. Further analysis will be carried out on the cloud-based platform provided by NHLBI BioData Catalyst using data from all the four cohorts to validate the previous analysis and expand to related phenotype such as transferrin, iron-overload status. Discussion: In this study we identified common and rare variants that associate with serum ferritin concentration. The results from this pilot study point to novel gene variants that might contribute to iron overload in SCD patients and serve as new biomarkers. Future analysis is needed to determine whether the identified variants can also help with therapeutics and outcome prediction for early stages of SCD-associated iron overload. Our findings will be useful for the future treatment of SCD patients and design of novel SCD therapeutics. ACKNOWLEDGMENTS: This work was supported by NIH Research Grants (1P50HL118006, and 1R01HL125005). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosures Gordeuk: Imara: Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy, Research Funding; Ironwood: Research Funding; Novartis: Consultancy. Telen:CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Forma Therapeutics: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlycoMimetics Inc.: Consultancy.

Sickle cell disease (SCD) is a hemoglobinopathy that affects more than 100,000 individuals in the United States. The disease is characterized by the presence of sickle hemoglobin and recurrent episodes of pain. Some individuals with SCD experience frequent hospitalizations and a high burden of pain. The role of central mechanisms in SCD pain has not been explored. Twenty-five adolescents and young adults with SCD underwent functional magnetic resonance imaging. Participants were stratified into groups with high pain or low pain based on the number of hospitalizations for pain in the preceding 12 months. Resting state functional connectivity was analyzed using seed-based and dual regression independent component analysis. Intrinsic brain connectivity was compared between the high pain and low pain groups, and association with fetal hemoglobin, a known modifier of SCD, was explored. Patients in the high pain group displayed an excess of pronociceptive connectivity such as between anterior cingulate and default mode network structures, such as the precuneus, whereas patients in the low pain group showed more connectivity to antinociceptive structures such as the perigenual and subgenual cingulate. Although a similar proportion of patients in both groups reported that they were on hydroxyurea, the fetal hemoglobin levels were significantly higher in the low pain group and were associated with greater connectivity to antinociceptive structures. These findings support the role of central mechanisms in SCD pain. Intrinsic brain connectivity should be explored as a complementary and objective outcome measure in SCD pain research. Perspective Altered connectivity patterns associated with high pain experience in patients with sickle cell disease suggest a possible role of central mechanisms in sickle cell pain. Resting state brain connectivity studies should be explored as an effective methodology to investigate pain in SCD.

Emerging evidence indicates that microRNA control and modulate immunity. MicroRNA have not been investigated in acquired aplastic anemia, a T-cell-mediated immune disease. Analysis of 84 microRNA expression levels in CD4(+) and CD8(+) T cells of patients with aplastic anemia revealed concurrent down-regulation of miR-126-3p, miR-145-5p, miR-223-3p, and miR-199a-5p (3-fold change, P0.05) in both T-cell populations, which were unique in aplastic anemia compared to other hematologic disorders. MiR-126-3p and miR-223-3p were down-regulated in CD4(+) T effector memory cells, and miR-126-3p, miR-145-5p, and miR-223-3p were down-regulated in CD8(+) T effector memory and terminal effector cells. Successful immunosuppressive therapy was associated with restoration to normal expression levels of miR-126-3p, miR-145-5p, and miR-223-3p (2-fold change, P0.05). In CD4(+) and CD8(+) T cells in aplastic anemia patients, MYC and PIK3R2 were up-regulated and proved to be targets of miR-145-5p and miR-126-3p, respectively. MiR-126-3p and miR-145-5p knockdown promoted proliferation and increased interferon-γ and granzyme B production in both CD4(+) and CD8(+) T cells. Our work describes previously unknown regulatory roles of microRNA in T-cell activation in aplastic anemia, which may open a new perspective for development of effective therapy. Clinicaltrials.gov identifier: NCT 01623167.

