scholarly journals In Vivo Effects of LSD1 Inhibition By Small Chemical Inhibitors in Sickle Cell Mice

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 968-968
Author(s):  
Cuong Le ◽  
Greggory Myers ◽  
Alawi Habara ◽  
Natee Jearawiriyapaisarn ◽  
Mary Lee ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Small Molecule ◽  
Conflicts Of Interest ◽  
General Interest ◽  
Cell Disease ◽  
Erythroid Progenitor ◽  
Chemical Inhibitors ◽  
In Vivo Effects

Abstract For the past decades the principal strategy for treating the β-globinopathies [sickle cell disease (SCD) and β-thalassemia] has been to devise methods for increasing the levels of fetal hemoglobin (HbF) in adult erythroid cells, as a strong clinical literature has demonstrated that high HbF levels ameliorate disease pathophysiology. We originally reported the lysine-specific histone demethylase 1 (LSD1) as a new protein involved in the regulation of the fetal γ-globin genes. Inhibition of LSD1 using either RNAi or by the momoamine oxidase inhibitor tranylcypromine in primary human erythroid progenitor cells induces HbF to therapeutic levels. Thus, inhibition of LSD1 activity presents a novel exploratory avenue as a therapeutic strategy to treat SCD. Subsequent studies using another inhibitor RN-1 showed that RN-1 treatment of SCD mice results in increased HbF synthesis and leads to effective improvement of many aspects of the disease pathology normally associated with sickle cell disease. Here we report studies designed to examine the in vivo effects of some additional, publically available small molecule chemical inhibitors of LSD1 on HbF synthesis and erythroid physiology in SCD mice. In control SCD mice treated with DMSO only, the number of HbF positive cells was about 2.5% of total in RBC, while in SCD mice exposed to GSK-LSD1, or OG-L002, there was a statistically significant increase in the percentage of HbF positive cells after 4 weeks of treatment (up to 8%, or 6% respectively). Furthermore, sickled RBCs and reticulocytes are significantly reduced while the number of mature erythrocytes increased markedly in the peripheral blood of GSK-LSD1, or OG-L002 treated SCD mice. These findings suggest that GSK-LSD1 and/or OG-L002 could be potentially new HbF inducers based on LSD1 inhibition. We believe that these experiments will be of wide general interest and will be the foundation for development of small molecule therapeutics with which to treat hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Regulating Transcriptional Co-Activator PGC-1α for the Treatment of Sickle Cell Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2358-2358
Author(s):  
Alawi Habara ◽  
Cuong LE ◽  
George J Murphy ◽  
David H.K. Chui ◽  
Martin H. Steinberg ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Globin Genes ◽  
Cell Disease ◽  
Erythroid Progenitor

Abstract Sickle cell disease (SCD) is the most common inherited human hematologic disease, which causes hemolytic anemia, pain, disability, progressive multi-organ damage and early mortality. Clinical studies have shown that increased synthesis of fetal hemoglobin (HbF) in sickled erythroid cells leads to diminished severity of many clinical features of SCD. Therefore, therapeutic agents that can increase HbF production will be of benefit to SCD patients. Hydroxyurea (HU) is the FDA-approved therapeutic for treatment of SCD, but not all patients respond favorably or adequately. Therefore, other methods of targeting HbF are highly desired, particularly those that act by different mechanisms that might be used in combination with HU or alone (for those who do not tolerate HU). We recently identified PPARγ co-activator (PGC-1α) as a new protein involved in the regulation of the fetal globin genes. Forced overexpression of PGC-1α in vitro by adenovirus infection in bone marrow cells from SCD mice resulted in significantly increased human γ- and murine εy- and βh1-globin genes. Furthermore, up-regulation of PGC-1α by a small molecular agonist (Compound Z) in human umbilical cord blood-derived erythroid progenitor (HUDEP-1) cells markedly increases γ-globin gene expression and HbF synthesis. The highest response was achieved when HUDEP-1 cells were treated with 5µM Compound Z for 2 days, which results in 66.6% HbF+ cells compared to vehicle control (29.5% HbF+ cells). The effect of Compound Z in inducing HbF was further validated in erythroblasts derived from cultures of normal adults' CD34+ cells as well as in iPSC-derived sickle erythroblasts (SS24 cells). These data suggest that modulating PGC-1α activity may be effectively applied to the treatment of SCD since enhanced HbF synthesis would alleviate pathophysiological effects of SCD. Disclosures No relevant conflicts of interest to declare.

