Pathogenesis Of Sickle Cell Disease Research Articles

Evaluation of peripheral blood inflammatory biomarkers in sickle cell disease with and without retinopathy

Background The aim of this study was to evaluate the clinical significance of blood-cell associated inflammation markers in patients with sickle cell disease (SCD) and sickle cell retinopathy (SCR).Methods Neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), monocyte to lymphocyte ratio (MLR), systemic immune inflammation index (SIII), systemic inflammation response index (SIRI), systemic inflammation modulation index (SIMI) and aggregate systemic inflammation index (AISI) were calculated. This study included 45 healthy controls (Group 1) and 100 SCD (Group 2). Patients in Group 2 were then divided into two groups: without SCR (Group 3) and with SCR (Group 4), and patients with SCR (Group 4) were further divided into two groups: non-proliferative sickle cell retinopathy (NPSCR) (Group 5) and proliferative sickle cell retinopathy (PSCR) (Group 6).Results The mean values for NLR, PLR, SIII, SIRI, AISI, and SIMI were significantly higher in Group 2 compared to Group 1 (p = 0.011 for NLR, p = 0.004 for SIII, and p < 0.001 for others). Furthermore, AISI and SIMI parameters demonstrated statistically significant discriminatory power to distinguish Group 5 from Group 6 (p = 0.0016 and p = 0.0006, respectively).Conclusion Given the critical role of inflammatory mechanisms in the pathogenesis of SCD and its related complications, the assessment of blood-cell-associated inflammatory markers may present a pragmatic and advantageous approach to the clinical oversight and therapeutic intervention of SCD.

Evaluation of Genes and Molecular Pathways Involved in Pathogenesis of Sickle Cell Anemia: A Bioinformatics Analysis and Future Perspective

Background: Sickle cell disease (SCD) is one of the hematological disorders characterized by a defect in the structure and function of globin chains. Hereditary factors play an important role in the pathogenesis of SCD. We aimed to investigate the genes and pathways related to the pathogenesis of SCD. Methods: Microarray dataset was downloaded from the Gene Expression Omnibus (GEO) database. LIMMA package of R-software was used to detect UP and Down regulations between SCD and control subjects. Enrichment analysis and Protein-protein interaction (PPI) networks were performed using GeneCodis4 software and GeneMANIA database, respectively. PrognoScan database was used to evaluate the relationship between the hub genes and patients' survival. Results: Overall, 447 DEGs were identified in SCD patients compared to control subjects. Out of 447 DEGs, 345 genes were up-regulated and 102 genes were down-regulated. Effective hub genes in SCD pathogenesis include SLC4A1, DTL, EPB42, SNCA, and TOP2A. In addition, hub genes had a high diagnostic value. Conclusion: Evaluation of hub genes in SCD can be used as a diagnostic panel to detect high-risk patients. In addition, by identifying the UP and Down stream pathways, treatment strategies in the monitoring and treatment of patients can be designed.

Lower Arginine Bioavailability, Increased FeNO Levels, and Airway Resistance on Impulse Oscillometry Are Characteristics of Asthma in Children and Young Adults with Sickle Cell Disease.

Background and Objectives: Data on characteristics of asthma in children with sickle cell disease (SCD) is conflicting. Recently, the L-arginine pathway has gained attention in the pathogenesis of asthma and SCD. This study aimed to determine the distinctive clinical and laboratory features and the role of arginine metabolism in asthmatic children with SCD. Materials and Methods: A total of 52 children and adolescents with SCD, including 24 with asthma (SCD-A) and 28 without asthma (SCD-NA), and 40 healthy controls were included. A questionnaire, atopy tests, fractional exhaled nitric oxide (FeNO), and lung function tests were employed. Serum metabolites of the arginine pathway were measured. The results of the three groups were compared. Results: The demographic characteristics and atopy markers of the three groups were similar. FEV1%, FEV1/FVC, MMEF%, and total lung capacity (TLC%) values of SCD-A patients were not significantly different from the SCD-NA group, but they were significantly lower than the values measured in the controls. FeNO values greater than 35 ppb were present only in the SCD-A group. In impulse oscillometry, median resistance values at 5 Hz (R5)% were higher in both SCD subgroups than in healthy controls (p = 0.001). The (R5-20/R5)% values were higher in the SCD-A group (p = 0.028). Serum arginine levels and arginine bioavailability indices were significantly lower in the SCD-A group than in the SCD-NA group and healthy controls (p = 0.003 and p < 0.001). Conclusions: Asthma in children with SCD was not associated with atopy or low FEV1/FVC levels. However, lower arginine bioavailability and higher FeNO levels differentiated asthma in patients with SCD. High R5% and (R5-20/R5)% values indicated increased airway resistance in SCD, with a predominance of small airway disease in asthmatics.

