Sickle Erythrocytes Research Articles

The antisickling agent, 5-hydroxymethyl-2-furfural: Other potential pharmacological applications.

For the last two decades, the aromatic aldehyde 5-hydroxymethyl-furfural (5-HMF) has been the subject of several investigations for its pharmacologic potential. In 2004, the Safo group reported that 5-HMF has potent antisickling activity by targeting and ameliorating the primary pathophysiology of hypoxia-induced sickling of erythrocytes (red blood cells [RBC]). Following the encouraging outcome of the preclinical and phase I/II clinical studies of 5-HMF for the treatment of sickle cell disease (SCD), there have been multiple studies suggesting 5-HMF has several other biological or pharmacologic activities, including anti-allergic, antioxidant, anti-hypoxic, anti-ischemic, cognitive improvement, anti-tyrosinase, anti-proliferation, cytoprotective, and anti-inflammatory activities. The wide range of its effects makes 5-HMF a potential candidate for treating a variety of diseases including cognitive disorders, gout, allergic disorders, anemia, hypoxia, cancers, ischemia, hemorrhagic shock, liver fibrosis, and oxidative injury. Several of these therapeutic claims are currently under investigation and, while promising, vary in terms of the strength of their evidence. This review presents the research regarding the therapeutic potential of 5-HMF in addition to its sources, physicochemical properties, safety, absorption, distribution, metabolism, and excretion (ADME) profiles.

Morphological Factors and Forces Acting in Normal and Sickled Erythrocytes in Sickle Cell Anemia: A Mathematical Model

We carried out a theoretical study that considers the morphological factors and forces acting on a normal and a sickled blood cell. Sickle cell disease is associated with vaso-occlusion which creates blood flow crises as a result of loss in shape memory of the normal red blood cell. Certain forces and chemical compositions acting on the cell could retain the shape memory. The problem of a second order partial differential equation modeled was solved to get the forces and chemical composition required to restore the sickled red blood cells back to its original shape. The results gotten showed that, increase in chemical reaction increased the RBC growth by creating a stretching force that causes the sickle cell to regain elasticity with improved oxygen.

Genetic variants associated with white blood cell count amongst individuals with sickle cell disease.

Sickle cell disease (SCD) is a Mendelian disorder characterized by a point mutation in the β-globin gene that leads to sickling of erythrocytes. Several studies have shown that absolute neutrophil count is strongly associated with clinical severity of SCD, suggesting an apparent role of white blood cells (WBC) in SCD pathology. However, the mechanism by which genetic variants lead to WBC count differences in SCD patients remains unclear. Genome-wide association (GWA) analyses were carried out amongst a cohort of 2409 Brazil SCD participants. Association of WBC count and genetic markers were investigated in homozygous sickle cell anaemia participants and compound heterozygous sickle cell haemoglobin C participants. GWA analysis showed that variants in genes TERT, ACKR1, and FAM3C are associated with WBC count variation. The well-studied association between WBC count and Duffy null phenotype (variant in ACKR1) in healthy populations was replicated, reinforcing the influence of the SNP rs2814778 (T>C) in WBC count. Genetics plays an important role in regulating WBC count in patients with SCD. Our results point to possible mechanisms involved in WBC count variation and as increased WBC count is associated with more severe SCD, these results could suggest potential therapeutic targets for individuals with SCD.

Lactated Ringer vs Normal Saline Solution During Sickle Cell Vaso-Occlusive Episodes.

Sickle cell disease (SCD), a clinically heterogenous genetic hemoglobinopathy, is characterized by painful vaso-occlusive episodes (VOEs) that can require hospitalization. Patients admitted with VOEs are often initially resuscitated with normal saline (NS) to improve concurrent hypovolemia, despite preclinical evidence that NS may promote erythrocyte sickling. The comparative effectiveness of alternative volume-expanding fluids (eg, lactated Ringer [LR]) for resuscitation during VOEs is unclear. To compare the effectiveness of LR to NS fluid resuscitation in patients with SCD and VOEs. This multicenter cohort study and target trial emulation included inpatient adults with SCD VOEs who received either LR or NS on hospital day 1. The Premier PINC AI database (2016-2022), a multicenter clinical database including approximately 25% of US hospitalizations was used. The analysis took place between October 6, 2023, and June 20, 2024. Receipt of LR (intervention) or NS (control) on hospital day 1. The primary outcome was hospital-free days (HFDs) by day 30. Targeted maximum likelihood estimation was used to calculate marginal effect estimates. Heterogeneity of treatment effect was explored in subgroups. A total of 55 574 patient encounters where LR (n = 3495) or NS (n = 52 079) was administered on hospital day 1 were included; the median (IQR) age was 30 (25-37) years. Patients who received LR had more HFDs compared with those who received NS (marginal mean difference, 0.4; 95% CI, 0.1-0.6 days). Patients who received LR also had shorter hospital lengths of stay (marginal mean difference, -0.4; 95% CI, -0.7 to -0.1 days) and lower risk of 30-day readmission (marginal risk difference, -5.8%; 95% CI, -9.8% to -1.8%). Differences in HFDs between LR and NS were heterogenous based on fluid volume received: among patients who received less than 2 L, there was no difference in LR vs NS; among those who received 2 or more L, LR was superior to NS. This cohort study found that, compared with NS, LR had a small but significant improvement in HFDs and secondary outcomes including 30-day readmission. These results suggest that, among patients with VOEs in whom clinicians plan to give volume resuscitation fluids on hospital admission, LR should be preferred over NS.

Respiratory management of acute chest syndrome in children with sickle cell disease.

