Synthetic Heparan Sulfate Compounds Attenuate Vascular Complications Associated with Sickle Cell Disease

Abstract In sickle cell disease (SCD), a mutation of the β-globin gene leads to abnormal polymerization of hemoglobin, resulting in formation of sickled red blood cells, hemolytic anemia, and vaso-occlusive crisis (VOC). These primary events produce clinical complications and trigger additional multiple pathologies driven by chronic oxidative stress, sterile inflammation, and activation of coagulation. Heparins, highly sulfated forms of heparan sulfate (HS), are a group of polysaccharide compounds with great variance in structure. In addition to their anticoagulant effect, heparins have anti-adhesive and anti-inflammatory properties. These are determined by both sulfation pattern and length of the polysaccharide chain, which influence the ability to bind HMGB-1, histones, and P-selectin (Psel). This heterogeneity together with the short half-life and dosing regimen based on anticoagulant activity, limit the use of heparins as anti-adhesive and anti-inflammatory agents. To overcome these limitations, we previously developed a chemoenzymatic approach to synthesize a structurally defined HS oligosaccharides and demonstrated their ability to reduce sterile inflammation in animal models by binding HMGB-1 and histones. In the present study, we investigated the compound's anti-Psel properties in vitro and in a mouse model of SCD. First, using a Psel inhibition ELISA assay, we determined that a heptadecasaccharide (17-mer) is the minimum polysaccharide chain length required for inhibition of Psel binding to Sialyl Lewis X polyacrylamide. Based on these data, we synthesized three 18-mer compounds with a different sulfation position on each monosaccharide ring (NS2S, NS6S and NS2S6S). To obtain 18-mers with no anticoagulant activity, we omitted 3-O-sulfation of glucosamine, which is important for binding antithrombin III (confirmed by anti-FXa activity assay). In the Psel inhibition ELISA assay, all compounds demonstrated dose dependent (0.1 - 1000 µg/mL) anti-Psel activity comparable to that observed for low molecular weight heparin (LMWH). Psel is a key molecule mediating VOC by promoting formation of multicellular aggregates. Therefore, we evaluated the effect of 18-mers on Psel-mediated platelet/leukocyte aggregates (PLA) formation ex vivo. Leukocytes and platelets isolated from healthy donors were stimulated with PMA (100 nM) for 1 hour or thrombin (5 µg/mL) for 30 minutes, respectively, then incubated with vehicle, 18-mer compounds, or LMWH (0.5, 5, 50 and 500 µg/mL) for 15 minutes. After incubation, cells were combined to allow PLA formation for 15 minutes and analyzed by flow cytometry. At the highest tested 18-mer concentration, all compounds attenuated PLA formation. However only NS2S6S, the most highly sulfated compound, showed significant inhibition at all concentrations. NS2S6S decreased PLA formation to 82.8% 90.8%, 76.3% and 68.3%, lowest to highest concentrations respectively (p<0.01 for all concentrations versus vehicle). LMWH demonstrated significant decreases only at the two highest concentrations (82.1% and 63.8%, p<0.001). The number of circulating PLA was increased in sickle Townes HbSS mice by 6.1-fold (p<0.01) compared to non-sickle Townes HbAA controls. In ex vivo experiments, addition of NS2S6S (1 mg/ml) to the HbSS blood decreased PLA formation to 66.4% (p=0.03) compared to untreated HbSS blood. Finally, we determined the effect of NS2S6S on heme-induced microvascular stasis in Townes HbSS mice. Sickle mice were implanted with a dorsal skinfold chamber to visualize dermal microvessels. PBS or NS2S6S (3 mg/kg, s.c.) were injected 15 min before infusion of heme (1.2 µmol/kg, iv). 0ne, 2, 3 and 4 hours after heme infusion, microvascular stasis was observed in 31.5, 20.5, 18.9 and 14.2% of preselected vessels in PBS treated sickle mice, and NS2S6S treatment reduced that numbers to 10.9, 6.2, 3.1 and 3.2%, respectively (p<0.01 for all time points). In summary, we showed that NS2S6S prevents Psel dependent formation of PLA ex vivo and reduces heme-induced stasis in sickle mice. Together with previously described anti-HMGB1 and anti-histone effects, this compound is a good candidate for multi-modal therapy to mitigate the pathophysiology of SCD. However, like LMWH, NS2S6S has a short half-life which makes prophylactic treatment of SCD patients impractical. Studies to extend the half-life of HS are currently ongoing in our group. Disclosures Xu: Glycan Therapeutics: Current Employment. Belcher: Mitobridge/Astellas: Consultancy, Research Funding; CSL Behring: Research Funding. Vercellotti: CSL Behring: Research Funding; Mitobridge, an Astellas Company: Consultancy, Research Funding. Liu: Glycan Therapeutics: Current Employment.

