Severe painful vaso-occlusive crises and mortality in a contemporary adult sickle cell anemia cohort study.

BACKGROUND: Frequent painful vaso-occlusive crises (VOCs) were associated with mortality in the Cooperative Study of Sickle Cell Disease (CSSCD) over twenty years ago. Modern therapies for sickle cell anemia (SCA) like hydroxyurea are believed to have improved overall patient survival. The current study sought to determine the relevance of the association between more frequent VOCs and death and its relative impact upon overall mortality compared to other known risk factors in a contemporary adult SCA cohort. METHODS: Two hundred sixty four SCA adults were assigned into two groups based on patient reported outcomes for emergency department (ED) visits or hospitalizations for painful VOC treatment during the 12 months prior to evaluation. RESULTS: Higher baseline hematocrit (p = 0.0008), ferritin (p = 0.005), and HDL cholesterol (p = 0.01) were independently associated with 1 or more painful VOCs requiring an ED visit or hospitalization for acute pain. During a median follow-up of 5 years, mortality was higher in the ED visit/hospitalization group (relative risk [RR] 2.68, 95% CI 1.1-6.5, p = 0.03). Higher tricuspid regurgitatant jet velocity (TRV) (RR 2.41, 95% CI 1.5-3.9, p \u3c 0.0001), elevated ferritin (RR 4.00, 95% CI 1.8-9.0, p = 0.001) and lower glomerular filtration rate (RR=2.73, 95% CI 1.6-4.6, p \u3c 0.0001) were also independent risk factors for mortality. CONCLUSIONS: Severe painful VOCs remain a marker for SCA disease severity and premature mortality in a modern cohort along with other known risk factors for death including high TRV, high ferritin and lower renal function. The number of patient reported pain crises requiring healthcare utilization is an easily obtained outcome that could help to identify high risk patients for disease modifying therapies. TRIAL REGISTRATION: ClinicalTrials.gov NCT00011648 http://clinicaltrials.gov

The immunopathological landscape of human pre-TCRα deficiency: From rare to common variants.

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre–a T cell receptor (pre-TCRa) expression. Low circulating naive ab T cell counts at birth persisted over time, with normal memory ab and high gd T cell counts. Their TCRa repertoire was biased, which suggests that noncanonical thymic differentiation pathways can rescue ab T cell development. Only a minority of these individuals were sick, with infection, lymphoproliferation, and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naive ab T cell counts but high gd T cell counts. Although residual pre-TCRa expression drove the differentiation of more ab T cells, autoimmune conditions were more frequent in these patients compared with the general population

Iron Status Is Inversely Correlated with Circulating Red Cells in Patients with Sickle Cell Anemia

Abstract Introduction: Sickle cell anemia (SCA, HbSS) arises from the presence of sickle hemoglobin (HbS, a consequence of a single base mutation in HBB gene). Upon deoxygenation, deoxy-HbS polymerizes with detrimental effects on shape, function, and lifespan of the erythrocytes. Because the delay time of sickling is inversely proportional to the mean cellular hemoglobin concentration (MCHC), decreasing the concentration of HbS is an attractive therapeutic approach. We recently demonstrated that dietary iron restriction decreases MCHC with a concomitant improvement in hematologic parameters and a decreased propensity to sickle in the Townes murine model of sickle cell anemia. Data describing the relationship between iron status, MCHC, and hematologic parameters in SCA patients are limited to isolated case series that do not account for alpha thalassemia, a known modifier of SCA severity. Based on our observations in mice, we hypothesize that decreased iron availability is associated with improved hematologic parameters in SCA patients in the absence of alpha thalassemia. We investigated the relationship between MCHC and red cell count, as well as transferrin saturation and red cell count, in healthy volunteers and sickle cell patients without alpha thalassemia. Methods: A database consisting of 795 patients with sickle cell disease (all genotypes) attending the National Institutes of Health between 2001 and 2015 was queried (see table). Data were filtered for sickle genotype, whether on hydroxyurea therapy, transfusion status (HbA), alpha thalassemia genotype, and relevant hematologic and/or iron panels obtained on the same visit. Data from healthy volunteers were obtained from the National Institutes of Health Biomedical Translational Research Information System (BTRIS) database filtering for those without detectable HbS that had hematologic and iron panel data obtained on the same visit. Correlations were analyzed by the Pearson correlation coefficient, r. p-values &amp;lt; 0.05 were considered statistically significant. Results: Analyses were performed on patients with HbSS without detectable HbA that were not taking hydroxyurea and did not have alpha thalassemia. Thirty-nine of these patients had MCHC and red cell counts obtained on the same visit. Data indicate that MCHC is inversely correlated with red cell count in these patients (r=-0.423; p = 0.007). Importantly, no significant relationship between MCHC and red cell count was observed in healthy volunteers (n=40; r=-0.125; p =0.438). Transferrin saturation is directly correlated with circulating red cells in healthy volunteers (n=65; r=0.359; p=0.0033). However, in patients with sickle cell disease transferrin saturation has an inverse relationship with red cell count (n=53; r=-0.3131; p=0.0225). Conclusions: Transferrin saturation and MCHC have are inversely correlated with circulating red cell counts in patients with sickle cell disease. These results support the concept that decreased iron availability improves hematologic parameters in sickle cell disease. Future studies will focus on pharmacologic means to achieve this end. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare. </jats:sec