Acute chest syndrome (ACS) is a leading cause of respiratory distress and hospitalisation in children with sickle cell disease (SCD). The aetiology is multifactorial and includes fat embolism, venous thromboembolism, alveolar hypoventilation and respiratory infections, with the latter being particularly common in children. These triggers contribute to a vicious cycle of erythrocyte sickling, adhesion to the endothelium, haemolysis, vaso-occlusion and ventilation-perfusion mismatch in the lungs, resulting in the clinical manifestations of ACS. The clinical presentation includes fever, chest pain, dyspnoea, cough, wheeze and hypoxia, accompanied by a new pulmonary infiltrate on chest radiography. Respiratory symptoms may overlap with those of acute asthma, which may be difficult to distinguish. Patients with ACS may deteriorate rapidly; thus prevention, early recognition and aggressive, multidisciplinary team management is essential. In this narrative review, we highlight the current evidence regarding the epidemiology, pathophysiology, treatment and preventative strategies for ACS, focusing on the aspects of major interest for the paediatric pulmonologist and multidisciplinary team who manage children with SCD.

TSH Receptor Reduces Hemoglobin S Polymerization and Increases Deformability and Adhesion of Sickle Erythrocytes.

SCD is a hereditary disorder caused by genetic mutation in the beta-globin gene, resulting in abnormal hemoglobin, HbS that forms sickle-shaped erythrocytes under hypoxia. Patients with SCD have endocrine disorders and it was described that 7% of these patients have clinical hypothyroidism. Recent studies have shown that mature erythrocytes possess TSH receptors. Thus, we aimed to assess the effects of TSH on SCD erythrocytes. The experiments were conducted using different concentrations of TSH (1, 2, 3, and 5 mIU/L). In HbS polymerization assay, erythrocytes were exposed to TSH in hypoxia to induce polymerization, and measurements were taken for 30 minutes. The deformability assay was made using Sephacryl-S 500 columns to separate deformable from nondeformable cells. Static adhesion test utilized thrombospondin to assess erythrocyte adhesion in the presence of TSH. TSH at all contractions were able to reduce polymerization of HbS and increase deformability. The static adhesion of erythrocytes at the lowest concentrations of 1 and 2 mIU/L were increased, but at higher contractions of 3 and 5 mIU/L, static adhesion was not modulated. The results suggest that TSH has potential involvement in the pathophysiology of sickle cell disease by inhibiting HbS polymerization, positively modulating deformability and impacting static adhesion to thrombospondin.

Computing Sickle Erythrocyte Health Index on quantitative phase imaging and machine learning

Sickle cell disease (SCD) is a genetic disorder characterized by abnormal hemoglobin and deformation of red blood cells (RBCs), leading to complications and reduced life expectancy. This study developed an in vitro assessment, the Sickle Erythrocyte Health Index, using quantitative phase imaging (QPI) and machine learning to model the health of RBCs in people with SCD. The health index combines assessment of cell deformation, sickle-shaped classification, and membrane flexibility to evaluate erythrocyte health. Using QPI and image processing, the percentage of sickle-shaped cells and membrane flexibility were quantified. Statistically significant differences were observed between individuals with and without SCD, indicating the impact of underlying pathophysiology on erythrocyte health. Additionally, sodium metabisulfite led to an increase in sickle-shaped cells and a decrease in flexibility in the sickle cell blood samples. Based on these findings, two approaches were used to calculate the Sickle Erythrocyte Health Index: one using hand-crafted features and one using learned features from deep learning models. Both indices showed significant differences between non-SCD and SCD groups and sensitivity to changes induced by sodium metabisulfite. The Sickle Erythrocyte Health Index has important clinical implications for SCD management and could be used by providers when making treatment decisions. Further research is warranted to evaluate the clinical utility and applicability of the Sickle Erythrocyte Health Index in diverse patient populations.

Death due to sickle cell crisis: a case report.

Sickle cell disease (SCD) is the most common hereditary hemoglobinopathy worldwide. It results in characteristic acute and chronic findings on postmortem computed tomography (PMCT), macroscopic and microscopic examinations. While the diagnostic imaging and macroscopic features are not specific for SCD on their own, when coupled with microscopic features such as sickled erythrocytes and evidence of chronic venous congestion (i.e., Gamna-Gandy bodies), these clues can help alert forensic pathologists to the presence of SCD. Despite the prevalence of the disease and the constellation of findings alluded to above, SCD is not often explored in forensic pathology literature. This case demonstrates classic acute and chronic features of SCD on PMCT, macroscopic and microscopic examinations. It explores the pathophysiology leading to sudden and unexpected death in a person with SCD and possible pitfalls in attribution of cause of death.

Clearance of VWF by hepatic macrophages is critical for the protective effect of ADAMTS13 in sickle cell anemia mice

AbstractAlthough it is caused by a single-nucleotide mutation in the β-globin gene, sickle cell anemia (SCA) is a systemic disease with complex, incompletely elucidated pathologies. The mononuclear phagocyte system plays critical roles in SCA pathophysiology. However, how heterogeneous populations of hepatic macrophages contribute to SCA remains unclear. Using a combination of single-cell RNA sequencing and spatial transcriptomics via multiplexed error-robust fluorescence in situ hybridization, we identified distinct macrophage populations with diversified origins and biological functions in SCA mouse liver. We previously found that administering the von Willebrand factor (VWF)–cleaving protease ADAMTS13 alleviated vaso-occlusive episode in mice with SCA. Here, we discovered that the ADAMTS13-cleaved VWF was cleared from the circulation by a Clec4f+Marcohigh macrophage subset in a desialylation-dependent manner in the liver. In addition, sickle erythrocytes were phagocytized predominantly by Clec4f+Marcohigh macrophages. Depletion of macrophages not only abolished the protective effect of ADAMTS13 but exacerbated vaso-occlusive episode in mice with SCA. Furthermore, promoting macrophage-mediated VWF clearance reduced vaso-occlusion in SCA mice. Our study demonstrates that hepatic macrophages are important in the pathogenesis of SCA, and efficient clearance of VWF by hepatic macrophages is critical for the protective effect of ADAMTS13 in SCA mice.