Impact of Hydroxyurea on Clinical and Biological Parameters of Sickle Cell Anemia in Children in Abidjan.

Background: The lives of individuals affected by sickle cell disease are marked by painful crises sometimes accompanied by complications. Curative treatments such as bone marrow transplantation or gene therapy exist, but are not currently performed in Côte d'Ivoire. Treatment with hydroxyurea remains an effective alternative. The aim of our study is to contribute to improving the management of children with sickle cell disease.&#x0D; Methods: We conducted a prospective observational study from November 2017 to April 2019 at the at the Yopougon University Hospital. Children aged 5 to 15 years experiencing at least 3 vaso-occlusive crises (VOC) per year were included in the study after obtaining informed and written consent from their parents. Each patient received a daily dose of 15mg/kg of hydroxyurea.&#x0D; Results: The mean age of the children was 9 years. More than 75% of patients were homozygous SSFA2 major sickle cell individuals. After 6 months on hydroxyurea, our study observed rates of 84.4%, 100%, and 97.8%, respectively, for the absence of vaso-occlusive crises, hospitalization, and transfusion. Biologically, from M0 to M12 the mean hemoglobin level increased significantly, from 7.24 to 8.55 g/dL; white blood cell (WBC) and platelet counts decreased; Fetal hemoglobin (Hb F) increased significantly from 10.3% to 19.7%. Biochemical parameters within normal ranges, except for a moderate treatment-related increase in transaminases.&#x0D; Conclusion: The induction of fetal hemoglobin (Hb F) production through hydroxyurea intake is the primary mechanism by which hydroxyurea modifies the pathogenesis of sickle cell disease

Inflammation and autoimmunity are interrelated in patients with sickle cell disease at a steady-state condition: implications for vaso-occlusive crisis, pain, and sensory sensitivity.

ClinicalTrials.gov, Identifier: NCT05045820.

Comprehensive Insights into Eosinophil Interactions in Sickle Cell Anemia Severity

Sickle Cell Anemia (SCA) is a prevalent inherited blood disorder characterized by abnormal hemoglobin leading to erythrocyte deformation and consequential vaso-occlusive events. While the primary focus has traditionally centered on the role of red blood cells in SCA pathophysiology, recent investigations have unveiled the intricate involvement of eosinophils in modulating disease severity. Eosinophils, classically recognized for their role in parasitic infections and allergic responses, have emerged as significant contributors to the inflammatory milieu and vascular dysfunction observed in SCA. This paper aims to provide an extensive understanding of the multifaceted interactions between eosinophils and the pathogenesis of sickle cell anemia, emphasizing their impact on disease severity. Insights into eosinophil biology, including their cytokine release profile, granule protein activity, and endothelial interactions, underscore their dual role in both propagating and modulating inflammation in the context of SCA. The complex interplay between eosinophils and the inflammatory microenvironment in SCA dictates disease severity, warranting a deeper understanding of these interactions for the development of targeted therapeutic strategies. In conclusion, this comprehensive review consolidates current knowledge regarding eosinophil involvement in sickle cell anemia severity, emphasizing the necessity for further investigations into specific eosinophilic mechanisms. A deeper comprehension of eosinophilic contributions to SCA pathophysiology presents a pathway toward novel therapeutic modalities, ultimately enhancing the management and prognosis of individuals afflicted with this challenging hematologic disorder.