Effects of Flavonoid Quercetin on Thrombo-Inflammatory Processes in Patients with Sickle Cell Disease

Abstract Introduction: Altered erythrocyte physiology in Sickle Cell Disease (SCD) results in severe vascular complications, including vaso-occlusion (VOC) along with various other manifestations, such as thrombosis and end organ damage. Moreover, platelet activation endothelial dysfunction and fibrin formation, are critical processes implicated in thrombo-inflammatory vasculopathy in SCD. Since flavonoids intake is associated with lowered adverse cardiovascular disease outcomes and cancer associated thrombosis, we are evaluating the effects of the flavonoid Isoquercetin (IQ) on thrombo-inflammatory biomarkers in patients with SCD (NCT:04514510). Here we report the mechanistic effects of Quercetin (Que), an aglycone that differs from IQ by the absence of 3-linked glucoside moiety, on markers of erythrocyte and platelet activation in patients with SCD. Methods: We studied patients with SCD (Genotype SS, mean age: 39 yrs, N=8) and ethnic matched controls (Genotype AA, mean age: 42 yrs, N=8). Erythrocyte reactive oxygen species (ROS) was measured in washed red cells by flow cytometry using the fluorescent probe 2', 7'-dichlorofluorescein diacetate (DCFDA) in the presence or absence of Quercetin (Que) (100 µM). Effects of in vitro erythrocyte ROS induction by hydrogen peroxide (H 2O 2) and ROS inhibition by N-acetyl-L-cysteine (NAC) were evaluated. Baseline and agonist-induced (ADP 20 µM) platelet P-selectin expression in vitro in the presence or absence of Que (100 µM) was assessed by flow cytometry. Results: At baseline, SS erythrocytes demonstrated elevated levels of ROS reflected by greater florescent intensity with DCFDA when compared with erythrocytes from ethnic matched controls (SS mean= 1884, SD: 508.8 vs. AA mean: 1604, SD: 554.5; p value= 0.3798). Stimulation of AA erythrocytes in vitro with H 2O 2 led to accumulation of ROS that approached levels similar to those exhibited by SS erythrocytes at baseline (AA mean: 1890, SD: 748.3). Pre-treatment of SS and AA erythrocytes in vitro with the antioxidant NAC exhibited minimal effects on ROS in SS erythrocytes (SS baseline+NAC mean: 1942, SD: 526.8, p value 0.4609 and SS H 2O 2+NAC mean: 1889, SD: 437.4, p value 0.5469) but lowered ROS levels in both baseline and H 2O 2 treated AA erythrocytes (AA baseline+NAC mean: 1442, SD: 453.9, p value 0.1563 and AA H 2O 2+NAC mean: 1581, SD: 518.7, p value 0.078). Pre-treatment of SS and AA erythrocytes in vitro with Que substantially lowered erythrocyte ROS in patients and controls at the baseline (SS baseline ROS with Que mean: 1582, SD: 542.0, p value 0.0078; AA baseline ROS with Que mean: 1341, SD: 490.9, p value 0.0313; Fig. 1A), and after H 2O 2 stimulation (SS H 2O 2 induced ROS+Que mean: 1677, SD: 572.5, p value 0.0156; AA H 2O 2 induced ROS+Que mean: 1417, SD: 512, p value 0.0078). Pre-treatment of erythrocytes with a combination of Que and NAC did not reveal synergistic anti-oxidant activity (SS NAC+Que mean: 1616, SD: 464.8; AA NAC+Que mean: 1333, SD: 524.1). We simultaneously investigated the effects of Que on platelet P-selectin expression in the same subjects. As expected, baseline platelet surface P-selectin expression was higher in SS patients compared with controls (SS N=5, mean: 1.48%, Max: 4.9%, Min: 0.03%; AA N=4, mean: 0.89%, Max: 2.3%, Min: 0%; p value 0.55). Stimulation of platelet rich plasma (PRP) with ADP increased surface P-selectin expression in comparison with baseline, in both patients and controls (SS N=5, mean: 18.04%, Max: 40.4%, Min: 5.8%, p value 0.0047; AA N=4, mean: 20.60%, Max: 43.8%, Min: 5.4%; p value 0.0140). Pre-treatment of PRP with Que prior to ADP stimulation revealed a trend toward decreased platelet surface P-selectin expression in both patients and controls (SS N=5, mean: 10.49%, Max: 24.8%, Min: 3.1%; AA N=4, mean: 12.09%, Max: 21.9%, Min: 3.4%; Fig 1B) although this did not return to baseline. Conclusion: The flavonoid Quercetin appears to have a beneficial effect in lowering erythrocyte ROS and platelet surface P-selectin and warrant further evaluation as a strategy to ameliorate thrombo-inflammatory pathophysiology in sickle hemoglobinopathies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare. </jats:sec