The Effect of Voxelotor on Erythrocyte Deformability, Neutrophil Phenotype and Endothelial Activation

Introduction Sickle cell disease (SCD) is characterized by chronic intravascular hemolysis, caused by polymerization of abnormal sickle hemoglobin. The ongoing intravascular hemolysis leads to release of cell-free heme and iron, resulting in widespread oxidative damage, inflammation, neutrophil activation, endothelial activation and hypercoagulability, all contributing to vaso-occlusive events and end organ damage. Voxelotor, a small molecule reversibly binding to hemoglobin S (HbS) increases its oxygen affinity, thereby decreasing HbS polymerization and sickling, resulting in decreased hemolysis. The decrease in erythrocyte sickling might lead to improved erythrocyte deformability and decreased adhesion, while reductions in cell-free heme and labile plasma iron levels might result in decreased neutrophil activation, adhesion and aging and decreased endothelial activation. The aim of this study is to assess the effects of voxelotor on erythrocyte deformability, neutrophil activation, adhesion and aging and endothelial activation. Methods SCD patients (HbSS and HbSβ 0-thalassemia), with hemoglobin (Hb) ≤ 10.5 g/dL were treated with voxelotor 1500 mg in a single arm interventional study. Blood was collected at baseline and after 3 months of treatment. Erythrocyte deformability was assessed by ektacytometry (Lorrca, RR mechatronics), measuring elongation index (EI) and point of sickling (PoS). EI max and EI min indicate the highest and lowest erythrocyte deformability upon oxygenation and deoxygenation respectively. PoS is the oxygen tension at which 5% reduction of the EI max is observed. Antigen expression of neutrophil markers for activation, adhesion and aging were assessed with flow cytometry. Reactive oxygen species (ROS) production of neutrophils was measured by Amplex Red Assay (Molecular probes), measuring hydrogen peroxide (H 2O 2) production under unstimulated and stimulated (zymosan, streptozotocin (STZ), factor-n-formyl-MET-LEU-Phe (fMLP), Phorbol myristate acetate (PMA)) and platelet-activating factor-fMLP (PAF-fMLP)). Endothelial activation was assessed by measuring von Willebrand Factor antigen (vWF-Ag) using enzyme-linked immunosorbent assay (ELISA) (Glostrup). Prothrombin fragment 1+2 (F 1+2), thrombin-antithrombin complexes (TAT) and D-dimer were measured using ELISA. Data are presented as median with interquartile range (IQR), unless stated otherwise. A P value of <0.05 was considered significant. Results 15 SCD patients (11 HbSS, 4 HbSβ 0-thalassemia), 5 female, median age 35 years (IQR 27-46)) completed 3-month follow-up. Hb levels increased significantly after 3 months of treatment (from 7.4 (6.9 - 8.1) g/dL to 9.7 (7.9 - 11.0) g/dL, p=0.002), corresponding to significant decreases in LDH and bilirubin levels and reticulocyte counts as markers of hemolysis (p<0.05). PoS decreased significantly after 3 months (from 20.1 (17.5 - 26.1) mmHg to 16.1 (10.3 - 18.1) mmHg, p=0.006). Both EI max and EI min increased after 3 months of treatment (from 0.33 (0.24 - 0.42) to 0.38 (0.28 - 0.48), p=0.029 and from 0.13 (0.07 - 0.26) to 0.21 (0.16 - 0.41), p=0.007 respectively). Markers of neutrophil activation, adhesion and aging were generally lower after 3 months of treatment, though the differences were not statistically significant (Figure 1 A-C). Neutrophil ROS production in both unstimulated and stimulated condition remained unchanged (Figure 1D). After 3 months of treatment vWF-Ag significantly decreased (from 252 (187 - 284) % to 183 (153-205) %, p = 0.033). No changes were observed in markers of coagulation activation (F 1+2 and TAT, and D-dimer). Conclusion Voxelotor significantly improved erythrocyte deformability as suggested by decreased PoS and increased EI max and EI min, potentially leading to improved rheological properties. While markers of neutrophil activation seem to decrease, the differences are not statistically significant, possibly due to the relatively small sample size. A reduction in vWF-Ag was observed possibly reflecting diminished endothelial activation, due to a decrease in intravascular hemolysis.