Summary Background Increased life expectancy in sickle cell disease (SCD) has resulted in greater recognition of the consequences of repeated intravascular vaso-occlusion and chronic haemolysis to multiple organ systems. Aim To report the long-term consequences of liver dysfunction in SCD. Methods A cohort of SCD patients was prospectively evaluated at the National Institutes of Health (NIH) Clinical Center. The association of mortality with liver enzymes, parameters of liver synthetic function and iron overload was evaluated using Cox regression. Results Exactly, 247 SCD patients were followed up for 30 months of whom 22 (9%) died. After controlling for predictors, increased direct bilirubin (DB), ferritin, alkaline phosphatase and decreased albumin were independently associated with mortality. In a multivariable model, only high DB and ferritin remained significant. Ferritin correlated with hepatic iron content and total blood transfusions but not haemolysis markers. Forty patients underwent liver biopsies and 11 (28%) had fibrosis. Twelve of 26 patients (48%) had portal hypertension by hepatic venous pressure gradient (HVPG) measurements. All patients with advanced liver fibrosis had iron overload; however, most patients (69%) with iron overload were without significant hepatic fibrosis. Ferritin did not correlate with left ventricular dysfunction by echocardiography. DB correlated with bile acid levels suggesting liver pathology. Platelet count and soluble CD14 correlated with HVPG indicating portal hypertension. Conclusions Ferritin and direct bilirubin are independently associated with mortality in sickle cell disease. Ferritin likely relates to transfusional iron overload, while direct bilirubin suggests impairment of hepatic function, possibly impairing patients’ ability to tolerate systemic insults.

Rationale: Patients with sickle cell disease (SCD) have markers of chronic inflammation, but the mechanism of inflammation and its relevance to patient survival are unknown. Objective: To assess the relationship between iron, inflammation, and early death in SCD. Methods and Results: Using peripheral blood mononuclear cell transcriptome profile hierarchical clustering, we classified 24 patients and 10 controls in clusters with significantly different expression of genes known to be regulated by iron. Subsequent gene set enrichment analysis showed that many genes associated with the high iron cluster were involved in the toll-like receptor system (TLR4, TLR7, and TLR8) and inflammasome complex pathway (NLRP3, NLRC4, and CASP1). Quantitative PCR confirmed this classification and showed that ferritin light chain, TLR4, and interleukin-6 expression were >100-fold higher in patients than in controls ( P P P Conclusions: Gene expression markers of high intracellular iron in patients with SCD are associated with markers of inflammation and mortality. The results support a model in which intracellular iron promotes inflammatory pathways, such as the TLR system and the inflammasome, identifying important new pathways for additional investigation.

Background Voxilaprevir (VOX; GS-9857) is a pangenotypic HCV NS3/4A protease inhibitor (PI) with potent antiviral activity against HCV genotypes (GTs) 1–6 and improved coverage of GT1 NS3 resistance-associated substitutions (RAS) associated with other HCV PIs. In a 3-day Phase Ib monotherapy study in patients infected with HCV GT1a, 1b, 2, 3 and 4, VOX was well-tolerated and resulted in maximal mean viral load reduction >3 log10 IU/ml at the 100 mg dose across all genotypes evaluated. This report characterizes the HCV NS3 RAS in the study. Methods The NS3 gene was amplified and successfully deep sequenced using MiSeq for 66 patients at baseline and 61 patients post-baseline using 15% and 1% assay cutoffs. Results With a 15% assay cutoff, pretreatment HCV NS3 RAS were present in the HCV of 38% (9/24) of patients with GT1a and 5% (1/19) with GT3a; there were no pre-treatment NS3 RAS present in patients with GT1b ( n=6), GT2 ( n=7) or GT4 ( n=4). In patients with and without pretreatment NS3 RAS, ≥3.4 log10 mean maximal viral load reductions over 3 days of VOX administration were observed. The majority of patients did not have detectable treatment-emergent NS3 RAS and only 12% (7/53) and 26% (14/53) had emergent NS3 RAS using 15% and 1% cutoffs, respectively. No NS3 RAS were detected in patients with GT2 or GT4. A156T or A156V were the most prevalent emergent NS3 RAS in patients with GT1a or GT1b infection, but were not observed in patients with GT3 infection. Conclusions The lack of selection of NS3 RAS in the majority of patients demonstrates a high resistance barrier for VOX. ClinicalTrails.gov identifier NCT02185794.

Patients with sickle cell disease (SCD) are occasionally prescribed systemic corticosteroids to treat steroid-responsive conditions. Additionally, use of systemic corticosteroids for sickle cell pain episodes and acute chest syndrome is under investigation. We report 4 patients with SCD who developed severe vaso-occlusive events following the administration of systemic steroids. We also review similar cases from the literature and suggest measures for reducing the potential risk associated with use of systemic corticosteroids in this group of patients. We conclude that corticosteroids should be used with caution in patients with SCD.