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.

Real-world observational study on the long-term effect of L-glutamine treatment on renal parameters of adult and pediatric patients with sickle cell disease.

Sickle cell disease (SCD) is a rare genetic blood condition affecting millions worldwide. Oxidative stress is a key player in the pathogenesis of SCD and its comorbid consequences. Renal function impairment is a common complication of SCD in both pediatric and adult patients with serious consequences leading to increased risk of mortality. In this observational real-world study, we are reporting the long-term (120 weeks) renal function in 10 patients treated with L-glutamine. Ten patients (4 pediatric and 6 adults), with confirmed diagnoses of SCD (HbSS genotype), were enrolled, these included four patients from Qatar with Arab Indian haplotype and six patients from French Guiana with African haplotype. All patients were treated with L-glutamine oral powder (~0.3 g/kg body weight, Endari®) twice daily for 120 weeks. Clinical events and laboratory parameters (renal function, hemoglobin, reticulocytes, and lactate dehydrogenase [LDH]) were measured at baseline, 48, and 120 weeks. The study showed that with L-glutamine treatment there were improvements in renal and hematological parameters with no vaso-occlusive crisis at both 48-and 120-week follow-up time points in all 10 patients. Improvements were seen in the albumin creatinine ratio (ACR) from baseline to 48 weeks (mean [Standard deviation SD] ACR: -4.19 [9.81] mg/g) and 120 weeks (mean [SD] ACR: -12.31 [21.09] mg/g). Mean (SD) increase in hemoglobin concentrations from baseline to 48 weeks and 120 weeks was 0.72 (1) g/dL and 1.41 (0.79) g/dL, respectively. Mean (SD) reticulocyte counts and LDH levels decreased from baseline to 48 weeks (mean [SD] change from baseline to 48 weeks, reticulocyte counts: -40.30 [101.58] × 109 cells/L; LDH levels: -259 [154.93] U/L) and 120 weeks (mean [SD] change from baseline to 120 weeks, reticulocyte counts: -58.30 [128.38] × 109 cells/L; LDH levels: -344.80 [274.63] U/L). This is one of the first studies that assessed the long-term renal outcomes in SCD using L-glutamine. L-glutamine improved the renal function in patients with SCD along with improvements in clinical outcomes and hemolysis, from 48 weeks and sustained through 120 weeks of treatment.