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GTx011, a Potent Allosteric Modifier of Hemoglobin Oxygen Affinity, Prevents RBC Sickling in Whole Blood and Prolongs RBC Half-Life in Vivo in a Murine Model of Sickle Cell Disease

Blood ◽

10.1182/blood.v124.21.217.217 ◽

2014 ◽

Vol 124 (21) ◽

pp. 217-217 ◽

Cited By ~ 1

Author(s):

Kobina Dufu ◽

Donna Oksenberg ◽

Chengjing Zhou ◽

Athiwat Hutchaleelaha ◽

David R. Archer

Keyword(s):

Flow Cytometry ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Half Life ◽

Ex Vivo ◽

Cell Disease ◽

Employment Equity ◽

Hypoxic Conditions ◽

Equity Ownership

Abstract Sickle cell disease (SCD) is caused by a point mutation in the β-globin gene leading to production of hemoglobin S (HbS) that polymerizes under hypoxic conditions with subsequent formation of sickled red blood cells (RBCs). We have developed a novel small molecule, GTx011, which attains effective concentrations in blood upon oral dosing in multiple species. GTx011 increases the affinity of oxygen (O2) for HbS, delays in vitro HbS polymerization and prevents sickling of isolated RBCs under hypoxic conditions. We report here that GTx011 prevents in vitro sickling of RBCs in blood from sickle cell patients. Moreover, in a murine model of sickle cell disease (Townes SS mice), GTx011 prevents ex vivo sickling of RBCs and prolongs RBC half-life. We previously reported that GTx011 prevents sickling of isolated sickle cell RBCs (SSRBCs) subjected to a fixed hypoxic condition (pO2 of ~30 mm Hg) for 30 min. For a more physiologically relevant evaluation, we determined the anti-sickling activity of GTx011 in blood under variable hypoxic conditions over a shorter duration of time. Sickling of SSRBCs in blood was evaluated using a combination of hemoximetry and morphometric measurements. Whole blood from sickle cell patients was modified in vitro with GTx011 prior to hemoximetry. Conversely, blood from SS mice with GTx011 orally dosed acutely or chronically for 10-12 days was used for hemoximetry. SSRBCs were harvested during hemoximetry at various O2 tensions and immediately fixed in a deoxygenated solution of 2% glutaraldehyde/PBS prior to morphological quantitative analysis with CellVigene software or imaging flow cytometry (AMNIS ImageStreamX MkII). To evaluate the effect of GTx011 on RBC half-life in SS mice, N-hydroxysuccinimide biotin was injected into SS mice on day 5 of chronic dosing, producing a pulse-label. Flow cytometry was performed using fluorescently labeled streptavidin to determine the decay of biotinylation and RBC half-life. Reticulocyte counts were measured at different intervals during the dosing regimen by determining the percentage of blood cells that were Ter-119+, Thiazole-Orange+ and CD45- by flow cytometry. In a dose-dependent manner, GTx011 decreased the p50 value of human blood indicating an increase in Hb-O2 affinity. In parallel, GTx011 dose-dependently reduced the number of sickled SSRBCs under all hypoxic conditions (pO2 of <40 mm Hg) evaluated. Moreover, at an O2 tension mimicking typical hypoxic conditions in tissue capillaries (40 mm Hg), 300 µM of GTx011 was sufficient to prevent sickling of human SSRBCs in whole blood (20% Hct). Similarly, ex vivo sickling analysis indicated that, relative to blood from vehicle-treated SS mice, blood from GTx011-treated SS mice showed a pronounced reduction in the number of sickled RBCs under hypoxic conditions with a concurrent reduction in p50. For example, at a pO2 of 10 mm Hg, 19% of SSRBCs in blood from GTx011-treated mice sickled ex vivo compared with 56% in blood from vehicle-treated SS mice. In SS mice chronically dosed with GTx011, a prolongation of the RBC half-life from 2.4 days to 3.8 days was achieved together with a marked decrease in reticulocyte count. This increase in RBC half-life and accompanying reduction in reticulocyte count was observed in mice with GTx011 concentrations in blood that corresponded to >30% calculated Hb target occupancy. Taken together, these data suggest that GTx011 has the potential to be a beneficial therapeutic agent for the chronic treatment of SCD. Table SS mice RBC half life Reticulocytes Sickled RBCs Hemoximetry Chronic treatment, PO, BID, 10-12 days (Days) (%) (% at 10 mm Hg) p20 (mm Hg) p50 (mm Hg) Vehicle-treated 2.4 53 56 18 32 GBT440-treated (100mg/kg) 3.8 32 19 4.5 21 Disclosures Dufu: Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Zhou:Global Blood Therapeutics: Research Funding. Hutchaleelaha:Global Blood Therapeutics: Employment, Equity Ownership. Archer:Global Blood Therapeutics: Consultancy, Research Funding.

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Successful Plerixafor-Mediated Mobilization, Apheresis, and Lentiviral Vector Transduction of Hematopoietic Stem Cells in Patients with Severe Sickle Cell Disease

Blood ◽

10.1182/blood.v130.suppl_1.990.990 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 990-990

Author(s):

John F Tisdale ◽

Francis J. Pierciey ◽

Rammurti Kamble ◽

Julie Kanter ◽

Lakshmanan Krishnamurti ◽

...

Keyword(s):