Background— Fetal hemoglobin (HbF) is the major modifier of the clinical course of sickle cell anemia. Its levels are highly heritable, and its interpersonal variability is modulated in part by 3 quantitative trait loci that affect HbF gene expression. Genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) in these quantitative trait loci that are highly associated with HbF but explain only 10% to 12% of the variance of HbF. Combining SNPs into a genetic risk score can help to explain a larger amount of the variability of HbF level, but the challenge of this approach is to select the optimal number of SNPs to be included in the genetic risk score. Methods and Results— We developed a collection of 14 models with genetic risk score composed of different numbers of SNPs and used the ensemble of these models to predict HbF in patients with sickle cell anemia. The models were trained in 841 patients with sickle cell anemia and were tested in 3 independent cohorts. The ensemble of 14 models explained 23.4% of the variability in HbF in the discovery cohort, whereas the correlation between predicted and observed HbF in the 3 independent cohorts ranged between 0.28 and 0.44. The models included SNPs in BCL11A , the HBS1L-MYB intergenic region, and the site of the HBB gene cluster, quantitative trait loci previously associated with HbF. Conclusions— An ensemble of 14 genetic risk models can predict HbF levels with accuracy between 0.28 and 0.44, and the approach may also prove useful in other applications.

GTP cyclohydrolase (GCH1) is rate limiting for tetrahydrobiopterin (BH4) synthesis, where BH4 is a cofactor for nitric oxide (NO) synthases and aromatic hydroxylases. GCH1 polymorphisms are implicated in the pathophysiology of pain, but have not been investigated in African populations. We examined GCH1 and pain in sickle cell anemia where GCH1 rs8007267 was a risk factor for pain crises in discovery (n = 228; odds ratio [OR] 2.26; P = 0.009) and replication (n = 513; OR 2.23; P = 0.004) cohorts. In vitro, cells from sickle cell anemia subjects homozygous for the risk allele produced higher BH4. In vivo physiological studies of traits likely to be modulated by GCH1 showed rs8007267 is associated with altered endothelial dependent blood flow in females with SCA (8.42% of variation; P = 0.002). The GCH1 pain association is attributable to an African haplotype with where its sickle cell anemia pain association is limited to females (OR 2.69; 95% CI 1.21-5.94; P = 0.01) and has the opposite directional association described in Europeans independent of global admixture. The presence of a GCH1 haplotype with high BH4 in populations of African ancestry could explain the association of rs8007267 with sickle cell anemia pain crises. The vascular effects of GCH1 and BH4 may also have broader implications for cardiovascular disease in populations of African ancestry.

Background Hydroxyurea (HU) reduces vaso-occlusive crises (VOC) and other complications of sickle cell anaemia (SCA). Alpha-thalassaemia is a known modifier of SCA. Studies on the efficacy of HU in SCA patients with α-thalassaemia have yielded varying results. Objective To determine the effect of α-thalassaemia in response to HU therapy in the Multicenter Study of Hydroxyurea (MSH) cohort. Methods We compared the laboratory parameters and VOC incidence in the MSH cohort stratified by the presence or the absence of α-thalassaemia. Results Hydroxyurea showed significant (P = 0.001 for all baseline vs. follow-up comparisons) treatment effect on red cell indices irrespective of α-globin gene deletion. The magnitude of the HU-related changes was similar for mean corpuscular volume (MCV) (no α-thalassaemia 13 fl and α-thalassaemia 13 fl) and mean corpuscular haemoglobin (MCH) (no α-thalassaemia 4 pg and α-thalassaemia 4 pg) in both groups. Foetal haemoglobin (HbF) and F-cells also increased significantly with HU treatment in both groups. Total haemoglobin increased after HU treatment in both groups, but the increase was smaller and not statistically significant in patients with α-thalassaemia. In contrast, HU-related reduction in VOCs was more pronounced in patients with α-thalassaemia (VOC incidence rate ratio HU/placebo: 0.63 for α-thalassaemia and 0.54 for no α-thalassaemia (P for interaction 0.003). Conclusion Hydroxyurea decreases VOCs in SCA patients with and without α-thalassaemia, and the degree of VOC reduction was more pronounced in the patients with alpha-thalassaemia. Despite the lower baseline values, changes in standard laboratory parameters such as MCV and HbF percent remain useful in monitoring HU therapy in the presence of α-thalassaemia.