Fetal Hemoglobin Decrease during Voxelotor Treatment

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene leading to the formation of hemoglobin S (HbS). The polymerization of deoxygenated HbS is the first event in the molecular pathogenesis of SCD resulting in sickling of red blood cells (RBC), and hemolytic anemia which has been shown to be associated with organ damage and early death. Hemolytic anemia is defined by an accelerated rate of destruction RBC resulting in a lowered Hb level and a reduced gas transport capacity. Because Hb S in its oxy (R) conformation do not polymerize, drugs modifying hemoglobin (Hb) to increase the proportion of oxy-HbS have been propose to inhibit HbS polymerization. Voxelotor is such a molecule, which allosterically modifies Hb-oxygen affinity improving anemia and reducing hemolysis. However, the impact of Voxelotor on HbF levels is unknown. We describe here variations of %HbF and Mean corpuscular HbF (MCHbF) in a cohort of homozygous SCD patients treated with Voxelotor in our Sickle Cell Referral center. We conducted a prospective register study to analyze clinical and biological evolution of SCD patients treated with Voxelotor at 1500mg taken orally once daily. From December 2021 through December 2022, 25 SCD adult patients were followed during a median of 7 months [1-12]. Among them, HbF values were analyzable for 15 patients (SS gentype n=14; women in 73%; median age of 50 years [24-61]) who completed 6 months of Voxelotor with no transfusion during the same period; and with a stable dose of hydroxyurea (HU), for at least 3 months before inclusion and during the study in case of patients treated by HU (n=10). The dose of Voxelotor was stable for 13/15 patients and was decreased in 2 patients due to skin allergy (n=1) and moderate digestive intolerance. One patient died after 6 months of treatment in a context of end-stage renal failure and severe pulmonary hypertension not related to Voxelotor treatment. Mean corpuscular HbF (MCHbF) was calculated (MCHbF (pg) = MCH x %HbF). HbF percentage was measured by high performance liquid chromatography (Biorad, Variant II, beta-thal dual program). All participants provided written informed consent (CPP Erythropedie). From baseline to 6 months of follow up mean Hb (g/dL) concentration increased from 7.4 [5.4-8.5] to 8 [5-11.9] (p=0,0003; Figure A); absolute reticulocyte count (ARC ; Giga/L) decreased from 158 to 145 (p=0,005), %HbF decreased by 63% in all patients from 7,1% to 3,3% (p=0,008; Figure B) and MCHbF (pg) decreased from 3,36 to 1,03 (p=0,001; Figure D). A decrease in % HbF was observed from the first 15 days of treatment in both groups of patients treated or not with HU (Figure C) ; no changes in mean corpuscular volume (p=0.15) was observed in patients treated with HU during follow up. The HbF decrease in SCD patients treated with Voxelotor could be explained by different mechanisms: - Reduction of stress erythropoiesis which is due to an increase in erythroid output in response to anemic stress associated with a reduced expression of BCL11A, a transcriptional repressor that inhibits adult-stage HbF expression. This hypothesis is supported by the reduction in reticulocytes in patients treated with Voxelotor - The loss of an HbF-related survival advantage for SCD RBCs, while the normal RBC lifespan is approximatively 120 days, in SCD RBCs it is only a sixth of it. HbF is a major modulator of SCD and prolonge SCD RBCs survival. Its protective effect relies on its quantity and distribution among the RBC population, which is often heterogeneous in SCD patients. Protection against polymerisation of Voxelotor is probably more homogeneous among RBCs. As a result, RBCs containing low levels of HbF, which would normally disappear, have a prolonged lifespan. Similarly, it was presumed that sickling of erythroblasts could lead to ineffective terminal erythroid differentiation with an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. However we found no significant difference for index of ineffective erythropoiesis (soluble transferrin receptor / absolute reticulocyte count) before and after 6 months of treatment. The question raised is therefore that of the risk of a rebound effect upon discontinuation of Voxelotor, which has a short half-life, during the time it takes for HbF to increase. To better study this hypothesis, distribution of HbF/RBC will be studied in the HEMOPROVE trial (NCT05199766) currently in progress.

A preliminary study of phosphodiesterases and adenylyl cyclase signaling pathway on red blood cell deformability of sickle cell patients.

Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by chronic anemia, intravascular hemolysis, and the occurrence of vaso-occlusive crises due to the mechanical obstruction of the microcirculation by poorly deformable red blood cells (RBCs). RBC deformability is a key factor in the pathogenesis of SCD, and is affected by various factors. In this study, we investigated the effects of adenylyl cyclase (AC) signaling pathway modulation and different phosphodiesterase (PDE) modulatory molecules on the deformability and mechanical stress responses of RBC from SCD patients (HbSS genotype) by applying 5Pa shear stress with an ektacytometer (LORRCA). We evaluated RBC deformability before and after the application of shear stress. AC stimulation with Forskolin had distinct effects on RBC deformability depending on the application of 5Pa shear stress. RBC deformability was increased by Forskolin before shear stress application but decreased after 5Pa shear stress. AC inhibition with SQ22536 and protein kinase A (PKA) inhibition with H89 increased RBC deformability before and after the shear stress application. Non-selective PDE inhibition with Pentoxifylline increased RBC deformability. However, modulation of the different PDE types had distinct effects on RBC deformability, with PDE1 inhibition by Vinpocetine increasing deformability while PDE4 inhibition by Rolipram decreased RBC deformability after the shear stress application. The effects of the drugs varied greatly between patients suggesting some could benefit from one drug while others not. Developing drugs targeting the AC signaling pathway could have clinical applications for SCD, but more researches with larger patient cohorts are needed to identify the differences in the responses of sickle RBCs.