Sickle Cell Disease ◽

Board Of Directors ◽

Sickle Cell ◽

Research Funding ◽

Ex Vivo ◽

Cell Disease ◽

Employment Equity ◽

Advisory Committees ◽

Cd34 Cells ◽

Equity Ownership

Abstract Background Patients with severe sickle cell disease (SCD) may benefit from β-globin gene transfer into autologous hematopoietic stem cells (HSC). Successful HBB gene transfer requires vector-mediated transduction of primitive HSCs. Steady-state bone marrow (BM) is the default HSC source in patients with SCD. Normal human BM contains up to 30% CD34+CD19+ pro-B cells and other lineage-committed cell types (CD34dim) that will not contribute to improved long-term erythropoiesis via gene therapy; these cells mobilize at low rates. CD34+ cell yields from BM harvest (BMH) are typically lower than those after mobilization and peripheral blood (PB) apheresis; multiple rounds of BMH may be required to obtain adequate cell doses for autologous gene therapy (GT) protocols. As G-CSF can cause life-threatening SCD complications and is contraindicated, plerixafor, a CXCR4 receptor antagonist, may accomplish HSC mobilization without the neutrophil or endothelial activation that elicit vaso-occlusion. We modified the protocol for the HGB-206 phase 1 study of LentiGlobin GT in severe SCD (NCT02140554) to assess HSC mobilization with plerixafor alone, followed by apheresis and transduction of mobilized cells. We also characterized BM-derived and plerixafor-mobilized HSC populations from patients with SCD. Methods HGB-206 is a phase 1 study of LentiGlobin Drug Product (DP), which contains autologous HSCs transduced ex vivo with the betibeglogene darolentivec (BB305) lentiviral vector, in patients with severe SCD (defined as a history of recurrent vaso-occlusive crisis [VOC], acute chest syndrome, stroke, or tricuspid regurgitant jet velocity of >2.5 m/s). Patients in group B receive 240 µg/kg plerixafor followed 4-6 hours later by apheresis, processing ~3 total blood volumes to collect backup HSCs. If < 1.5 x 106 CD34+ cells are collected, patients undergo a second day of apheresis. Cells collected in excess of those required for backup in case of graft failure are transduced with BB305 lentiviral vector for exploratory analyses. Group B patients then proceed to BMH to obtain cells for clinical DP manufacture. Group C will receive DP manufactured from mobilized PB. Mass cytometry (CyTOF) was used to analyze ex vivo cultured CD34+ cells with over 35 cell surface markers. Results To date, 3 patients have undergone plerixafor mobilization. Patients had a transient 1.5- to 3-fold increase in peak white blood cell and absolute neutrophil levels after plerixafor. Peak absolute CD34+ cell counts in PB were 170, 58, and 160 x 106 CD34+ cells/liter. A total of 15.3, 5.6, and 9.0 x 106 CD34+ cells/kg were collected in a single day of apheresis, and no subsequent apheresis or mobilization was required. In the same study, a mean of 5.0 (range 0.3-10.8) x 106 CD34+ cells/kg were collected per BMH (N=21). The mobilization and apheresis procedures had an acceptable toxicity profile. No dose-limiting toxicities were observed after plerixafor dosing. One patient had a single VOC approximately 48 hours after receiving plerixafor; this patient also experienced VOCs of similar severity after BMH. In contrast, after 21 BMHs in 9 patients, 18 ≥ grade 3 AEs were reported in 6 patients, primarily pain. Ex vivo cultured CD34+ cells isolated from BMH consisted of an average of 41.0% (17.3%-50.7%) CD34dim cells, with 16%-50% of the CD34dim cells expressing lymphoid markers. In contrast, ex vivo cultured CD34+ cells isolated from plerixafor mobilized PB contained an average of 8.2% (1.5-19.5%) CD34dim cells. Similar drug product vector copy numbers were obtained after research-scale transduction of CD34+ cells from marrow and PB from the same patient. Conclusion Initial results suggest that obtaining adequate doses of CD34+ cells from plerixafor-mobilized PB of patients with SCD may be safe and feasible, without the life-threatening complications associated with G-CSF, and with fewer, less invasive procedures compared with BMH. PB-derived CD34+ cells may contain lower proportions of lineage-committed CD34+ cells than BM-derived cells from patients with SCD. Cells collected by BMH and PB mobilization/apheresis appear to have an equivalent transduction efficiency. Together these results indicate that it may be possible to use plerixafor-only mobilization in clinical studies of autologous HSC GT in SCD. Results of mobilization, apheresis, and DP manufacturing at clinical scale for additional patients will be available for presentation. Disclosures Pierciey: bluebird bio: Employment. Kanter: American Society of Hematology (Sickle Cell Disease Guideline Panel): Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; MUSC: Other: The site PI for sponsored research conducted at MUSC who receives funds from: Novartis, bluebird bio, GBT, Sancillo, Apopharma, Pfizer; NHLBI (sickle cell disease research advisory committee): Membership on an entity's Board of Directors or advisory committees, Research Funding; Sancillo: Research Funding; Apopharma: Research Funding; Pfizer: Research Funding; GBT: Research Funding; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kwiatkowski: Novartis: Research Funding; Bluebird Bio: Research Funding; Apopharma: Research Funding; Agios: Consultancy, Honoraria; Ionis: Consultancy, Honoraria. Thompson: Novartis: Consultancy, Research Funding; bluebird bio: Consultancy, Research Funding; Baxalta: Research Funding; Celgene: Consultancy, Research Funding. Shestopalov: bluebird bio: Employment, Equity Ownership. Bonner: bluebird bio: Employment, Equity Ownership. Joseney-Antoine: bluebird bio: Employment, Equity Ownership. Asmal: bluebird bio: Employment, Equity Ownership. Walters: bluebird bio: Research Funding; ViaCord Processing Lab: Other: Medical Director; Sangamo Therapeutics: Consultancy; AllCells, Inc: Other: Medical Director.

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Control of HIV-1 Infection By Ferroportin Q248H Mutation

Blood ◽

10.1182/blood-2019-128685 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 953-953

Author(s):

Namita Kumari ◽

Seyed Mehdi Nouraie ◽

Hatajai Lassiter ◽

Asrar Ahmad ◽

Kathryn Anastos ◽

...