A new class of highly potent NS5A inhibitors with an unsymmetric benzimidazole-difluorofluorene-imidazole core and distal [2.2.1]azabicyclic ring system was discovered. Optimization of antiviral potency and pharmacokinetics led to the identification of 39 (ledipasvir, GS-5885). Compound 39 (GT1a replicon EC50 = 31 pM) has an extended plasma half-life of 37–45 h in healthy volunteers and produces a rapid >3 log viral load reduction in monotherapy at oral doses of 3 mg or greater with once-daily dosing in genotype 1a HCV-infected patients. 39 has been shown to be safe and efficacious, with SVR12 rates up to 100% when used in combination with direct-acting antivirals having complementary mechanisms.

Therapeutic induction of fetal hemoglobin (HbF) is one of the most promising approaches to ameliorate the severity of hemoglobinopathies such as β-thalassemia and sickle cell disease (SCD). Among HbF induction agents, hydroxyurea (HU) was approved by FDA to use for the treatment of SCD. However, there is variability in HU response among SCD patients. Individual genetic variants are mostly influenced in differences in pharmacological responsiveness to drug. We previously reported that the small guanosine triphosphate (GTP)-binding protein, secretion-associated and RAS-related (SAR1A) protein was a specific HU-inducible gene. The single nucleotide polymorphisms (SNPs) in SAR1A promoter also contributed to inter-individual differences in regulation of HbF expression and SCD patient responses to HU. Additionally, microRNAs (miRNAs) have been identified as potential key genes that regulate HbF induction and related with the clinical heterogeneity of SCD. Here, we demonstrate that SNPs within SAR1A coding regions are associated with differences in individual responses to HU therapy and potentially influenced in miRNAs binding. In order to determine SNPs in SAR1A coding regions, we sequenced all 8 exons of SAR1A gene in 32 SCD patients. Three (rs56090714, rs3812693, rs56381518)and twenty-four (rs78341510, rs114346554, rs72807054, rs1370644731, rs1491135303, rs1412150420, rs1423653432, rs1480964347, rs1479076497, rs1180306451, rs1482823291, rs1275470720, rs201493587, rs1470556171, rs2394643, rs80028936, rs7919647, rs115340990, rs15801, rs1046747, rs79535872, rs7653, rs1280408553, and rs10586) variants were identified in codon 1 and 8, respectively. Interestingly, codon 2 was found a novel mutation at position 119, C>A. No mutation was detected in codon 3, 4, 5, 6 and 7. Among these SNPs, rs7919647 at codon 8 was highest frequency (96.9%) in SCD patients. Next, we analyzed the association of SNPs and clinical and laboratory profiles using multiple regression. The rs56381518, rs1479076497, rs1180306451, rs1482823291, rs2394643 and rs115340990 showed significant association with total Hb levels after HU treatment in SCD patients. Only rs1180306451 was associated with absolute HbF levels (P= 0.0161). While no SNPs were observed significant association with HbF, F-cell or F-reticulocyte levels. In addition, the potential miRNAs binding to SNPs at 3'UTR regions were determined by using MicroSNiPer. We found miRNAs that may bind to SNPs as shown in Table 1. miR-625-5p, miR-5003-3p, miR-1236-5p, miR4271, miR-345-3p, miR4725-3p, miR-378a-3p, miR-548q and miR-135a-3p were previously identified only in mild-SCD patients. Furthermore, it has been reported that miR-1200 and miR-19b-1-5p were differentially expressed in high HbF levels condition. Our findings highlight the importance of genetic variants in SAR1A codon region that may predict the hydroxyurea response in SCD patients and miRNAs role in clinical heterogeneity of SCD. Disclosures No relevant conflicts of interest to declare.

Objectives Health care providers influence parental vaccination decisions. Over 90% of parents report receiving vaccine information from their child's health care provider. The majority of parents of vaccinated children and children exempt from school immunization requirements report their child's primary provider is a good source for vaccine information. The role of health care providers in influencing parents who refuse vaccines has not been fully explored. The objective of the study was to determine the association between vaccine-related attitudes and beliefs of health care providers and parents. Methods We surveyed parents and primary care providers of vaccinated and unvaccinated school age children in four states in 2002–2003 and 2005. We measured key immunization beliefs including perceived risks and benefits of vaccination. Odds ratios for associations between parental and provider responses were calculated using logistic regression. Results Surveys were completed by 1367 parents (56.1% response rate) and 551 providers (84.3% response rate). Parents with high confidence in vaccine safety were more likely to have providers with similar beliefs, however viewpoints regarding disease susceptibility and severity and vaccine efficacy were not associated. Parents whose providers believed that children get more immunizations than are good for them had 4.6 higher odds of holding that same belief compared to parents whose providers did not have that belief. Conclusions The beliefs of children's health care providers and parents, including those regarding vaccine safety, are similar. Provider beliefs may contribute to parental decisions to accept, delay or forgo vaccinations. Parents may selectively choose providers who have similar beliefs to their own.