A Novel, Rapid, and Accurate Quantitative Hydroxyurea Assay

Introduction: Hydroxyurea is a highly effective treatment for children and adults with sickle cell anemia (SCA) and is now considered standard-of-care therapy in high-resource countries. There is wide interpatient variability in the optimal dose, which is defined as the daily dose that provides maximal induction of protective fetal hemoglobin to decrease erythrocyte sickling while causing only mild marrow suppression. The time and cost of hydroxyurea dose titration are barriers to its widespread use in low-resource settings. The titration process required to identify an optimal dose can be abbreviated by measuring individualized hydroxyurea pharmacokinetics (PK) to guide personalized precision dosing, which could be especially useful in low-resources settings with limited laboratory monitoring. However, measurement of hydroxyurea in serum and other physiological fluids is not commonly performed during routine clinical treatment. Current methodology to quantify hydroxyurea concentrations requires complex and time-consuming chemical derivatization followed by detection by colorimetric spectrophotometry, high performance liquid chromatography (HPLC), or liquid chromatography mass spectrometry (LC-MS). To consider performing PK-guided dosing in low-resource settings, a simple, accurate, and efficient hydroxyurea assay is needed. Methods: A commercially available open-system HPLC unit for hemoglobin separation (SmartLifeLC, PolyLC Inc, Columbia MD) was equipped with a 3.5-micron, 4.6 mm × 50 mm Zorbax Eclipse XDB-C18 column with matching guard column (Agilent Technologies, Santa Clara, CA); mobile phases of 46% methanol and 100% acetonitrile; and a 200-600nm multi-wavelength detector. The chemical derivatization process was modified from previously published techniques that use xanthydrol (Sigma-Aldrich, St. Louis MO) for detection of primary amides. N-methylurea (Sigma-Aldrich) was included as an internal standard. Hydroxyurea (Sigma-Aldrich) was diluted in whole blood collected from healthy controls to create a calibration curve encompassing the typical pharmacological range of hydroxyurea at 80, 40, 20, 10, and 5 mg/mL. Quality Control (QC) samples were prepared at 50, 25, 12.5, and 6.25 mg/mL to validate the calibration curve before testing patient samples. Blood was applied to 20mL volumetric absorptive microsampling devices (Mitra, Trajan Scientific and Medical, Torrance CA) and dried for future elution and analysis. HPLC results were compared to LC-MS results to assess accuracy. Results: Blood was eluted from the Mitra microsampling device and derivatized in a xanthydrol solution before HPLC analysis. Using a 10-minute run with 210nm wavelength detection, the xanthydrol-modified hydroxyurea reproducibly yielded a linear calibration curve with r 2 >0.95 (Figure), and the actual concentrations were within 20% of the theoretical values. Repeated testing of control and patient samples revealed a typical intra-day coefficient of variation <15% and inter-day coefficient of variation <20%. The agreement between nominal values and assay results determined for a wide range of QC samples from both methods exceeded the FDA-recommended threshold of 67% for chromatographic assay validation with 86% agreement on LC-MS and 85% with the new method. The entire elution and derivatization procedure could be completed using dried Mitra blood samples in less than 60 minutes. Conclusions: Quantitative hydroxyurea measurement of blood or serum can now be accomplished using a novel, rapid, HPLC technique that features xanthydrol-based derivatization of hydroxyurea, an internal standard, and UV wavelength detection. This technique is accurate, reproducible, and relatively simple, and thus deemed suitable for hydroxyurea concentration measurements needed for PK-guided dosing calculations for children with SCA living in low-resource settings. This novel technique will be added to the multi-country Realizing Effectiveness Across Continents with Hydroxyurea (REACH, NCT01966731) trial that will feature PK-guided precision dosing for young children with SCA starting hydroxyurea treatment.

Assessing Real-Time Sickling Kinetics in Individual Cells from Erythrocyte Populations of Subjects with Different HbA, HbS and HbF Percentages

INTRODUCTION: Polymerization of deoxygenated sickle hemoglobin (HbS) leads to erythrocyte sickling. Treatment in sickle cell disease (SCD) aims to reduce the risks of red blood cell (RBC) sickling and associated complications including, but not limited to vaso-occlusive crises (VOC). There is substantial promise in developing therapies targeting HbS polymerization that consequently decrease RBC sickling, a fundamental pathophysiological driver of SCD. Nonetheless, there are limited options available for reliable diagnostic tools for the assessment of disease status and therapeutic outcomes. A robust and easy-to-use system to assess population-scale sickling kinetics and to monitor responses to Hb-modifying therapies in clinical trials for the clinical management of patients may be beneficial. We have developed a dynamic sickling assay (DSA) that uses an enzymatic oxygen scavenging system to provide tight control over the rate and depth of induced-hypoxia. DSA is able to differentiate samples based on endogenous Hb expression and can be used to monitor sickling kinetics pre- and post HbS-modifying therapies, including hematopoietic stem cell transplantation (HSCT). METHODS: SCD and sickle cell trait (SCT) subjects were recruited under the RBC Health Initiative IRB protocol number FF-RBC-003. Four hematopoietic stem cell transplant (HSCT) SCD subjects were enrolled in NIH haploidentical HSCT protocol 17-H-0069. Diluted blood samples were supplemented with protocatechuic acid (PCA), followed by protocatechuate 3,4-dioxygenase (PCD) enzyme inducing hypoxia at a predetermined rate. The sample was then injected into a microfluidic chamber, and resulting RBC morphological changes were observed and captured with time-lapse photography. Image analysis identified and quantified RBC using an AI-trained SICKLE image analysis software. HbAS (n=3), HbSS with HbA (post-transfusion, n=3) and HbSS samples containing 3.1 to 34.5% HbF (n=9) were processed with DSA to assess the effects of hemoglobin distribution on sickling kinetics.Pre- and post-HSCT samples were run to ascertain the effect on sickling kinetics at baseline, 3M, 6M and 12M post-HSCT. Sickling profiles were constructed enabling a comprehensive assessment of RBC sickling and treatment outcomes. The reported parameters include: mPOS@5% (time to reach 5% induced sickling, min), mPOS@50% (time to reach 50% maximum induced sickling,min), rate of sickling (the maximum sickling rate, %/min), maximum induced sickling (the highest induced sickling percentage, %), AUC at 10min (area under the curve@10min of DSA, %∙minute) RESULTS: DSA inter-run CVs are 6.9% for mPOS@5%, 3.8% for mPOS@50%, 2.7% for max sickling, 4.2% for AUC and 10.8% for the rate of sickling. Statistically significant correlations are observed between %HbS and rate of sickling, max sickling, mPOS@5%, and AUC at 10min. As percentages of Hb increased in transfused SCD patient samples, there were noticeable correlations between the delay in mPOS@5% and decreased rate of sickling, maximum sickling, and AUC10. Similarly, as HbA percentages increased in SCT samples, a decrease in AUC10 and max sickling were observed. Transfused and SCT samples did not show statistical significance due to sample size limitations. However, there was a strong correlation in delay of mPOS@5% and mPOS@50%, as well as a decrease in the rate of sickling and AUC10 with increasing percentages of HbF in samples (Table 1). Comparing pre-HSCT (BL) sample sickling kinetics to 3, 6 and 12 month post-HSCT samples showed decreases in the rate of sickling (from 44.4 to 0.6%), maximum sickling (from 45-23%), decreases in AUC and delays in mPOS (Table 2). CONCLUSIONS: This study found significant linear relationships between %HbS and sickling parameters.As HbA percentage increases in transfused SCD and SCT samples, improvements in sickling parameters are observed. There is a strong correlation between increased HbF percentage and improved DSA parameters. Comparative data analysis from patients pre- and post-HSCT also show improvement in sickling parameters. Overall, DSA provides a simple, robust method for assessing responses to Hb differences and monitoring SCD therapies such as gene-editing.