Effects of Quercetin on Neutrophil Extracellular Trap Formation in Sickle Cell Disease

Abstract Introduction: Sickle Cell Disease (SCD) is characterized by chronic inflammation with innate immune cell activation, especially observed in neutrophils. Emerging evidence implicates the imbalance between neutrophil extracellular trap (NET) formation and degradation as having a central role in the pathophysiology of thromboinflammation and venous thrombosis. Although NETosis and NET formation influences venous thromboembolism (VTE) pathophysiology, little is known about baseline and agonist-induced NETosis in SCD. We hypothesized that systemic neutrophil activation would lead to higher baseline and agonist induced NETosis in SCD and would influence phenotypic variability. To test this hypothesis, we assessed baseline and agonist induced NETosis in patients with SCD and ethnic matched controls. We also investigated the anti-inflammatory effects of flavonoid Quercetin on neutrophil activation. Methods: Neutrophils negatively selected from citrate anticoagulated blood using an immunomagnetic bead based kit (MACSxpress® Miltenyi Biotec) were either fixed immediately to assess baseline NETosis or stimulated with fMLP (1 µM) for 1 hour to assess agonist-induced NETosis. To study flavonoid anti-inflammatory effects, neutrophils were pretreated with Quercetin (100 µM) for 30 min prior to fixation and fMLP stimulation. NETosis was assessed by flow cytometry. Extracellular DNA extrusion on neutrophils was detected by gating the neutrophil population staining with Sytox green. Sytox green positive neutrophils that were positive for both myeloperoxidase (MPO) and tri-Citrullinated Histones (H3Cit) were defined as undergoing NETosis. In some experiments, NET formation was independently confirmed by image flow cytometry (AMNIS). Results: Subjects included SCD patients (genotype SS n=11) and ethnic matched controls (genotype AA, n=11) with a median age of 49 years (p=0.58) and a predominance of males (70%). All SCD patients were at least 60 days remote from an acute painful vaso-occlusive crisis or blood transfusion and were receiving hydroxyurea. The white cell and absolute neutrophil counts were higher in SCD patients (mean ± SD 8.77 ± 1.52 and 5.07 ± 1.78 x 10 9/L) when compared with controls (mean ± SD 5.33 ± 1.05 and 2.8 ± 0.95 x 10 9/L). Subsequent data are presented as median percentages with interquartile ranges (IQR). A subgroup of the study population demonstrated spontaneous NETosis (27%; SS = 4; AA = 4) and were therefore excluded from our analysis. Contrary to expectations, SCD patients exhibited a lower percentage of NETosis at baseline compared to controls (20 % (11, 36) vs. 33 % (15, 58); p=0.22). Similarly, neutrophils from SCD patients exhibited lower agonist-induced NETosis compared to controls (42% (19, 47) vs. 51% (37, 70); p=0.15) (Fig 1 A and B) Pretreatment of neutrophils from SCD patients with Quercetin appeared to inhibit basal levels of NETosis (6%, (2, 26) vs. 20% (11, 36) p=0.08) although this effect was not appreciable in controls (33% (11, 58) vs. 33% (15, 58) p=0.41) (Fig 1 C and D). Neutrophils from SCD patients that were pretreated with Quercetin and then stimulated with fMLP demonstrated significantly reduced NETosis compared to untreated neutrophils (17.1% (10, 38) vs 41.7% (19, 47) p=0.007) although this effect was not significant in controls (35% (17, 72) vs 50.7% (37, 70) p=0.11) Fig 1 E and F. Our ongoing experiments will demonstrate the effects of more specific inhibitors of neutrophil activation (e.g. R406) in human and mouse models of SCD. Conclusion: These preliminary data suggest lowered NETosis in SCD patients despite neutrophil activation in the systemic inflammatory environment that are partially explained by hydroxyurea treatment. The results also support further evaluation of anti-inflammatory therapies to reduce neutrophil activation in SCD and ameliorate thrombo-inflammatory disease pathology. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare. </jats:sec