scholarly journals Lung Vaso-Occlusion in Sickle Cell Disease Mediated By Arteriolar Neutrophil-Platelet Micro-Emboli

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 159-159
Author(s):  
Margaret F. Bennewitz ◽  
Ravi Vats ◽  
Egemen Tutuncuoglu ◽  
Mark T. Gladwin ◽  
Prithu Sundd
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Acute Chest Syndrome ◽  
Cell Disease ◽  
Fab Fragments ◽  
Pulmonary Arteriole ◽  
Biophysical Mechanism

Abstract Background: Vaso-occlusive crisis (VOC) is the primary reason for emergency medical care by sickle cell disease (SCD) patients. SCD patients hospitalized with VOC often develop acute chest syndrome (ACS), a form of acute lung injury, suggesting a role for pulmonary vaso-occlusion in the onset of ACS. However, the cellular, molecular and biophysical mechanism of pulmonary vaso-occlusion is unknown. Methods: SCD transgenic or non-sickle control mice were intravenously (IV) challenged with 2 to 3 ng of bacterial lipopolysaccharide (LPS). Fluorescent anti-mouse Ly-6G and CD49b mAbs were administered IV for in vivo staining of circulating neutrophils and platelets, respectively. Multiphoton excitation enabled quantitative fluorescence intravital lung microscopy (qFILM) was used to determine the molecular mechanism of pulmonary vaso-occlusion in live mice. Function-blocking anti-mouse P-selectin mAb (Fab fragments) was administered IV to assess the role of platelet P-selectin in promoting pulmonary vaso-occlusion. Results: A nanogram dose of IV LPS selectively triggered pulmonary vaso-occlusion in SCD but not control mice. Remarkably, pulmonary vaso-occlusion involved occlusion of the pre-capillary pulmonary arteriole bottle-necks (junction of an arteriole and capillaries) by large neutrophil-platelet embolic aggregates. IV administration of Fab fragments of function blocking anti-P-selectin mAb led to the resolution of pulmonary vaso-occlusion, which was primarily mediated by the attenuation of large neutrophil-platelet aggregates into smaller aggregates that are not stopped by the arteriolar bottle-necks. Conclusion: These results establish the relevance of neutrophil-platelet aggregation in pulmonary arterioles in promoting pulmonary vaso-occlusion in SCD and also highlight the therapeutic potential of inhibiting platelet P-selectin to prevent ACS in SCD patients hospitalized with VOC. Acknowledgments: This study was supported by 1R01HL128297-01 (P.S.), AHA 11SDG7340005 (P.S.), VMI startup funds (P.S.). M.F.B. was supported by NIH-NHLBI training grant T32HL110849 and NIH-NHLBI F32 NRSA 1F32HL131216-01. Disclosures No relevant conflicts of interest to declare.

Quantitative Analysis of Small Molecular Weight Thiols and Disulfides in Blood from a Sickle Cell Disease Patient Infused with N-Acetyl-L-Cysteine

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2662-2662
Author(s):  
Shelby A Cate ◽  
Tahsin Ozpolat ◽  
Junmei Chen ◽  
Colette Norby ◽  
Barbara A. Konkle ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Small Molecule ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Antioxidant Properties ◽  
Disease Patient ◽  
Cell Disease ◽  
Mixed Disulfides