Keyword(s):

African American ◽

Viral Load ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Research Funding ◽

Ex Vivo ◽

Cell Disease ◽

Hiv 1 ◽

Transcription And Replication ◽

Iron Export

BACKGROUND: We recently showed that patients with Sickle Cell Disease (SCD), a hereditary hemolytic disorder, have low incidence of HIV-1 infection [1] and reduced ex vivo HIV-1 infection [2]. PBMC from SCD patients exhibited increased expression of iron export protein, ferroportin and reduced cellular iron levels leading to CDK2 inhibition, reduced SAMHD1 phosphorylation and increased expression of IkBα. Ferroportin expression is regulated by liver-produced hepcidin that facilitates ferroportin internalization and degradation. Ferroportin Q248H mutation has an allele frequency of 2.2-13.4% in African populations. We previously reported reduced sensitivity of ferroportin Q248H mutant to physiologic hepcidin concentrations in patients with sickle cell disease [3]. OBJECTIVES: To analyze the effect of ferroportin Q248H mutation on HIV-1 infection in vitro and in disease progression among a cohort of HIV-1 infected African-American women. METHODS: HEK293 cells were used to express ferroportin Q248H mutant and test cellular ferritin and intracellular labile iron using calcein-AM. Confocal microscopy was used to visualize ferroportin expression. HIV-1 transcription was measured in 293T cells transfected with HIV-1 LTR-Luciferase vector and Tat expressing vector. Ex vivo infection was analyzed in monocyte-derived macrophages infected with VSVg-pseudotyped HIV-1 virus. Ferroportin Q248H mutation was genotyped using Thermo Fisher probe (C_25753769_10) and genotyping services at University of Utah. RESULTS: We observed reduced intracellular iron in ferroportin Q248H expressing cells compared to WT ferroportin even when the cells were treated with hepcidin. In the absence of hepcidin, both WT ferroportin and Q248H ferroportin efficiently inhibited HIV-1 transcription and replication. Hepcidin induced HIV-1 transcription and replication in the cells with WT ferroportin but not Q248H mutant ferroportin. HIV-1 replication was reduced in primary macrophages obtained from patients with ferroportin Q248H mutation. To test whether expression of ferroportin Q248H offered protection from HIV-1 infection, we analyzed a cohort of HIV-1 infected women (WIHS). We genotyped 970 African-American subjects of whom 628 were HIV-1 infected and 342 were non-infected. The prevalence of Q248H hetero or homozygote mutations was 7.0% in non-infected and 11.8% among HIV-1 infected individuals (Odds Ratio=1.77, p=0.02). Analysis of HIV viral load showed significant lower viral load in the subjects with ferroportin Q248H mutation compared to WT. CONCLUSIONS: Our findings point to the contribution of iron metabolism in HIV-1 restriction and the potential role of the ferroportin Q248H mutation in the regulation of HIV-1 infection in vivo. ACKNOWLEDGMENTS: This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005, 5G12MD007597 and P30AI087714). We thank Women's Interagency HIV-1 study (WIHS) for sharing DNA samples and providing access to the clinical data. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. REFERENCES: Nouraie M, Nekhai S, Gordeuk VR. Sickle cell disease is associated with decreased HIV but higher HBV and HCV comorbidities in U.S. hospital discharge records: a cross-sectional study. Sex Transm Infect. 2012;88(7):528-533. Kumari N, Ammosova T, Diaz S, et al. Increased iron export by ferroportin induces restriction of HIV-1 infection in sickle cell disease. Blood Adv. 2016;1(3):170-183. Nekhai S, Xu M, Foster A, et al. Reduced sensitivity of the ferroportin Q248H mutant to physiological concentrations of hepcidin. Haematologica. 2013;98(3):455-463. Disclosures Anastos: NINR: Research Funding; NHGRI: Research Funding; NICHD: Research Funding; NIMH: Research Funding; NHLBI: Research Funding; NCI: Research Funding; NIAID: Research Funding; NINDS: Research Funding; NIDCR: Research Funding; NIMHD: Research Funding; NLM: Research Funding; Fogarty: Research Funding; NIDDK: Research Funding; NIA: Research Funding; NIAAA: Research Funding; NIDA: Research Funding.

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Restriction of HIV-1 Infection in Sickle Cell Disease and Trait

Blood ◽

10.1182/blood-2018-99-117029 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2337-2337

Author(s):

Namita Kumari ◽

Miguel de Mulder ◽

Javed Khan ◽

Asrar Ahmad ◽

Songping Wang ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Research Funding ◽