A Review of the Relationship between the Immune Response, Inflammation, Oxidative Stress, and the Pathogenesis of Sickle Cell Anaemia.

Sickle cell anaemia (SCD) is a life-threatening haematological disorder which is predominant in sub-Saharan Africa and is triggered by a genetic mutation of the β-chain haemoglobin gene resulting in the substitution of glutamic acid with valine. This mutation leads to the production of an abnormal haemoglobin molecule called haemoglobin S (HbS). When deoxygenated, haemoglobin S (HbS) polymerises and results in a sickle-shaped red blood cell which is rigid and has a significantly shortened life span. Various reports have shown a strong link between oxidative stress, inflammation, the immune response, and the pathogenesis of sickle cell disease. The consequence of these processes leads to the development of vasculopathy (disease of the blood vessels) and several other complications. The role of the immune system, particularly the innate immune system, in the pathogenesis of SCD has become increasingly clear in recent years of research; however, little is known about the roles of the adaptive immune system in this disease. This review examines the interaction between the immune system, inflammation, oxidative stress, blood transfusion, and their effects on the pathogenesis of sickle cell anaemia.

Association of Vascular Endothelial Growth Factor gene Polymorphism 2578C/A with Vaso-occlusive Crisis in sickle disease

Objectives: to investigate the contribution of vascular endothelial growth factor (VEGF) variants 2578 C/A (rs699947) to vaso-occlusive crisis (VOC) pathogenesis in sickle cell disease (SCD) patients.

A critical role for altered red cell cation permeability in pathogenesis of sickle cell disease and other haemolytic anaemias.

The aetiology of sickle cell disease is well known, but pathogenesis is complicated and details remain uncertain. A thorough understanding may suggest novel ways for designing more effective therapies. One area of importance, covered here in Nader et al., is the altered cation permeability of sickle cells and how the co-ordinated operation of a number of membrane transport proteins contributes to disease progression, all driven by the initial event of HbS polymerisation. There are echoes here of the cation leaks of hereditary stomatocytosis. Nader et al. propose a central role for PIEZO1, a novel mechanosensitive channel found in red cells, which may be aberrantly activated in sickle cells following HbS polymerisation and which may have potential as a novel target for future chemotherapies. Commentary on: Nader et al. Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner. Br J Haematol 2023;202:657-668.

Mathematical Analysis for the Flow of Sickle Red Blood Cells in Microvessels for Bio Medical Application.

Sickle cell disease (SCD) is an inherited monogenic disease which is characterized by distorted red blood cells (RBCs) that cause vaso-occlusion and vasculopathy. In the pathogenesis of SCD, polymerized hemoglobin turn RBCs into fragile, less deformable cells, and are subsequently more susceptible to endothelial adhesion after deoxygenation. Presently, electrophoresis and genotyping are used as routine tests for diagnosis of SCD. These techniques are expensive and require specialized laboratories. Lab-on-a-chip technology is a low-cost microfluidics-based diagnostic tool which holds significant promise for rapid screening of RBC deformability. To explore the sickle RBC mechanics for screening purposes, we present a mathematical model for the flow of single RBC with altered rheological properties and slip effect on capillary wall in microcirculation. We consider single-file flow of cells through the axis symmetrical cylindrical duct, applying lubrication theory as plasma trapped between successive red blood cells. The rheological parameters used from published literature for normal RBC and corresponding variation has been taken for the purpose of this simulation to present the condition of the disease. An analytical solution has been found for realistic boundary conditions and results are simulated using MATLAB. We found that the height of plasma film in the capillary increases with increase in cell deformability and compliance which affects the forward flow velocity in the capillary. Rigid RBCs with increased adhesion between cell and capillary wall shows reduction in velocity and occurrence of vaso-occlusion events in extreme conditions. These rheological properties of the cells coupled with microfluidics mechanics can mimic the physiological condition and provides unique insights with novel possibilities for the design of microfluidics base diagnostic kit towards effective therapeutic intervention of SCD.