Abstract N-acetyl-L-cysteine (NAC) is an FDA approved drug used to treat acetaminophen overdose or as a mucolytic agent in respiratory disorders. The commonly accepted mechanism of action is that NAC undergoes deacetylation to cysteine, which is then used to synthesize glutathione (GSH), a major intracellular antioxidant. Like other thiol-containing compounds, NAC can also act as a reducing agent to break protein disulfide bonds or as a scavenger of reactive oxygen species. Due to its antioxidant properties, NAC has been proposed as a potential treatment for many diseases associated with oxidative stress, including sickle cell disease (SCD), neurological disorders, infectious diseases, and cancers. Though NAC has been widely studied, a full understanding of the mechanism by which NAC is effective in vivo has been limited by challenges in accurately quantifying NAC and its metabolites. As part of a clinical trial of NAC therapy in SCD, we have developed a liquid chromatography-mass spectrometry (LC-MS) based assay to quantify small molecule free thiols and disulfides using isotopically labeled internal standards. We applied this method to quantify small molecular thiols and disulfides in whole blood, red blood cells, and plasma from a SCD patient before (pre) and at 1, 8, 24 and 72 hr time points of intravenous administration of NAC at a dose of 300 mg/kg (a bolus infusion of 150 mg/kg for 1 hour followed by 150 mg/kg given over the next 7 hr). The cysteine concentration in whole blood increased to 286 μM at 1 hr from 97 μM at baseline, indicating that NAC is indeed rapidly metabolized (deacetylated) to cysteine. Interestingly, although cysteine concentration in RBCs increased over 4 fold at 1 hr and remained high compared to baseline, the highest concentration of total GSH in blood was observed at 24 hr (743 μM compared to 494 μM at baseline). Intracellular availability of cysteine is known as a rate-limiting step for GSH synthesis, and the delayed accumulation of GSH may suggest that NAC is involved in the extracellular deficit of reducing equivalents before it serves as a substrate in GSH synthesis. To explore this possibility, we quantitated NAC and its oxidation products, homo- and mixed disulfides. We found that total NAC concentration reached 1.58 mM in whole blood at 1 hr, but 44% of NAC was oxidized to N-acetyl-cystine (NAC-ss) or formed mixed disulfides with GSH (GS-ss-NAC) and Cys (Cys-ss-NAC), whereas the NAC used for infusion contained less than 0.5% in the oxidized form (NAC-ss). Concurrent with the formation of NAC disulfides, the levels of oxidized GSH (GSSG, GS-ss-Cys) and cysteine (cystine) were significantly decreased. These observations suggest that NAC administration of SCD not only increases GSH levels by raising the cysteine concentration, but also directly functions as an antioxidant to reduce oxidative stress. SCD patients are known to have low levels of GSH and frequently experience oxidative stress. NAC treatment is likely to address both issues. We plan to analyze the effects of NAC on blood small molecule thiol concentrations in several more SCD patients. Disclosures No relevant conflicts of interest to declare.

Targeting Neutrophil Aging and the Microbiota for the Treatment of Sickle Cell Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 279-279
Author(s):  
Dachuan Zhang ◽  
Grace Chen ◽  
Deepa Manwani ◽  
Arthur Mortha ◽  
Chunliang Xu ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Leukocyte Adhesion ◽  
Cell Disease ◽  
Fecal Transplantation ◽  
Penicillin V ◽  
Blood Disorder ◽  
Prophylaxis Therapy