Ex Vivo ◽

Additional Restriction ◽

Cell Disease ◽

Restriction Factors ◽

Intracellular Iron ◽

Hiv 1

Abstract BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) replication is controlled by host intrinsic antiviral restriction factors some of which are counteracted by HIV-1 accessory proteins. We recently showed that ex vivo HIV-1 infection is suppressed in PBMCs obtained from Sickle Cell Disease (SCD) patients. The inhibition was mediated in part by SAMHD1 and NF-κB inhibitor, IkBα and triggered by ferroportin, an iron export protein. Ferroportin expression reduced intracellular iron levels and inhibited cellular CDK2 activity leading to reduction of SAMHD1 phosphorylation and increased expression of IkBα. OBJECTIVES: The study was designed to further clarify the mechanism of HIV-1 inhibition in SCD and identify additional HIV-1 restriction factors that may contribute to the restriction mechanism. METHODS: A customized array was utilized to determine the expression of restriction factors in SCD PBMCs. The shRNA-mediated knockdowns of the identified genes were used to further validate the role of these factors in HIV-1 replication in cultured and primary cells. HIV-1(IIIB strain) and VSVG-pseudotyped pNL4-3.Luc.R-E-virus was used to analyze HIV-1 replication. RESULTS: Overexpression of ferroportin in THP-1 cells led to increased expression of HO-1 and p21 and reduced phosphorylation of SAMHD1. Inhibition of HO-1 and p21 by small molecules increased HIV-1 replication in SCD PBMCs suggesting that these factors contributed to the restriction mechanism. Knocking down SAMHD1 in SCD PBMC did not fully restore HIV-1 replication pointing to additional restriction factors. Analysis of HIV-1 restriction factors in SCD PBMCs using customized array showed increased expression of APOBECs, TRIMs, CH25H, CPSF6, CTR9, EIF2ak2, IFI16, MX2, PML and RTF1 mRNAs. We next tested the effect of SCD trait on HIV-1 infection ex vivo and in vivo. PBMCs obtained from SCD trait individuals showed restricted HIV-1 infection with HIV-1(IIIB strain). To further validate these findings, we compared 9 trait (HbAS) HIV-1 infected individuals with 107 non-SCD (HbAA or HbAC) HIV-1 infected individuals from Howard University HIV clinic. HIV-1 viral load and levels of HIV-1 env and gag were significantly lower in HIV-1 infected SCD trait subjects. Expression of HO-1, p21 were increased and RNR2 expression was reduced in SCD trait PBMCs. Small molecule inhibitors of HO-1 and p21 induced HIV-1 replication in SCD trait PBMCs. CONCLUSIONS: Our findings point to HO-1 and p21 as factors that, in addition to SAMHD1 and IKBα, mediate HIV-1 restriction in SCD. In SCD trait, HO-1, p21 and RNR2 play a key role in ex vivo HIV-1 restriction. Thus HIV-1 infection is deregulated not only in SCD patients but also in SCD trait individuals. The HIV-1 restriction is mediated by iron-activated antiviral restriction factors. ACKNOWLEDGMENTS: This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005, 5G12MD007597, 1UM1AI26617 and P30AI087714). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosures Nekhai: NIMHD, NIH: Research Funding; NHLBI, NIH: Research Funding; NIAID, NIH: Research Funding.

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3057 – MATURATION OF EX VIVO-CULTURED HUMAN ERYTHROCYTES AND SICKLE CELL DISEASE MODELLING USING CRISPR-CAS9

Experimental Hematology ◽

10.1016/j.exphem.2020.09.074 ◽

2020 ◽

Vol 88 ◽

pp. S55

Author(s):

Yelena Boccacci ◽

Guillaume Margaillan ◽

Nellie Dumont ◽

Mathieu Drouin ◽

Yannick Doyon ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Ex Vivo ◽

Human Erythrocytes ◽

Disease Modelling ◽

Cell Disease

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Increased Release of Soluble MD-2 in Sickle Cell Disease and Its Role in Pro-Inflammatory Signaling in Endothelial Cells

Blood ◽

10.1182/blood-2019-122912 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 208-208

Author(s):

Ping Zhang ◽

John D Belcher ◽

Julia Nguyen ◽

Fuad Abdulla ◽

Gregory M Vercellotti

Keyword(s):