Impaired Hemoglobin Clearance By Liver Sinusoidal Endothelium Promotes Liver Damage in Sickle Cell Disease

Sickle cell disease (SCD) is a monogenic disorder that affects 100,000 African Americans and millions of people worldwide. Intra-erythrocytic polymerization of sickle hemoglobin (HbS) promotes erythrocyte sickling, impaired rheology, ischemia and hemolysis, leading to the development of progressive liver injury in SCD. Liver resident macrophages and monocytes are known to enable the clearance of HbS, however, the role of liver sinusoidal endothelial cells (LSECs) in HbS clearance and liver injury in SCD remains unknown. Using real-time intravital ( in vivo) imaging of the liver as well as primary culture of human and mice LSECs, we show for the first time that liver injury is associated with accumulation of HbS and iron in the LSECs, leading to the development of senescence in LSECs. Hepatic monocytes were observed to attenuate LSEC-senescence by accelerating HbS clearance in the liver of SS mice, however, this protection was impaired in P-selectin-deficient SCD mice secondary to reduced monocyte recruitment in the liver. These findings are the first to suggest that LSECs contribute to HbS clearance and LSEC-senescence promotes progressive liver injury in SS mice. Our results reveal a novel insight into the pathogenesis of hemolysis induced chronic liver injury in SCD caused by LSEC senescence. Identifying the regulators of LSEC mediated HbS clearance may lead to new therapies to prevent the progression of liver and other organ injuries in SCD.

Evaluation of Antisickling Activity and Antibacterial Potential of Methanolic Root Extract of Ximenia americana Linn. (Olacacaea)

Aims: This research work was done in order to evaluate invitro antisickling activity, and antibacterial potentials of the methanolic root extract of Ximenia americana, Linn. Olacaceae. Study Design: It is an experimental research work. Place and Duration of Study: Department of Microbiology and Biotechnology, National Institute of Pharmaceutical Research and Development (NIPRD), Idu, Abuja, Nigeria. The study was carried out between December, 2019 to June, 2020. Methodology: Evaluation of the antisickling activity of the methanolic root extract of Ximenia americana was carried out in two-fold; viz. the ability of the plant extract to inhibit or prevent sickling (antisickling effect) of erythrocytes from blood samples obtained from sickle cell disease (SCD) volunteers on one hand, and the ability of the extract to reversed pre-sickled erythrocytes using standard protocols. The antibacterial assay was done using microtiter broth dilution method to determine the minimum inhibitory concentration (MIC) on some selected encapsulated bacterial (E. coli, Salmonella typhi, Staphylococcus aureus, Pseudomonas mirabilis, Pseudomonas aeruginosa, Bacillus subtilis, and Klebsiella pneumonia) that have been reported to caused complications in SCD. Results: The methanolic root extract of Ximenia americana showed a concentration dependent antisickling activity across concentration gradient. The percent sickling inhibition was dose-dependent (0.05 mg = 60 %, 0.5 mg = 62 %, 1 mg = 88 %, 2 mg = 87 %, and 4 mg = 90 %), this result was statistically significant (p < 0.05 vs control) when compared to the untreated group (25 %). Similarly, the result of the sickle red cell reversal by the test extract demonstrated a percent dose-dependent reversibility (0.05 mg = 36 %, 0.5 mg = 40 %, 1 mg = 48 %, 2 mg = 58 %, and 4 mg = 63 %), a result that was significantly (p < 0.5 vs control) compared to the control (19 %). The antimicrobial assay result showed that, the sensitivity of the selected microorganisms to the plant extract was concentration dependent. Staphylococcus aureus and Bacillus subtilis showed better sensitivity to the extract with higher zones of inhibition (8 mm and 7 mm) at 80 mg/mL concentration. Conclusion: Methanolic root extract of Ximenia americana L demonstrated a significant dose-dependence antisickling and sickle cell reversal activities, with p < 0.05 vs control. Also, the extract showed sensitivity against some selected bacteria isolates as a result possess antibacterial activity against susceptible microorganism.

Impaired hemoglobin clearance by sinusoidal endothelium promotes vaso-occlusion and liver injury in sickle cell disease.

Sickle cell disease (SCD) is a monogenic disorder that affects 100,000 African-Americans and millions of people worldwide. Intra-erythrocytic polymerization of sickle hemoglobin (HbS) promotes erythrocyte sickling, impaired rheology, ischemia and hemolysis, leading to the development of progressive liver injury in SCD. Liver-resident macrophages and monocytes are known to enable the clearance of HbS; however, the role of liver sinusoidal endothelial cells (LSEC) in HbS clearance and liver injury in SCD remains unknown. Using real-time intravital (in vivo) imaging in mice liver as well as flow cytometric analysis and confocal imaging of primary human LSEC, we show for the first time that liver injury in SCD is associated with accumulation of HbS and iron in the LSEC, leading to senescence of these cells. Hemoglobin uptake by LSEC was mediated by micropinocytosis. Hepatic monocytes were observed to attenuate LSEC senescence by accelerating HbS clearance in the liver of SCD mice; however, this protection was impaired in P-selectin-deficient SCD mice secondary to reduced monocyte recruitment in the liver. These findings are the first to suggest that LSEC contribute to HbS clearance and HbS-induced LSEC senescence promotes progressive liver injury in SCD mice. Our results provide a novel insight into the pathogenesis of hemolysis-induced chronic liver injury in SCD caused by LSEC senescence. Identifying the regulators of LSEC-mediated HbS clearance may lead to new therapies to prevent the progression of liver injury in SCD.