Abstract Sickle cell disease (SCD) is a genetic blood disorder characterized by repeated episodes of vaso-occlusion, in which neutrophils play a primary function through their recruitment in inflamed venules and interactions with sickle red blood cells (sRBCs) via activated αMβ2 integrin (Mac-1). Intravital microscopy analyses revealed considerable heterogeneity in Mac-1 activation on adherent neutrophils, suggesting that subsets of neutrophils differ markedly in their pro-inflammatory activity (Nat Med 2009; 15:384). Recently, we found that aged neutrophils, marked by CD62Llo CXCR4hi, represented an overly active subset exhibiting enhanced Mac-1 activation (Blood 2013; 122:324). To further characterize aged neutrophils, we compared their transcriptome with control and in vivo- activated neutrophils. Gene set enrichment analyses revealed that aged neutrophils up-regulated several pathways that were also enhanced during activation, including integrin, leukocyte adhesion, toll-like receptor (TLR), and NFκB signalling pathways (P < 0.05), suggesting a contribution by exogenous inflammatory mediators. Based on the expression profile, we hypothesized that microbiota-derived signals may influence neutrophil aging in the circulation. To test this idea, we analyzed aged neutrophil numbers in antibiotics (ABX)-treated and germ-free (GF) mice, in which the gut microbiota was largely depleted or deficient, respectively. We found significant reductions of aged neutrophil numbers in both models, and the numbers could be significantly restored by either lipopolysaccharide (LPS) gavage or fecal transplantation (Ctrl / ABX / ABX+LPS: 98±14 / 36±11 / 107±25 cells per μl blood; SPF / GF / GF-FT: 56±12 / 4±1 / 15±4 cells per μl blood; P < 0.05). We next investigated whether microbiota-driven neutrophil aging was mediated by TLRs and Myd88 signalling using chimeric mice reconstituted with a mixture of WT and Myd88, TLR4 or TLR2-deficient hematopoietic cells. Interestingly, we observed significant reductions in the aged subset of Myd88, TLR4 or TLR2-deficient neutrophils compared to WT neutrophils in the same mice, and in their capacity to capture fluosphere beads that specifically bound to activated Mac-1 (WT / LysM-cre/Myd88-flox: 10.4±0.3 / 7.0±0.6 %, 1.0±0.1 / 0.4±0.1 bead per cell; WT / Tlr4-/-: 10.3±0.2 / 6.0±0.2 %, 1.0±0.1 / 0.4±0.1 bead per cell; WT / Tlr2-/-: 10.0±0.4 / 6.8±0.5 %, 1.3±0.2 / 0.8±0.1 bead per cell; P < 0.01). Analysis of SCD mice (BERK) revealed that the aged neutrophil population was significantly increased (by >10-fold) compared to hemizygous (SA) mice, and the expansion was completely abrogated by microbiota depletion (SA / SCD (SS)-Ctrl / SS-ABX: 71±11 / 981±261 / 124±27 cells per μl blood, P < 0.01). We then challenged SA, untreated and ABX-treated SCD mice with TNF-α to induce acute vaso-occlusion. SCD mice exhibited significant increases in neutrophil adhesion, Mac-1 integrin activation and heterotypic interactions with RBCs compared to SA mice, all of which were markedly reduced by microbiota depletion (SA / SS-Ctrl / SS-ABX: 6.4±0.6 / 18.1±1.0 / 11.2±1.0 adherent neutrophil per vessel; 0.5±0.1 / 1.0±0.2 / 0.3±0.1 bead per cell; 0.1±0.1 / 0.5±0.1 / 0.2±0.1 RBC interaction per vessel per min; P < 0.05), resulting in significantly enhanced blood flow and prolonged survival in ABX-treated SCD mice (P < 0.05). Most interestingly, the splenomegaly of SCD mice was significantly reduced, and liver damage including fibrosis, necrosis and inflammation was dramatically alleviated in ABX-treated SCD mice (P < 0.05). Finally, we evaluated whether the numbers of circulating aged neutrophils were altered in patients with SCD. As Penicillin V antibiotic prophylaxis therapy is recommended for children < 5 years or older patients with immune defects to prevent life-threatening infections, we determined aged neutrophil numbers in this patient population. We found that SCD patients exhibited a marked increase in the numbers of circulating aged neutrophils compared to healthy controls, which were significantly reduced in patients on Penicillin V prophylaxis (Ctrl / SS / SS-PV: 261±40 / 1175±205 / 439±87 cells per μl blood, P < 0.05). Our results raise the possibility that targeting neutrophil aging and the microbiota may have broad implications in our understanding of sickle cell disease pathogenesis and its management that should be further studied in clinical trials. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effect of PSI697, a Small Molecule Inhibitor of P-Selectin, in the Townes Model of Sickle Cell Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3391-3391 ◽  
Author(s):  
Reema Jasuja ◽  
Sunita Patel Hett ◽  
Neelu Kaila ◽  
Debra Pittman
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Small Molecule ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Cell Disease ◽  
Tissue Ischemia ◽  
Multicellular Aggregates ◽  
Surgical Preparation ◽  
Platelet Aggregates