Whole genome sequence-based haplotypes reveal a single origin of the 1393 bp <i>HBB</i> deletion

BackgroundMutations of HBB give rise to two prevalent haemoglobin disorders—sickle cell disease (SCD) and β-thalassaemia. While SCD is caused by a single base substitution, nearly 300 mutations that downregulate expression of HBB have been described. The vast majority of β-thalassaemia alleles are point mutations or small insertion/deletions within the HBB gene; deletions causing β-thalassaemia are very rare. We have identified three individuals with haemoglobin Sβ0-thalassaemia in which the β0-thalassaemia mutation is caused by a large deletion.ObjectiveTo use whole genome sequence data to determine whether these deletions arose from a single origin.MethodsWe used two approaches to confirm unrelatedness: pairwise comparison of SNPs and identity by descent analysis. Eagle, V.2.4, was used to generate phased haplotypes for the 683 individuals. The Neighbor-Net method implemented in SplitsTree V.4.13.1 was used to construct the network of haplotypes.ResultsAll three deletions involved 1393 bp, encompassing the β-promoter, exons 1 and 2, and part of intron 2, with identical breakpoints. The cases were confirmed to be unrelated. Haplotypes based on 29 SNPs in the HBB cluster showed that the three individuals harboured different βS haplotypes. In contrast, the haplotype harbouring the 1393 bp deletion was the same in all three individuals.ConclusionWe suggest that all the reported cases of the 1393 bp HBB deletion, including the three cases here, are likely to be of the same ancestral origin.</jats:sec

Bone Marrow Characterization in Sickle Cell Disease: Inflammation and Stress Erythropoiesis Lead to Suboptimal CD34 Recovery Compared to Normal Volunteer Bone Marrow