Impact of Vaso-Occlusive Crises on the Transcriptomes of Patients with Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is an inherited, multisystem blood disorder that affects millions of people worldwide and is particularly common among those of African descent. Vaso-occlusive crises (VOCs) or acute pain events involving complex interactions among sickled erythrocytes, endothelial cells, leukocytes, and activated platelets are hallmark symptoms of SCD. Over time, episodic bouts of acute VOC may develop into chronic pain, defined as pain most days for 6 months. At present, there are no biomarkers that distinguish between acute and chronic pain. Here, we explore differences in gene expression during VOC events in a cohort of pediatric patients with SCD. The objective of this study was to examine transcriptome alterations in CD45+ cells of individuals with SCD during a VOC event compared with steady state (SS) in patients with or without chronic pain. Methods: Patients were aged <21 years, with HbSS or HbSβ 0 genotypes. Informed consent was obtained for all patients. CD45+ cell (lymphohematopoietic lineage) samples were collected from 158 patients with SCD under SS conditions (at the maximum tolerated dose of hydroxyurea; some patients were also on metformin) during a VOC event, and at a follow-up visit after a VOC. RNA sequencing (RNA-Seq) was performed using a 101-base pair, paired-end protocol run on the NovaSeq 6000 system. We applied supervised normalization of microarrays to adjust the normalized gene expression values for the effect of multiple covariates. ToppGene Suite was used for over-representation analysis (ORA; Chen et al. Nucleic Acid Res 2009). Analysis of differentially expressed genes was performed using limma software and gene set enrichment analysis using the Molecular Signature Database. Results: A summary of the study cohort is presented (Table). Most patients (98.7%) were on hydroxyurea. We collected RNA-Seq data from unpaired and paired samples at SS and during a VOC. After adjusting for age, sex, chronic pain status, and metformin status, analyses of RNA-Seq data showed upregulated genes linked to TNFα signaling via NFΚB, IL-6/JAK/STAT3 signaling, complement activation, and coagulation were enriched in CD45+ cells during VOCs compared with SS (Figure). Differentially expressed genes were also compared with whole blood transcriptome samples collected at baseline and during a VOC event as reported by Creary et al (Sci Rep 2020). Approximately 290 upregulated and 33 downregulated genes were shared in both studies. ORA analysis of the shared genes revealed upregulation of pathways such as neutrophil degranulation and complement activation during a VOC event. Analysis of samples from patients without chronic pain at SS indicated similar immune-related pathways, such as complement activation and interferon α response, were upregulated during a VOC event. In contrast, analysis of samples from patients reporting chronic pain revealed no significant differences in gene expression between a VOC event and SS. Conclusions: Samples collected during a VOC event showed significant changes in the transcriptome of CD45+ cells compared with SS in pediatric patients who had the most severe disease, or sickle cell anemia characterized by HbSS and HBSβ 0/+ genotypes, without a diagnosis of chronic pain, with multiple pathways altered by VOC. However, when VOC transcriptomes in patients with chronic pain were compared with SS, these pathways were not significantly altered. Our pediatric patient population had low rates of chronic pain; therefore, our sample size was limited. Additional analyses of SS and VOC transcriptomes, particularly in patients with chronic pain, will expand our understanding of the impact of VOC events on transcriptomes in individuals with and without chronic pain. This may lead to identification of quantitative biomarkers for a VOC, facilitating testing of new therapies for the treatment of pain in people with SCD.

Targeted Degradation of the Wiz Transcription Factor for Gamma Globin De-Repression

Sickle cell disease (SCD) is a life-threatening genetic condition arising from a point mutation in the β-globin gene that causes hemoglobin polymerization and vaso-occlusive sickling of erythrocytes. Hereditary persistence or pharmacologic induction of gamma-globin (HBG1 or HBG2) expression leads to increases in fetal hemoglobin (HbF) that biophysically oppose hemoglobin polymerization. Indeed, the standard-of-care, hydroxyurea (HU), increases expression of HbF in the erythroblast, and in patients the magnitude of response (9-24% HbF) correlates with a reduction in pain crises and acute complications. However, the use of HU is limited by variable response, myelosuppression, GI side effects and carcinogenicity risk. New targeted therapeutic approaches to induce HbF are therefore being intently pursued, leveraging the increasingly detailed functional characterization of the globin locus. The recent discovery of BCL11A as a repressor of HBG has prompted the widespread innovation of genetically engineered hematopoietic stem cells that suppress BCL11A expression or function. While a major advance in SCD therapeutics, the complexity and clinical implementation of stem cell transplantation may limit access and impact, especially in developing countries where SCD burden is greatest. We therefore undertook to discover a small-molecule HbF inducer, focusing on transcription factors that repress gamma-globin expression. Although transcription factors as a target class have historically been considered "undruggable," recent mechanistic characterization of phthalimide medicines as serendipitous, cereblon (CRBN)-dependent degraders of IKZF1 and IKZF3 by our organization and others, suggested the plausible degradation of new zinc-finger containing transcription factors by this emerging class of molecular glues. Therefore, we elaborated a large library of CRBN-biased ligands for study in target-directed and phenotypic drug discovery campaigns. Here we report for the first time the phenotypic discovery and structure-based optimization of two molecular glue degraders of the transcription factor, WIZ, which lead to pronounced de-repression of HbF. Using a chemical biology approach, the first molecule, dWIZ-1, was identified by phenotypic screening of a CRBN-biased chemical library for compounds that induce HbF without deleterious effect on erythroid cell proliferation and differentiation. Subsequent target deconvolution by global proteomics revealed WIZ as the target. WIZ knockout using two independent sgRNA elevated the proportion of γ-globin mRNA, HbF protein levels and HbF + cells (Figure 1) in vitro in primary human erythroblasts, validating WIZ as a previously unrecognized repressor of HbF. Optimization of dWIZ-1 led to a second tool compound, dWIZ-2, which was suitable to assess WIZ degradation and HbF induction in vivo due to its superior pharmacokinetic properties. dWIZ-2 treatment increased the proportion of γ-globin mRNA, HbF + cells and HbF protein levels in vitro in sickle cell disease patient-derived erythroblasts (Figure 2). Because many IMiDs lack activity in rodents, we used a humanized mouse model to measure the impact of dWIZ-2 on WIZ degradation and HbF expression in human erythroblasts in vivo. NBSGW mice humanized with CD34 + hematopoietic stem and progenitor cells from healthy human donors were orally dosed once daily with dWIZ-2 for 21 days. dWIZ-2 treatment resulted in robust dose-dependent WIZ degradation and an increase in total HbF and the proportion of HbF + human erythroblasts in the bone marrow. To further study the kinetics of HbF induction in the peripheral blood, and assess the effects of dWIZ-2 on steady-state hematopoiesis, we orally dosed naïve healthy cynomolgus monkeys with vehicle or 30 mg/kg dWIZ-2, once daily for 28 days. We detected elevated levels of γ-globin mRNA in the blood on day 28, reaching up to 37% of β-like globins. Additionally, on day 28, we observed up to 95% HbF + reticulocytes.dWIZ-2 was well-tolerated based on the absence of notable clinical observations during the study and consistent body weight for all animals. There were no dWIZ-2-related changes in hematology, coagulation or clinical chemistry measurements, specifically including reticulocytes, hematocrit and neutrophil counts. These findings support the development of an oral WIZ degrader as a globally accessible therapeutic approach for SCD.