Abstract Chronic vaso-occlusion is a major cause of morbidity and mortality in patients with sickle cell disease (SCD). Persistent vaso-occlusion can damage lungs, liver, kidneys or brain and ultimately lead to end-organ dysfunction. Vaso-occlusive pain crisis is a complex multistep process, initiated by adhesion of fragile sickle erythrocytes and rigid neutrophils to hypoxic and inflamed endothelium. Large multicellular aggregates of blood cells, including platelets and sickled erythrocytes, form on these adherent activated neutrophils in the microcirculation, ultimately causing vascular occlusion and tissue ischemia. Inflammatory mediators, such as P-selectin, play a key role in mediating these heterocellular interactions and attract additional leucocytes to the site of occlusion. P- and E-selectin mediate rolling and tethering of blood cells on the endothelium. Here, we explore cellular and animal models of sickle cell disease to assess PSI-697, an orally active small molecule antagonist of P-selectin. PSI-697 inhibits P-selectin binding to P-selectin glycoprotein ligand-1 (PSGL-1) with an IC50 of 50-125 µM (Bedard et al, JPET, 2008). Townes SCD mice were used to assess the efficacy for PSI-697 in preventing vaso-occlusion in SCD. Mice were randomized to treatment with vehicle or PSI-697. Animals were treated in a prophylactic setting prior to the surgical preparation. Surgical preparation induces a well described acute inflammatory response in the microcirculation associated with neutrophil adhesion to the endothelium and formation of multicellular aggregates. Alexa-488 labeled Ly-6G neutrophil antibody and Dylight-649 labeled CD42c platelet antibody were injected to quantify adherent neutrophils and neutrophil-platelet aggregates. Cremaster microvasculature was observed by intravital microscopy. Mice treated with 100 mg/kg of P-selectin inhibitor PSI-697 showed a 55% reduction in adherent neutrophils and a 78% decrease in the number of neutrophil-platelet aggregates compared to vehicle treated animals. Neutrophils showed ~7 fold increase in rolling velocity in mice treated with P-selectin inhibitor. Our results demonstrate that prophylactic administration of PSI-697, a small molecule P-selectin antagonist, improved parameters associated with vaso-occlusion in Townes SCD mice. Disclosures No relevant conflicts of interest to declare.

scholarly journals HMGB1 As a Novel Platelet Agonist That Acts Synergistically with ADP to Activate Platelets in Sickle Cell Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1073-1073
Author(s):  
Deirdre Nolfi-Donegan ◽  
Sruti Shiva ◽  
Cheryl A Hillery
Keyword(s):  
Sickle Cell Disease ◽  
Synergistic Effect ◽  
Platelet Activation ◽  
Sickle Cell ◽  
Low Dose ◽  
Conflicts Of Interest ◽  
Statistical Tests ◽  
Cell Disease

Abstract Background: Sickle cell disease (SCD) is a proinflammatory and prothrombotic disorder that exhibits increased platelet activation. High mobility group box 1 (HMGB1) is a nuclear protein that can mediate inflammation when released from inflammatory or ischemic cells. HMGB1 is increased in many inflammatory disease states including SCD. Recent data suggests HMGB1 activates platelets and may work synergistically with potent platelet agonists such as collagen and thrombin, but little is known regarding HMGB1-platelet interactions in combination with weaker agonists like ADP, or in isolated platelets. Moreover, the effect of HMGB1 on platelet activation has not been evaluated in SCD. We hypothesized that the in vitro addition of low-dose recombinant HMGBI (rHMBG1) to isolated platelets will lower the threshold dose of physiologic agonists required to achieve platelet activation, and that this effect is exaggerated in SCD. Methods: Platelets were isolated from healthy controls (n=4) and patients with hemoglobin SS disease (SCD; n=5). The level of platelet activation was assessed after treatment with ADP at concentrations of 0 μM, 0.5 μM, 2 μM, and 5 μM with the addition of either low-dose rHMGB1 (10 μg/mL) or the same volume of vehicle. Percent platelet activation was measured via flow cytometry using PE antibody to GPIIb (CD41) to select for platelets, and PAC1 to detect the activation-dependent conformational change in integrin αIIbβ3 (GP IIb-IIIa). Platelet activation was interpreted as percent of platelets that bound PAC1. Data was analyzed using FlowJo software and nonparametric statistical tests. Results: Mean baseline platelet activation was 1.5% (range 0.4-3.3%) for control platelets and 7.3% (1.4-17.7%) for SCD platelets (p=0.19). In the SCD group, the addition of low-dose rHMGB1 (10 μg/mL) increased the mean percent of activated platelets from 7.3% to 26.5% (10.9-43%) (p=0.01). In comparison, mean activation of control platelets increased from only 1.5% to 19.5% (8.3-42.7%) after addition of rHMGB1 (p=0.12). Having illustrated that rHMGB1 can activate washed SCD platelets, we then compared the synergistic effect of rHMGB1 with ADP. There was increased platelet activation observed when ADP was added to rHMGB1 in SCD platelets: ADP 0.5 μM increased mean platelet activation from 13.8% (range 0.3-25.3%) to 54.4% (6.7-84.9%) with the addition of rHMGB1 (p=0.02); ADP 2 μM increased platelet activation from 14.1% (2.8-23.8%) to 56.2% (22.2-88.6%) with rHMGB1 (p=0.006); and ADP 5 μM increased platelet activation from 21.4% (2.5-30.1%) to 65.3% (31.7-85.9%) after adding rHMGB1 (p=0.004) (Fig 1; * and **, diamonds vs stars). We did not find a similar statistically significant synergistic effect in the control samples treated with ADP compared to combined HMGB1 + ADP, except at ADP dose 2 μM where platelet activation increased from 14.94% (4.6-28.6%) to 39.1% (19.5-56.0%) after the addition of rHMGB1 (p=0.04; Fig 1; #, square vs circle). Activation of platelets with just ADP was not different comparing control with SCD platelets (Fig 1; circles vs stars). Similarly, activation of platelets with both ADP and rHMGB1 was not significantly different comparing control with SCD platelets except for a trend at 0.5 μM ADP + rHMGB1 10 μg/mL with 19.29% (6.6-38.7) in controls vs 54.44% (6.7-84.9) in the SCD group (p=0.07) (Fig 1; diamonds vs squares). Summary: We found that rHMGB1 acts both independently and synergistically with ADP to increase platelet activation in SCD platelets. In our small cohort, SCD platelets had increased responsiveness to low dose-rHMGB1 compared to control platelets. Moreover, combining rHMGB1 with ADP greatly enhanced platelet activation in SCD but not control platelets. Our data suggest that SCD platelets are sensitized to HMGB1 in the presence of weaker agonists such as ADP. This heightened responsiveness of SCD platelets to HMGB1 may explain the enhanced platelet activation and inflammation associated with SCD in vivo. With further study, HMGB1 could be a target of clinical drug-directed therapy in SCD patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Engineering, Generation and Preliminary Characterization of a Humanized Porcine Sickle Cell Disease Animal Model