Background: Pulmonary hypertension (PH) in adults with sickle cell disease (SCD) is associated with early mortality, but no prior studies have evaluated quantitative relationships of mortality to physiological measures of pre- and postcapillary PH. Objectives: To identify risk factors associated with mortality and to estimate the expected survival in a cohort of patients with SCD with PH documented by right heart catheterization. Methods: Nine-year follow-up data (median, 4.7 yr) from the National Institutes of Health SCD PH screening study are reported. A total of 529 adults with SCD were screened by echocardiography between 2001 and 2010 with no exclusion criteria. Hemodynamic data were collected from 84 patients. PH was defined as mean pulmonary artery pressure (PAP) > 25 mm Hg. Survival rates were estimated by the Kaplan-Meier method, and mortality risk factors were analyzed by the Cox proportional hazards regression. Measurements and Main Results: Specific hemodynamic variables were independently related to mortality: mean PAP (hazard ratio [HR], 1.61; 95% confidence interval [CI], 1.05‐2.45 per 10 mm Hg increase; P ¼ 0.027), diastolic PAP (HR, 1.83; 95% CI, 1.09‐3.08 per 10 mm Hg increase; P ¼ 0.022), diastolic PAP 2 pulmonary capillary wedgepressure(HR,2.19;95%CI,1.23‐3.89per10mmHgincrease; P ¼ 0.008), transpulmonary gradient (HR, 1.78; 95% CI, 1.14‐ 2.79 per 10 mm Hg increase; P ¼ 0.011), and pulmonary vascular resistance (HR, 1.44; 95% CI, 1.09‐1.89 per Wood unit increase; P ¼ 0.009) as risk factors for mortality. Conclusions: Mortality in adults with SCD and PH is proportional to the physiological severity of precapillary PH, demonstrating its prognostic and clinical relevance despite anemia-induced high cardiac output and less severely elevated pulmonary vascular resistance.

Haemolytic anaemia is variable among patients with sickle cell anaemia and can be estimated by reticulocyte count, lactate dehydrogenase, aspartate aminotransferase and bilirubin levels. Using principal component analysis of these measurements we computed a haemolytic score that we used as a subphenotype in a genome-wide association study. We identified in one cohort and replicated in two additional cohorts the association of a single nucleotide polymorphism in NPRL3 (rs7203560; chr16p13·3) (P = 6·04 × 10(-07) ). This association was validated by targeted genotyping in a fourth independent cohort. The HBA1/HBA2 regulatory elements, hypersensitive sites (HS)-33, HS-40 and HS-48 are located in introns of NPRL3. Rs7203560 was in perfect linkage disequilibrium (LD) with rs9926112 (r(2) = 1) and in strong LD with rs7197554 (r(2) = 0·75) and rs13336641 (r(2) = 0·77); the latter is located between HS-33 and HS-40 sites and next to a CTCF binding site. The minor allele for rs7203560 was associated with the -∝(3·7) thalassaemia gene deletion. When adjusting for HbF and ∝ thalassaemia, the association of NPRL3 with the haemolytic score was significant (P = 0·00375) and remained significant when examining only cases without gene deletion∝ thalassaemia (P = 0·02463). Perhaps by independently down-regulating expression of the HBA1/HBA2 genes, variants of the HBA1/HBA2 gene regulatory loci, tagged by rs7203560, reduce haemolysis in sickle cell anaemia.

Generation of induced dopaminergic (iDA) neurons may provide a significant step forward towards cell replacement therapy for Parkinson’s disease (PD). To study and compare transcriptional programs of induced cells versus primary DA neurons is a preliminary step towards characterizing human iDA neurons. We have optimized a protocol to efficiently generate iDA neurons from human pluripotent stem cells (hPSCs). We then sequenced the transcriptomes of iDA neurons derived from 6 different hPSC lines and compared them to that of primary midbrain (mDA) neurons. We identified a small subset of genes with altered expression in derived iDA neurons from patients with Parkinson’s Disease (PD). We also observed that iDA neurons differ significantly from primary mDA neurons in global gene expression, especially in genes related to neuron maturation level. Results suggest iDA neurons from patient iPSCs could be useful for basic and translational studies, including in vitro modeling of PD. However, further refinement of methods of induction and maturation of neurons may better recapitulate full development of mDA neurons from hPSCs.