Hidden behind thromboinflammation: revealing the roles of von Willebrand factor in sickle cell disease pathophysiology.

This review provides an update on the pathophysiology of sickle cell disease (SCD) with a particular focus on the dysregulation of the von Willebrand factor (VWF) - ADAMTS13 axis that contributes to its pathogenesis. In discussing recent developments, we hope to encourage new and ongoing discussions surrounding therapeutic targets for SCD. Within the last 5 years, the role of VWF in the pathophysiology of SCD has been further elucidated and is now a target of study in ongoing clinical trials. The pathophysiology of SCD is multifaceted, as it involves systemwide vascular activation, altered blood rheology, and the activation of immune responses and coagulative pathways. The presence of VWF in excess in SCD, particularly in its largest multimeric form, greatly contributes to its pathogenesis. Understanding the molecular mechanisms that underly the presence of large VWF multimers in SCD will provide further insight into the pathogenesis of SCD and provide specific targets for therapy.

Sickle red blood cell-derived extracellular vesicles activate endothelial cells and enhance sickle red cell adhesion mediated by von Willebrand factor.

Endothelial activation and sickle red blood cell (RBC) adhesion are central to the pathogenesis of sickle cell disease (SCD). Quantitatively, RBC-derived extracellular vesicles (REVs) are more abundant from SS RBCs compared with healthy RBCs (AA RBCs). Sickle RBC-derived REVs (SS REVs) are known to promote endothelial cell (EC) activation through cell signalling and transcriptional regulation at longer terms. However, the SS REV-mediated short-term non-transcriptional response of EC is unclear. Here, we examined the impact of SS REVs on acute microvascular EC activation and RBC adhesion at 2 h. Compared with AA REVs, SS REVs promoted human pulmonary microvascular ECs (HPMEC) activation indicated by increased von Willebrand factor (VWF) expression. Under microfluidic conditions, we found abnormal SS RBC adhesion to HPMECs exposed to SS REVs. This enhanced SS RBC adhesion was reduced by haeme binding protein haemopexin or VWF cleaving protease ADAMTS13 to a level similar to HPMECs treated with AA REVs. Consistent with these observations, haemin- or SS REV-induced microvascular stasis in SS mice with implanted dorsal skin-fold chambers that was inhibited by ADAMTS13. The adhesion induced by SS REVs was variable and was higher with SS RBCs from patients with increased markers of haemolysis (lactate dehydrogenase and reticulocyte count) or a concomitant clinical diagnosis of deep vein thrombosis. Our results emphasise the critical contribution made by REVs to the pathophysiology of SCD by triggering acute microvascular EC activation and abnormal RBC adhesion. These findings may help to better understand acute pathophysiological mechanism of SCD and thereby the development of new treatment strategies using VWF as a potential target.

Polymorphism and Association of the E-selectin and ICAM-1-K469E Genes with Sickle Cell Disease in Congo

Introduction: E-selectin and ICAM-1 are cellular adhesion molecules that play important roles in the pathogenesis of Sickle Cell Disease (SCD), especially in the vaso-occlusion process. Numerous studies reported that single nucleotide polymorphisms in E-selectin and ICAM-1 genes may be associated with the clinical expression of several diseases. However, no evidence exists regarding the association with SCD. Objectives: Investigate the association of E-selectins (S128R and S149R) and ICAM-1K469E polymorphisms with SCD in Congolese patients. Methodology: This case-control study included 110 SCD patients in vaso-occlusive pain crisis and 120 controls (healthy non-sickle cell subjects). Polymorphisms were genotyped by PCR-RFLP method. The differences in genotype and allele frequencies between both groups were analyzed with the Fisher’s exact test. A P level of &lt;0.05 was considered significant. Results: The frequency of the ICAM-1-K469E EE genotype was significantly higher in SCD patients compared to controls (8.2% vs 0.8%; P=0.008). The odd ratio value (OR=11.89; 95% CI = 1.48-96.53; P=0.01) indicated a high association with SCD. Furthermore, the E allele was also significantly associated with SCD (OR=1.72; 95% CI=1.10-2.68; P=0.008). By contrast, no statistically significant differences were found between SCD patients and controls regarding the allele and genotype frequencies of E- selectin S128R and S149R polymorphisms. Conclusion: Our findings suggest that the ICAM-1-K469E polymorphism may be associated with SCD among Congolese patients, possibly with the vaso-occlusive crisis. The EE genotype and the E allele may be genetic risk factors. However, because of the small sample size, this report should be considered as exploratory and further studies are required to confirm these genetic associations with SCD.