2020 ◽  
Author(s):  
Tobias M. Franks ◽  
Sharie J. Haugabook ◽  
Elizabeth A. Ottinger ◽  
Meghan S. Vermillion ◽  
Kevin M. Pawlik ◽  
...  
Keyword(s):  
Animal Model ◽  
Sickle Cell Disease ◽  
Mouse Models ◽  
Sickle Cell ◽  
Cell Model ◽  
Globin Genes ◽  
Cell Disease ◽  
Small Vessels

AbstractMouse models of sickle cell disease (SCD) that faithfully switch from fetal to adult hemoglobin (Hb) have been important research tools that accelerated advancement towards treatments and cures for SCD. Red blood cells (RBCs) in these animals sickled in vivo, occluded small vessels in many organs and resulted in severe anemia like in human patients. SCD mouse models have been valuable in advancing clinical translation of some therapeutics and providing a better understanding of the pathophysiology of SCD. However, mouse models vary greatly from humans in their anatomy and physiology and therefore have limited application for certain translational efforts to transition from the bench to bedside. These differences create the need for a higher order animal model to continue the advancement of efforts in not only understanding relevant underlying pathophysiology, but also the translational aspects necessary for the development of better therapeutics to treat or cure SCD. Here we describe the development of a humanized porcine sickle cell model that like the SCD mice, expresses human ɑ-, β− and γ-globin genes under the control of the respective endogenous porcine locus control regions (LCR). We also describe our initial characterization of the SCD pigs and plans to make this model available to the broader research community.

scholarly journals Reconstruction of Sickle Cell Disease with Circulating Sickling Red Blood Cells in Novel Humanized Cytokines and Liver Mistrg Mice

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 29-30
Author(s):  
Yuanbin Song ◽  
Rana Gbyli ◽  
Liang Shan ◽  
Wei Liu ◽  
Yimeng Gao ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Mouse Model ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Red Cell ◽  
Cell Disease ◽  
Ineffective Erythropoiesis ◽  
Cd34 Cells