Endothelial Cells ◽

Sickle Cell Disease ◽

Western Blot ◽

Human Plasma ◽

Research Funding ◽

Heme Binding ◽

Cell Disease ◽

Inflammatory Signaling ◽

Tlr4 Signaling ◽

Potential Source

Sickle cell disease (SCD) is the most common hemoglobinopathy worldwide, resulting from a mutation in the beta globin gene. SCD has significant pathophysiological consequences -- hemolysis, inflammation, oxidative stress, hypercoagulability, endothelial dysfunction and painful vaso-occlusive crises. The latter can be precipitated by infection or other metabolic stressors. Hemolysis chronically exposes endothelial cells, leukocytes, and platelets to hemoglobin and heme that promote pro-inflammatory and prothrombotic phenotypes. We previously demonstrated that toll-like receptor 4 (TLR4) signaling is required for microvascular stasis induced by hemoglobin, heme, or lipopolysaccharide (LPS) in sickle mice. MD-2 is a glycoprotein, co-expressed with TLR4 at the surface of various cell types, principally myeloid and endothelial lineages. MD-2 also exists as a soluble plasma protein (sMD-2), mainly as a large disulfide-bound multimeric glycoprotein, as well as oligomers and monomers. sMD-2 binds LPS and confers TLR4 sensitivity to LPS . A marked increase in sMD-2 has been reported in plasma from patients with sepsis and rheumatoid arthritis. sMD-2 in SCD plasma has not been studied. Since SCD has a pro-inflammatory phenotype, we hypothesized that sMD-2 is increased in SCD plasma and promotes pro-inflammatory signaling of endothelial cells. We assessed plasma levels of sMD-2 by Western blot and found that sMD-2 was increased 1.7-fold in SS human plasma (n=8) compared to healthy AA plasma (p<0.05, n=7). In mice, plasma sMD-2 was increased 7.6-fold in Townes-SS sickle mice (n=9) compared to control Townes-AA mice (p<0.0002, n=7). In contrast, plasma CD14, another required component of LPS-TLR4 signaling, was not significantly different in SS humans (n=8) and SS mice (n=9) compared to AA controls (p<0.05). The liver is one potential source of sMD-2 in plasma. In mice, hepatic MD-2 mRNA was increased 2.1-fold in SS compared to AA (p<0.05, n=6). Activated vascular endothelium is another potential source and target of sMD-2 in plasma. It has been reported by other groups and confirmed by us that LPS induces sMD-2 secretion by human umbilical vein endothelial cells (HUVEC). To determine whether heme can induce sMD-2 secretion from endothelial cells, we treated HUVEC with heme (0-30 μM) for 18 hours and found heme increased sMD-2 in media in a dose-responsive manner. To determine if sMD-2 in plasma could activate TLR4 signaling in endothelial cells, we incubated HUVEC with 2% SS or AA human plasma for 18 hours and measured IL-8 in the media by ELISA. Media IL-8 concentration was 2.6-fold higher in HUVEC incubated with SS plasma compared to AA plasma (p<0.02, n=4). Tak242, a TLR4 signaling inhibitor, blocked IL-8 secretion by HUVEC + SS plasma. Since heme has been shown to activate TLR4 signaling, we examined whether heme could bind to sMD-2 in plasma using a heme-agarose pull-down assay. Human plasma was incubated with heme-agarose to pull down heme binding proteins, followed by Western blot for sMD-2 protein in the pellet. The blot confirmed that sMD-2 in plasma bound specifically to heme. When sMD-2 was removed from SS plasma using an anti-MD-2 affinity column, the sMD-2-depleted plasma reduced IL-8 secretion by HUVEC by 34.3% (p<0.002, n=4). Furthermore, when the high-affinity heme-binding protein hemopexin (10 μM) was added to SS plasma, IL-8 secretion by HUVEC was reduced by 31.6% (p<0.01, n=7). Next, we made recombinant human sMD-2 in CHO cells with protein-free ProCHO medium. UV/Vis absorption spectra (250-600 nm) and heme-agarose pull-down assays found there was heme bound to recombinant sMD-2 in the ProCHO medium. When recombinant sMD-2-heme was added to human AA plasma and incubated with HUVEC, IL-8 secretion increased 2.2-fold (p<0.004, n=3). TLR4 inhibitor Tak242 blocked this increase in IL-8 secretion. When hemopexin was added to the recombinant sMD-2-heme before adding it to AA plasma, IL-8 production was reduced 38% compared to non-hemopexin treated (p<0.01, n=7). In conclusion, these data indicate that sMD-2 is increased in SCD plasma, binds heme, and can stimulate endothelial cell IL-8 production through a TLR4-dependent mechanism. We speculate that sMD-2 bound to heme might play an important role in pro-inflammatory signaling by endothelium in SCD. Disclosures Belcher: Mitobridge, an Astellas Company: Consultancy, Research Funding. Vercellotti:Mitobridge, an Astellas Company: Consultancy, Research Funding.

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COVID Symptoms and COVID Anxiety in Sickle Cell Disease

Blood ◽

10.1182/blood-2020-143054 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 16-17

Author(s):