Radchip: A Laminin-Lined Red Blood Cell (RBC) Adhesion Device for Functional Characterization and Clinical Evaluation of Sickle RBCs at Steady-State

Sickle cell disease (SCD) is a genetic disorder caused by a point mutation in the beta-globin gene producing an abnormal hemoglobin (HbS) which, under deoxygenation, polymerizes and causes severe distortion of the red blood cell (RBC). RBCs may be dehydrated, poorly deformable and highly adhesive to the endothelium, with abnormal adhesion being of primary interest to this study. Recently, several new disease modifying drugs have been FDA approved, with more in development. These novel agents target specific aspects of SCD pathophysiology, including adhesion. Changes induced by such therapies are not always detectable by common conventional lab instruments. LORRCA (oxygen-gradient ektacytometry) is the gold-standard for deformability measurement; it has been well described both analytically and clinically in SCD. Adhesion and deformability have a dynamic, yet complementary relationship that we explore here. While these may behave in an interdependent manner, each determinant must be considered independently. There is a growing demand for innovative tools to evaluate rheological properties under physiological conditions, particularly microfluidic devices. However, many current devices are limited in their clinical utility due to, choice of ligand, complexity of design, or reliance on endothelial cell culture (typically HUVEC cells) which are difficult to use, vary with passage number, and do not represent adult SCD endothelium. We developed a Red blood cell Adhesion Chip (RAdChip) to model RBC to endothelial adhesion using laminin, a physiologically relevant ligand, to easily, effectively, and reproducibly measure RBC adhesion. Here, we describe our analytic and clinical validation of our device. Peripheral blood samples were collected in EDTA from individuals with SCD and healthy controls under an IRB approved protocol. RBCs were extracted via centrifugation, washed and resuspended in PBS to a 0.5% HCT. Microfluidic devices were fabricated using polydimethylsiloxane (PDMS). A 5ug/ml solution of recombinant laminin was applied to each channel, then blocked with 1% bovine serum albumin (BSA). Resuspended RBCs were introduced to the device through tubing connected to the inlet port. Harvard Apparatus syringe pumps were used to perfuse samples and wash channels at ~1dyne/cm 2. Adhesion was visualized with a Keyence inverted bright-field microscope at 20x (Itasca, IL). Adhered cells were counted within a 1.8mm 2 region of interest (Roi) using the iCLOTS automated cell quantification software (Fay et al, Nat comm accepted 7/2023). We measured adhesion index via RAdChip for common SCD genotypes: HbSS/SB0: (median (x͂) =756; range (r): 154-1889; n=26) HbSC/SB+ : (x͂ =299; r=: 114-731; n=11 ) HbAA: (x͂) =13; r= 5-90; n=7). RBC adhesion values were independent of age (p=0.65) and gender (p=0.63). An increase of 0.1 unit in EImin and EImax decreases RBC adhesion by 214 and 268 cells/mm 2, respectively. A 1mmHg increase in PoS increased adhesion by 19.5 cells/mm 2. sRBC adhesion had a significant positive association with vaso-occlusive events (VOE) (p=0.005), absolute neutrophil count (ANC) (p=0.005), and EImax (p<0.001). Notably, analytical validation of RAdChip produced a mean CV of 0.13 ± 0.94. RAdChip adds a key component of functional RBC evaluation that conventional lab equipment does not measure. We have analytically validated our device, demonstrating an excellent coefficient of variability. 44 patient samples, each examined in triplicate produced a mean CV of 0.13. We have demonstrated clinical validity of our device by establishing correlations with known markers of severity and pain event frequency. RBC adhesion values increase as the number of VOE events increase, reaffirming the belief that adhesion is a principal element in the multistep VOE cascade. In like manner, improvements in deformability are associated with decreased RBC adhesion, strengthening the interdependent nature of VOE molecular events. Recognizing the dynamic nature of SCD, it is necessary to explore sickle erythrocyte adhesion longitudinally to investigate changes before, during, and after VOE occurrences. Continued and increased integration of adhesion measurements with RAdChip and other rheology devices into routine SCD care may be helpful in assessing disease severity, identifying need for additional disease modifying agents, and assist with second line agent selection and monitoring.