In vivo models of human erythropoiesis with generation of circulating mature human red blood cells (huRBC) have remained elusive, limiting studies of primary human red cell disorders. In our prior study, we have generated the first combined cytokine-liver humanized immunodeficient mouse model (huHepMISTRG-Fah) with fully mature, circulating huRBC when engrafted with human CD34+ hematopoietic stem and progenitor cells (HSPCs)1. Here we present for the first time a humanized mouse model of human sickle cell disease (SCD) which replicates the hallmark pathophysiologic finding of vaso-occlusion in mice engrafted with primary patient-derived SCD HSPCs. SCD is an inherited blood disorder caused by a single point mutation in the beta-globin gene. Murine models of SCD exclusively express human globins in mouse red blood cells in the background of murine globin knockouts2 which exclusively contain murine erythropoiesis and red cells and thus fail to capture the heterogeneity encountered in patients. To determine whether enhanced erythropoiesis and most importantly circulating huRBC in engrafted huHepMISTRG-Fah mice would be sufficient to replicate the pathophysiology of SCD, we engrafted it with adult SCD BM CD34+ cells as well as age-matched control BM CD34+ cells. Overall huCD45+ and erythroid engraftment in BM (Fig. a, b) and PB (Fig. c, d) were similar between control or SCD. Using multispectral imaging flow cytometry, we observed sickling huRBCs (7-11 sickling huRBCs/ 100 huRBCs) in the PB of SCD (Fig. e) but not in control CD34+ (Fig. f) engrafted mice. To determine whether circulating huRBC would result in vaso-occlusion and associated findings in SCD engrafted huHepMISTRG-Fah mice, we evaluated histological sections of lung, liver, spleen, and kidney from control and SCD CD34+ engrafted mice. SCD CD34+ engrafted mice lungs showed an increase in alveolar macrophages (arrowheads) associated with alveolar hemorrhage and thrombosis (arrows) but not observed control engrafted mice (Fig. g). Spleens of SCD engrafted mice showed erythroid precursor expansion, sickled erythrocytes in the sinusoids (arrowheads), and vascular occlusion and thrombosis (arrows) (Fig. h). Liver architecture was disrupted in SCD engrafted mice with RBCs in sinusoids and microvascular thromboses (Fig. i). Congestion of capillary loops and peritubular capillaries and glomeruli engorged with sickled RBCs was evident in kidneys (Fig. j) of SCD but not control CD34+ engrafted mice. SCD is characterized by ineffective erythropoiesis due to structural abnormalities in erythroid precursors3. As a functional structural unit, erythroblastic islands (EBIs) represent a specialized niche for erythropoiesis, where a central macrophage is surrounded by developing erythroblasts of varying differentiation states4. In our study, both SCD (Fig. k) and control (Fig. l) CD34+ engrafted mice exhibited EBIs with huCD169+ huCD14+ central macrophages surrounded by varying stages of huCD235a+ erythroid progenitors, including enucleated huRBCs (arrows). This implies that huHepMISTRG-Fah mice have the capability to generate human EBIs in vivo and thus represent a valuable tool to not only study the effects of mature RBC but also to elucidate mechanisms of ineffective erythropoiesis in SCD and other red cell disorders. In conclusion, we successfully engrafted adult SCD patient BM derived CD34+ cells in huHepMISTRG-Fah mice and detected circulating, sickling huRBCs in the mouse PB. We observed pathological changes in the lung, spleen, liver and kidney, which are comparable to what is seen in the established SCD mouse models and in patients. In addition, huHepMISTRG-Fah mice offer the opportunity to study the role of the central macrophage in human erythropoiesis in health and disease in an immunologically advantageous context. This novel mouse model could therefore serve to open novel avenues for therapeutic advances in SCD. Reference 1. Song Y, Shan L, Gybli R, et. al. In Vivo reconstruction of Human Erythropoiesis with Circulating Mature Human RBCs in Humanized Liver Mistrg Mice. Blood. 2019;134:338. 2. Ryan TM, Ciavatta DJ, Townes TM. Knockout-transgenic mouse model of sickle cell disease. Science. 1997;278(5339):873-876. 3. Blouin MJ, De Paepe ME, Trudel M. Altered hematopoiesis in murine sickle cell disease. Blood. 1999;94(4):1451-1459. 4. Manwani D, Bieker JJ. The erythroblastic island. Curr Top Dev Biol. 2008;82:23-53. Disclosures Xu: Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Flavell:Zai labs: Consultancy; GSK: Consultancy.

scholarly journals Case Report: Acute Stroke in Young Adult Patient with Moyamoya Syndrome Secondary to Sickle Cell Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 13-13
Author(s):  
Oladipo Cole ◽  
Asia Filatov ◽  
Javed Khanni ◽  
Patricio Espinosa
Keyword(s):  
Case Report ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Moyamoya Disease ◽  
Conflicts Of Interest ◽  
Acute Chest Syndrome ◽  
African American Female ◽  
Moyamoya Syndrome ◽  
Cell Disease ◽  
Radiographic Findings