Abstract Introduction Gene therapy for sickle cell disease (SCD) requires modification of a high number of long term engrafting hematopoietic stem cells (LT-HSCs) sufficient to sustain production of the gene of interest at levels capable of overcoming the pathogenic HbSS phenotype. Unlike β-Thalassemia, the inflammatory bone marrow (BM) environment and stress erythropoiesis associated with SCD may have significant impacts on HSC quality and yield necessary for disease amelioration. Important work to optimize gene therapy through improvement in gene transfer efficiency, editing strategies, or transplant conditioning can only improve gene therapy in SCD if enough autologous HSCs are LT-HSCs, thus characterization of SCD BM and CD34+ HSCs is required. Collection type, storage, and delays in processing may further impact CD34+ recovery and should be investigated as a strategy to maximize LT-HSC recovery. Methods Twenty milliliters of BM from subjects with SCD (HbSS genotype) and normal volunteers was collected in different anticoagulants (Heparin, ACD-A) and processed immediately(day 0) or stored at 40C and processed the following day(day 1). After isolation via Ficoll density gradient centrifugation, the mononuclear (MN) layer was stained with antibodies against inflammatory markers (CD36, CD35, CD11b, CD62L, CD62P), non-MN cells (GPA, CD66b, CD41/61), or processed for CD34+ selection using a magnetic microbead CD34+ selection kit and stained for CD34, CD45, and GPA expression. Data were analyzed by conventional and imaging flow cytometry, the latter confirming post-CD34+ selection flow data and demonstrating antibody intensity as a characterization of HSC heterogeneity and progenitor lineage. Complete blood count and hemoglobin (Hb) electrophoresis were obtained at the time of BM collection. Statistical analyses were performed using unpaired t-tests. Results BM was collected from 18 subjects (16 with SCD; 11M; age 21-41 years). Median Hb (8.6 vs. 13.5 gm/dL, p&amp;lt;0.01) and white blood cell count (8.8 vs. 4.2 K/mcL, p&amp;lt;0.05) differed significantly between SCD and non-SCD subjects. Median percent sickle Hb in SCD subjects was 62%. Inflammatory markers and contamination with red cell and platelet markers in the post-Ficoll MN layer were higher in SCD vs. non-SCD BM regardless of anticoagulant (CD35 24% vs. 13%, p&amp;lt;0.05; CD36 22% vs. 11%, p&amp;lt;0.05; CD62P 16% vs. 3%, p&amp;lt;0.05; GPA 16% vs. 4%, p&amp;lt;0.05; CD41/61 19% vs. 3%, p&amp;lt;0.05), and trended higher on day 1 in SCD BM in both anticoagulants, significantly in Heparin (GPA 23% vs. 33% on day 1, p&amp;lt;0.05). Total CD45 expression was lower in SCD vs. non-SCD BM in both anticoagulants (p&amp;lt;0.05) and on day 0 (p&amp;lt;0.05) and 1 (p&amp;lt;0.01), with Amnis data confirming a higher CD34+CD45- population in SCD BM (4 ± 2% vs. 0.5 ± 0.3%, p&amp;lt;0.05). While there was no significant difference in total CD34+ cell count between SCD and non-SCD BM after selection post-Ficoll, there was a trend for lower CD34+ count in SCD in both anticoagulants (2.6x10^5 vs. 4.7x10^5, p=0.1). SCD CD34+ cells were characterized by higher GPA expression (28 ± 5% vs. 13 ± 3% in non-SCD BM, p&amp;lt;0.01) that worsened in Heparin on day 1 (22 ± 6.3% vs. 35 ± 12.4%, p&amp;lt;0.05). Image cytometry confirmed a majority of GPA expression in SCD BM is from single cell CD34+CD45+GPA+ and CD34+CD45-GPA+ HSCs in addition to red cell aggregates, with an increase in CD34+CD45-GPA+ HSCs on day 1 (10 ± 5% vs. 0.6 ± 0.2 % on day 0, p&amp;lt;0.05). Furthermore, the percentage of CD34hi HSCs was lower in SCD vs. non-SCD BM, with &amp;gt;50% SCD HSCs characterized as CD34dim (56% vs. 4% in non-SCD BM, p&amp;lt;0.001). Lastly, the purity of CD34+ selection worsened from day 0 to day 1 in SCD BM in heparin (94% vs. 68 ± 8%, p&amp;lt;0.05) and ACD-A (88% vs. 68 ± 0.7%, p&amp;lt;0.05). Conclusions SCD BM is characterized by increased inflammation and cell contamination in the MN layer regardless of anticoagulant that worsens over time in Heparin more significantly than in ACD-A. Compared to non-SCD BM, CD34+ HSC yield post-Ficoll is lower in SCD subjects, and is characterized by a larger proportion of CD34+CD45+GPA+ and CD34+CD45-GPA+ HSCs that rise with delays in processing. This indication of early differentiation along the erythroid lineage, with more than 50% of HSCs losing CD34+ intensity suggesting they are not LT-HSCs, suggests suppression of inflammation and stress erythropoiesis, combined with early cell processing may be critical for maximal HSC recovery necessary for successful gene therapy in SCD. Disclosures Luo: bluebird bio Inc.: Employment. Pierciey: bluebird bio: Employment. </jats:sec