Cerebral Hemodynamics in Infants with Sickle Cell Disease Indicate Presence of Early Hemodynamic Stress

Introduction: One of the most severe complications of sickle cell disease (SCD) is ischemic brain injury, caused by a combination of decreased oxygen transport delivery related to intravascular vaso-occlusion of sickle erythrocytes and/or cerebral vasculopathy. While newborn screening programs diagnose SCD shortly after birth, routine risk assessment for stroke typically starts with transcranial ultrasound doppler (TCD) at two years of age or later. Yet, little is known about how SCD affects cerebral hemodynamics during infancy, a crucial period for brain development. To address this gap, we used advanced quantitative near-infrared spectroscopy (qNIRS) to non-invasively monitor cerebral tissue hemoglobin concentration (HbT), oxygenation (SO 2), oxygen extraction fraction (cFTOE), blood flow (CBF), and oxygen metabolism (CMRO 2) in infants with SCD. Abnormal CBF and cFTOE indicate hemodynamic stress that may increase stroke risk. Our study aims to investigate the effects of SCD on cerebral oxygen transport in infancy and its associations with age and blood hemoglobin concentration (HgB). Methods: This is a subgroup analysis of a prospective, observational neuroimaging study of SCD at Boston Children's Hospital (NCT 04166526). Inclusion criteria of this subgroup are SCD patients age less than 24 months old at the time of enrollment who are not receiving chronic transfusions. Families read and signed informed consent before participating in research activities. Measurements were conducted with the patients' normally scheduled hematology appointments at Boston Children's Hospital. During each measurement session, bilateral frontal regions of the head were measured up to six times for 30 seconds each. qNIRS variables were compared with literature values obtained with the same optical technologies from a cohort of typically developed infants around 1 year old, equivalent to the median age in this study 1. Correlation analysis was used to analyze qNIRS variables with age and HgB from clinical lab tests. Results: To date, 8 (F/M: 4/4; HbSS/HbS/B +-thalassemia/HbSC: 6/1/1) patients were enrolled with a total of 16 measurements. The meanSD age at measurement was 11.5 5.23 months and the median (IQR) HgB was 9.4 (8.9-10) g/dL. Seven (44%) measurements were performed while patients were receiving treatment with hydroxyurea. There was no difference in HgB in the groups with and without hydroxyurea, though the treatment group was older (mean age: 15.4 vs 8.4 months, P=0.002). Group averages of qNIRS variables are summarized Table 1. Average cFTOE was 0.53 ± 0.07, significantly higher than the reference value of 0.32 (mean difference: 0.21, P<0.001), while the cohort's mean SO 2 of 45.78 ± 7.13% was significantly lower than the reference value of 68% (mean difference: -22.2, P<0.001). Furthermore, cFTOE inversely correlated with HgB (r= -0.63, P=0.04) while SO 2 increased with HgB (r= 0.63, P=0.04). HbT and cerebral blood volume (CBV) were also significantly lower than the reference values (mean difference of -24.8 μM and -0.75 ml/100g, P<0.001 and P<0.02, respectively) and positively correlated with age (r=0.51 and 0.57, P=0.04). CBF did not correlate with either age or HgB. Hemodynamic variables did not differ between groups with and without hydroxyurea, except for HbT and CBV likely secondary to the cohort age difference. Taken together, this suggests that CBF compensatory mechanisms were insufficient to prevent elevated oxygen extraction. Summary: These preliminary results suggest that inadequate oxygen transport to the brain secondary to SCD evokes a compensatory increase in cFTOE starting in early infancy. The progressive hemodynamic stress to brain health during the period of the brain's greatest growth and metabolic demand for oxygen is concerning because of the potential for injury as compensatory mechanisms become exhausted. Hydroxyurea did not reduce this stress, but may have prevented further declines. Recruitment is ongoing to increase the size of the cohort and to continue following patients' trajectories with age. We demonstrated the potential of qNIRS as biomarkers of cerebral hemodynamic compensation to determine when intervention is necessary and evaluate the effectiveness of a chosen intervention in individual patients. 1. Franceschini, M. A. et al. Assessment of infant brain development with frequency-domain near-infrared spectroscopy. Pediatr Res 61, 546-551, 2007.

Design, Synthesis, and Antisickling Investigation of a Thiazolidine Prodrug of TD-7 That Prolongs the Duration of Action of Antisickling Aromatic Aldehyde.

The synthetic allosteric effector of hemoglobin, TD-7 has been investigated as a potential therapeutic agent for the treatment of sickle cell disease. The pharmacologic activity of TD-7 is due to formation of a Schiff-base interaction between its aldehyde group and the two N-terminal αVal1 amines of hemoglobin, effectively inhibiting sickling of red blood cells. However, TD-7 faces a challenge in terms of poor oral bioavailability due to rapid in-vivo oxidative metabolism of its aldehyde functional group. To address this shortcoming, researches have explored the use of a L-cysteine ethyl ester group to cap the aldehyde group to form a thiazolidine aromatic aldehyde prodrug complex, resulting in the improvement of the metabolic stability of this class of compounds. This report details the synthesis of a thiazolidine prodrug of TD-7, referred to as Pro-7, along with a comprehensive investigation of Pro-7 functional and biological properties. In an in-vitro Hb modification and Hb oxygen affinity studies using normal whole blood, as well as erythrocyte sickling inhibition using sickle whole blood, Pro-7 exhibited a gradual onset but progressive increase in all activities. Additionally, in-vivo pharmacokinetic studies conducted with Sprague Dawley rats demonstrated that Pro-7 can undergo hydrolysis to release TD-7. However, the blood concentration of TD-7 did not reach the desired therapeutic level. These findings suggest that the incorporation of the L-cysteine ethyl ester group to TD-7 represents a promising strategy to enhance the metabolic stability of aromatic aldehydes that could lead to the development of a more effective drug for the treatment of sickle cell disease.