Moyamoya disease, well described in literature, is a chronic cerebrovascular occlusive disorder. It is characterized by progressive stenosis/occlusion of the terminal portions of the internal carotid arteries (ICA) and the proximal portions of the middle cerebral arteries (MCA). Less frequently described is Moyamoya syndrome, the name given to radiographic findings consistent with Moyamoya disease, but with an identifiable cause. The diseases associated with Moyamoya Syndrome include Sickle Cell Disease (SCD), Thalassemias, and Down's Syndrome to name a few. Common complications of Moyamoya include both ischemic and hemorrhagic strokes. Upon literature review, Moyamoya syndrome caused by SCD is not well described. When it is, the discussion is centered around the pediatric patient population and surgical management. Our case report describes a 22-year-old African American female with SCD who initially presented with Acute Chest Syndrome. Her hospital course was complicated by development of overt debilitating neurologic deficits. Subsequently, she was found to have Moyamoya Syndrome on neuroimaging. She was successfully treated with medical management without any surgical intervention. This case highlights the necessity of thorough examination, differential diagnosis, imaging findings, and consideration of predisposing syndromes in the work-up for Moyamoya syndrome; especially individuals with Sickle Cell Disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals Utility of Procalcitonin in Differentiating Acute Chest Syndrome from Vaso-Occlusive Crisis in Sickle Cell Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 10-11
Author(s):  
Satish Maharaj ◽  
Simone Chang ◽  
Karan Seegobin ◽  
Marwan Shaikh ◽  
Kamila I. Cisak
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Complete Blood Count ◽  
Conflicts Of Interest ◽  
Statistical Significance ◽  
Acute Chest Syndrome ◽  
Adult Males ◽  
Cell Disease ◽  
Unequal Variances ◽  
Recorded Data

Background: Acute chest syndrome (ACS) frequently complicates sickle cell disease (SCD) and is a leading cause of hospitalization and mortality. Many factors have been implicated in ACS, including infections, thrombosis, fat and pulmonary emboli. However, a clear etiology is not defined in 50% of the cases and ACS is considered a clinical endpoint for different pathogenic processes (Vichinsky et al 2000). The non-specific nature of ACS makes diagnostic tests challenging, and there are no serum tests clinical used to aid diagnosis. Procalcitonin (PCT) is a prohormone of calcitonin and serum PCT rises within hours of an inflammatory stimulus. PCT has clinical utility as a marker of severe systemic inflammation, infection, and sepsis (Becker et al. 2008). Few studies have evaluated PCT as a biomarker for ACS in patients presenting with vaso-occlusive crises (VOC). Two studies have reported no difference in PCT (Biemond et al. 2018 and Stankovic et al 2011), while one study reported higher PCT between ACS and VOC (Patel et al 2014). Methods: We retrospectively reviewed 106 patients with SCD who presented to the emergency department with fever and painful crises during 2015-2019. The patients were divided into two categories based on discharge diagnoses - patients with VOC only (n=88) and patients with ACS (n=18). Inclusion criteria for both groups were patients with SCD, 17 years and older and PCT measurement on presentation. Exclusion criteria were defined as patients who had received empiric antibiotics prior to PCT testing. Data collected on presentation included genotype, age, gender, complete blood count, PCT, creatinine, total bilirubin and hydroxyurea use. Length of stay was recorded. Data was analyzed between the two groups using descriptive statistics and accounting for unequal variances, withp-value set at 0.05 for significance. Results: Demographics and clinical characteristics are summarized in Table 1 (Figure). The sample included primarily adult males (77%), with about two-thirds on hydroxyurea. Genotype HbSS (73.6%) was most prevalent followed by HbSC (22.6%) and HbSβ (3.8%). The ACS group had a higher percentage of HbSS, lower use of hydroxyurea and higher mean bilirubin. Mean PCT for the ACS group was 0.52 ng/mL (range, 0.05-2.04), compared to 0.31 ng/mL (range, 0.02-6.82) in the VOC group; withp=0.084. ROC analysis showed a PCT&gt;0.5ng/mL had 39% sensitivity and 85% specificity for ACS in this sample. Conclusion: In this sample, PCT on presentation was higher in those with ACS compared to VOC, but this difference did not achieve statistical significance. Further study in a larger population would be useful to evaluate this finding. Disclosures No relevant conflicts of interest to declare.

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