Topical Sodium Nitrite Is Effective In Reducing Leg Ulcer-Associated Pain In Patients With Sickle Cell Disease

Abstract Background Chronic leg ulcers are a debilitating complication that affects individuals with SCD 10 times more frequently and at a much younger age than the general population. The underlying mechanism is not completely understood, and current therapies are not very effective. Severe pain at the ulcer site is often a greater concern to the patients than the status of the ulcer itself and a limiting factor in performing daily tasks and caring for the ulcer. We have previously reported the safety and tolerability of a Phase 1 study of topical sodium nitrite in patients with sickle cell disease and chronic leg ulcer in which escalating concentrations of sodium nitrite cream were applied twice a week to one ulcer per patient, for four weeks. We investigated pain and physical functioning in adults enrolled in this study. We hypothesized that there would be a significant reduction in the amount of pain at the ulcer site during the course of treatment sodium nitrite. In the subgroup of patients with more than one ulcer, we hypothesized that pain would be unchanged in the ulcers not treated with sodium nitrite. Patients and Methods Adult patients with sickle cell anemia consented to an IRB-approved FDA investigational New Drug protocol at the NIH Clinical Center, and completed a Brief Pain Inventory (BPI) before and after completion of therapy. Patients were asked to identify in detail the areas that were contributing to their overall pain status. Patients completed a 7-day opioid diary in which they were to record all medications taken, and frequency of consumption the week before and the week after completing the trial. In addition a Visual Analog pain Scale (VAS) score was obtained prospectively at each of seven patient encounters during the study for the study ulcer and each of the non study ulcers. A complete opioid usage history was collected before and after the four weeks of therapy to identify changes in opoid analgesic usage. Ulcer size was measured each time. Student T tests and one way ANOVA with Bonferroni correction were used for data analysis. Results Eighteen subjects (8 men and 10 women, ages 20 to 59 years); with active chronic leg ulcers were enrolled. Chronic daily opioid use occurred in 89% (16/18) and was attributed to localized pain at the ulcer site in 15 of them. Ulcers had been present for a median of 10 months (range 2 to 300 months); half of the patients had more than one ulcer. Baseline BPI pain severity and interference scores correlated with C reactive protein (CRP)(p=0.01), but not with ulcer age size, hydroxyurea use or transfusions, and % S. BPI scores and VAS scores of the treated ulcer improved significantly after use of topical sodium nitrite(p&lt;0.005 and &lt;001 respectively). In the subgroup that had more than one ulcer, VAS scores for non treated ulcers did not change significantly. Opioid analgesic use trended toward a decrease over time, but was not statistically significant for the entire group (392 mg before vs. 110 mg morphine equivalents after). Opioid analgesic use declined in 9 of 16 subjects, 3/16 increased (one had a new ulcer the last week of study, which was the source of the pain), six did not change. Ulcer size decreased after application of sodium nitrite, in a dose dependent manner, p=0.001. Changes in pain correlated with changes in size, and were dose dependent. Conclusions Patients with SCD and chronic ulcer experience significant pain at the ulcer site, which requires systemic narcotic therapy in most of them. Inflammation seems to be the main determinant of pain, not size or age of the ulcer or use of transfusions or hydroxyurea. Therapies that target ulcers, such as sodium nitrite, may reduce systemic opioid analgesic use and facilitate ulcer manipulation and care. Sodium nitrite may affect pain via mechanisms and at a dose that is independent from its healing properties. Disclosures: No relevant conflicts of interest to declare. </jats:sec