Wally R Smith ◽

Benjamin Jaworowski ◽

Shirley Johnson ◽

Thokozeni Lipato ◽

Daniel M Sop

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Research Funding ◽

Telephone Survey ◽

Cell Disease ◽

Weighted Mean ◽

The Us ◽

Associated Symptoms ◽

At Home

Background Even before the US upswing of the current COVID pandemic, the number of sickle cell disease (SCD) patients coming to hospitals and EDs appeared to fall drastically. This happened despite SCD patients having often been heavy utilizers of the ED and hospital for their iconic vaso-occlusive crises (VOC). Though ambulatory SCD clinics quick converted largely to telehealth in order to comply with stay-at-home orders designed to suppress person-to-person transmission, some SCD patients appeared to avoid care, delay care, or refuse doctors' invitations for care. Presumably patients did so out of COVID fears, but this has not been confirmed in the literature. Further, whether these patients had COVID symptoms but stayed at home has not been studied. As part of quality improvement (QI) to conduct COVID surveillance in an adult sickle cell program, we sought to explain and predict SCD health care utilization patterns we were observing, as well as to determine urgent physical and mental health needs of patients who appeared to be avoiding care. Methods Fifteen staff in the Adult Sickle Cell Medical Home at Virginia Commonwealth University, a large urban academic medical center, conducted a telephone survey ("wellness check"was used when we talked to patients) of all known adults with SCD over 19 days in 2020. A staff member confirmed the patient had SCD, asked permission to proceed, then asked about symptoms consistent with COVID-19. At the end of the telephone survey, respondents wer invited to complete an email survey of sickle cell and COVID-19 utilization attitudes (19-33 items, depending on the response pattern, either drawn from the National Health Interview Survey, from the Adult Sickle Cell Quality of Life Measurement quality of care survey, or drafted by the authors), the Sickle Cell Stress Survey-Adult (SCSS-A, a 10-item previously validated survey), and anxiety and depression (PHQ9 of the PRIME-MD). Results Of 622 adults approached by phone call, 353 responded to the following yes/no screening questions regarding the prior 14 days: fever over 100 F 0/353 (0.00%); cough 3/353(0.01%); difficulty breathing 0/353(0.00%); unexplained shortness of breath 2/353(0.01%); sore throat 2/353 (0.01%); unexplained muscle soreness 2/353(0.01%);contact with anyone who tested positive for COVID-19 2/353(0.01%); testing for COVID 19 6/353(0.02%). For QI purposes, we set a threshold of three or more COVID-associated symptoms or the presence of fever as criteria requiring intense telephone or in-person staff monitoring for the following week. Only three patients met criteria. A total of 219/353 had email surveys sent. Of 63 patients (28.8%) who returned email surveys by June 10, 2020, 35.9% had already managed a "pain attack" at home 4 or more times in the prior 12 months, and 45.5% of these said their bad ER experiences were very or somewhat important in that decision. In the prior 14 days, although 30/64 reported a crisis for at least one day, only 4/64 had visited the Emergency Department for pain. On a 0-10 scale, 21/61 patients endorsed "0" for worry that they would be COVID-infected by going for medical care (weighted mean 3.9), but 18/59 endorsed "10" for worry they were more at risk of COVID because of SCD (weighted mean 6.31), and 22/60 endorsed "10" for worry they would fare worse than others if COVID infected (weighted mean 6.97). Many patients forwent "needed" care (16/62) or delayed "needed" care by at least a day (36/61). Eleven patients met criteria for moderately severe to severe depression on the PHQ-9, and 28/63 somewhat or strongly agreed with the statement "death is always on the back of my mind" on the SCSS-A. Conclusions In adolescents and adults with SCD, many were already reticent to come to the ED for pain, but a significant portion reported delays or avoidance of needed care during the early stages of the US COVID pandemic, and few reported using the ED despite over half reporting at least one crisis day in 14. Patients nonetheless reported very few COVID-associated symptoms. Fears of COVID infection/susceptibility may limit visits for needed sickle cell care among adults. Acknowledgements: Mica Ferlis RN, FNP, Caitlin McManus, RN, FNP, Emily Sushko, RN, FNP, Justin West, RN, Kate Osborne, RN, Stefani Vaughan-Sams, Marla Brannon, BS, Nakeiya Williams, BS Disclosures Smith: GlycoMimetics, Inc.: Consultancy; Emmaeus Pharmaceuticals, Inc.: Consultancy; Novartis, Inc.: Consultancy, Other: Investigator, Research Funding; Global Blood Therapeutics, Inc.: Consultancy, Research Funding; Shire, Inc.: Other: Investigator, Research Funding; NHLBI: Research Funding; Patient-Centered Outcomes Research Institute: Other: Investigator, Research Funding; Health Resources and Services Administration: Other: Investigator, Research Funding; Incyte: Other: Investigator; Pfizer: Consultancy; Ironwood: Consultancy; Novo Nordisk: Consultancy; Imara: Research Funding; Shire: Research Funding.

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Clinical and Biomarker Predictors for Avascular Necrosis in Sickle Cell Disease

Blood ◽

10.1182/blood-2021-147829 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3091-3091

Author(s):

Michael Rabaza ◽

Maria Armila Ruiz ◽

Liana Posch ◽

Faiz Ahmed Hussain ◽

Franklin Njoku ◽

...

Keyword(s):

Risk Factors ◽

Sickle Cell Disease ◽

Board Of Directors ◽

Avascular Necrosis ◽

Sickle Cell ◽

Research Funding ◽

Medical Attention ◽

Cell Disease ◽

Advisory Committees ◽

Early Management

Abstract Introduction Sickle cell disease (SCD) affects 1 in 365 African Americans and approximately 25 million people world-wide. A common skeletal system complication is avascular necrosis (AVN), which can cause substantial pain and a reduced quality of life. While early management of AVN is focused on increasing range of motion with physical therapy and pain relief, there are no clear predictors for who is more likely to develop AVN and earlier institution of these preventive measure could help decrease disease progression. Vascular endothelial growth factor (VEGF) is a biomarker of endothelial injury and may indicate reduced vascular supply to the femoral or humeral head. Here we describe potential risk factors and biologic pathways for AVN in SCD, as understanding these may lead to improvements in future monitoring, early detection, and early intervention practices. Methods We investigated clinical and laboratory risk factors associated with AVN in a cohort of 435 SCD patients from our center. Blood samples, clinical, and laboratory data were collected at the time of enrollment during a clinic visit. Genotyping for alpha thalassemia was performed by PCR and the serum concentration of VEGF was measured by ELISA. AVN status was confirmed by review of the medical record and available imaging. We conducted a cross-sectional analysis comparing categorical and linear variables by AVN status using the chi-square and Kruskal-Wallis test, respectively. The independent association of the clinical and laboratory variables with AVN status was determined by logistic regression analysis. The initial model included variables with a P-value < 0.1 on univariate analysis and the final model was ascertained by stepwise forward and backward selection. Median values and interquartile range (IQR) are provided. Results The median age of the cohort was 32 (IQR, 24 - 43) years, 57% (250/435) were female, and 46% (198/435) were on hydroxyurea. AVN was observed in 34% (149/435) of SCD patients. SCD patients with AVN were older, had more frequent vaso-occlusive crises requiring medical attention, and had a higher body mass index (Table I) (P ≤ 0.002). We measured VEGF in 241 of the SCD patients with serum samples available at the time of enrolment. Serum VEGF concentrations trended higher in SCD patients with versus without AVN (420 vs. 359 pg/mL, respectively; P = 0.078). In the multivariate analysis model, AVN was independently associated with increased number of vaso-occlusive crises (OR 1.1, 95% CI: 1.0 - 1.14; P = 0.02), AST concentration (natural log OR 0.5, 95% CI: 0.2 - 0.9; P = 0.03), VEGF concentration (natural log OR 1.4, 95% CI: 1.0 - 1.9; P = 0.047), and tobacco use (OR 1.9, 95% CI: 0.9 - 3.7; P = 0.078). Discussion In conclusion, we demonstrate a high prevalence of AVN in an adult cohort of SCD patients. The presence of AVN was independently associated with a greater frequency of vaso-occlusive pain episodes, which may demonstrate a shared pathophysiology between AVN and vaso-occlusion that merits further investigation. We demonstrate that serum VEGF concentrations are higher in SCD patients with AVN and may be a clinical tool to identify those at high-risk and for earlier intervention for this complication. Figure 1 Figure 1. Disclosures Gordeuk: Modus Therapeutics: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Emmaus: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy. Saraf: Pfizer: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Alkaline Phosphatase Is Associated with Red Cell Alloimmunization in the Pulmonary Hypertension and Hypoxic Response (PUSH) Sickle Cell Disease Cohort

Blood ◽

10.1182/blood-2020-143036 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 20-20

Author(s):

Victoria Brooks ◽

Oluwalonimi Adebowale ◽

Victor R. Gordeuk ◽

Sergei Nekhai ◽

James G. Taylor

Keyword(s):

Risk Factors ◽

Alkaline Phosphatase ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Research Funding ◽

Severe Disease ◽

Case Control ◽

Red Cell ◽

Cell Disease ◽

History Of

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.

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Cardiac Injury and Microvascular Ischemia in Sickle Cell Disease

Blood ◽

10.1182/blood-2019-125445 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2286-2286

Author(s):

Kiranveer Kaur ◽

Ying Huang ◽

Subha Raman ◽

Eric H. Kraut ◽

Payal Desai

Keyword(s):

Sickle Cell Disease ◽

Myocardial Perfusion ◽

Sickle Cell ◽

Cardiac Mri ◽

Research Funding ◽

Cardiac Injury ◽

Microvascular Disease ◽

Elevated Troponin ◽

Cell Disease ◽

Troponin Elevation

Introduction: Myocardial ischemic injury remains an under recognized problem in patients with sickle cell disease (SCD), for which the exact prevalence remains undefined. SCD patients are known to have microvascular disease, impaired myocardial perfusion reserve and lack of typical epicardial vessel involvement based on prior data. Previous study at our institution has demonstrated that 3/22(13%) patients with clinically stable sickle cell disease had impaired myocardial perfusion reserve but no epicardial coronary artery disease. In this study, we will aim to learn prevalence of cardiac injury and microvascular ischemic disease. We will also evaluate for impact of these findings on overall survival (OS) of SCD patients. Methods: We conducted a retrospective chart review of patients with SCD seen at OSU Wexner Medical Center from July 2005 to July 2015 to identify patients who had elevated troponin-I level or cardiac MRI performed for chest pain. Clinical and laboratory data around the time of cardiac MRI and troponin elevation was collected. Abnormal MRI was defined in three ways: 1) Microvascular disease was defined by presence of subendocardial or myocardial perfusion defects and myocardial scarring. 2) Myocardial disease otherwise includes other findings suggestive but not specific for myocardial ischemia including left ventricular dysfunction, midmyocardial fibrosis, inflammation and regional wall motion abnormalities. 3) Abnormal MRI includes patients described in either 1) or 2). Kaplan-Meier (KM) method was used to evaluate the impact of microvascular disease defined in all 3 ways on OS. Proportional hazards model was fit to estimate the association between troponin elevation and OS, where troponin elevation was treated as a time-dependent variable and OS was measured from time of birth. Results: Sixty-nine (51% male; genotype Hb SS 75%, SC 16%, and Sβ-thal 9%) of 373 SCD patients had either abnormal troponin and/or had cardiac MRI done. Median age was 34 years (range 19-67 years). Of 238 patients who had troponin-I measured over this period, 18 % (n=42) had elevated troponin. 24 of 47 patients with cardiac MRI showed abnormalities described above specific for microvascular disease (n=14, 30%) and myocardial disease otherwise (n=10, 21%). We identified 22 patients with troponin measurement within 30 days before cardiac MRI. Elevated troponin levels predicted MRI abnormalities with sensitivity of 71% (95% confidence interval (CI) 42-92%) and specificity of 63% (95% CI 24-91%). The degree of troponin elevation did not correlate with the MRI abnormality. Hazard ratio of death in patients with elevated troponin was 5.1 (95% CI 2.7-9.6; p<0.0001). While the KM survival curves show lower OS in patients in abnormal MRI (p=0.74) and microvascular disease (p=0.42; Figure 1) group compared with normal MRI, the comparisons were not statistically significant. There was no difference in OS for patients with nonspecific myocardial disease findings (p=0.59). Conclusion: Over a 10-year period, the prevalence of cardiac injury as measured by elevated troponin was 18% (42/238) in patients with atypical chest pain. Among 47 patients who had cardiac MRI performed, 51% were abnormal with 30% having findings specific for microvascular cardiac disease. Troponin elevation appears to significantly increase the risk of all-cause mortality. Patient with microvascular and myocardial ischemic disease tend to have lower OS, but it did not reach statistical significance. This could be one of the potential contributing factors to high early mortality and sudden deaths in SCD patients. Further studies will be needed to elaborate on disease modifying interventions that impact survival in these patients. Disclosures Desai: Novartis: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Potomac: Speakers Bureau; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; University of Pittsburgh: Research Funding; Ironwood: Other: Adjudication Board.

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