Purine Nucleoside Phosphorylase Inhibition Attenuates the Progression of Anemia and Organ Damage in Sickle Cell Mice

Purine nucleoside phosphorylase (PNP) converts guanosine to guanine, inosine to hypoxanthine, and 2'-deoxyguanosine to guanine, and leads to the downstream formation of the pro-oxidant and vasculotoxic hypoxanthine and xanthine. Red blood cells are rich in PNP, yet PNP's role in the pathogenesis of sickle cell disease (SCD) is not known. Our group showed that patients with SCD have increased PNP release and activity, leading to accelerated guanosine/inosine metabolism and increased production of vasculotoxic hypoxanthine/xanthine (Bilan VP et al. 2018). Thus, we proposed that PNP may have a major pathogenic role in red blood cell sickling, hemolytic angioproliferation, cell aggregation and end organ damage, and that PNP inhibition may be beneficial in the treatment of SCD. The highly potent PNP inhibitor 8-aminoguanine is the product of the conversion of the endogenous guanosine metabolite and pro-drug 8-aminoguanosine (8-AG). We investigated the safety and efficacy of short-term (8 weeks) and chronic (20 weeks) treatment of Townes sickle mice (SS) and their non-sickling controls (AA) with 8-AG (60mg/kg/day administered ad libitum in the drinking water). We collected blood and urine samples at multiple intervals using metabolic cages. To determine 8-AG body disposition and PNP inhibition, urine samples were analyzed for purines by liquid chromatography - tandem mass spectrometry (LC-MS/MS). Both AA and SS mice tolerated 8-AG well with no evidence of toxicity. In both AA (n= 15) and SS (n=40) mice there was very efficient conversion of 8-AG that resulted in a 40-50-fold increase in urinary 8-aminoguanine concentrations (p<0.001). In 8-AG treated SS mice we observed reduced hypoxanthine levels, with an increased inosine/hypoxanthine ratio. Additionally, guanosine was increased, guanine was reduced, and an increased guanosine/guanine ratio was observed (p<0.05), suggesting potent PNP inhibition. In SS mice, the 8-week treatment with 8-AG significantly reduced hemoglobinuria (p<0.05) and albuminuria (p<0.05), attenuated left ventricular (LV, p<0.01) and right ventricular (RV, p<0.05) hypertrophy, limited hepatomegaly (p<0.001), and improved RV contractility (p<0.05), with a trend towards reduced lung weights and splenomegaly. In SS mice, 20-week treatment with 8-AG significantly slowed the progression of anemia as evidenced by attenuated age-related drops in hemoglobin and hematocrit levels, and it attenuated hemoglobinuria and albuminuria while significantly reducing splenomegaly (p=<0.05) and hepatomegaly (p<0.05, Figure). Semi-quantitative histopathological analysis suggested reduced iron deposition (Prussian blue stain, Figure), and reduced ischemic (H&E stain) and fibrotic (trichrome stain, Figure) injury in the spleen and liver from SS mice treated with 8-AG. Our data suggest that the PNP inhibitor 8-AG may have a pharmacological benefit in SCD and may warrant further investigation as a multi-pronged treatment approach for SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dietary Iron Restriction Alters Microbiota-Host Crosstalk and Protects Against Vaso-Occlusion and Organ Damage in Sickle Cell Disease Mice

Introduction: Sickle cell disease (SCD) is the most commonly inherited monogenic hemoglobinopathy and results from a mutation in the β-globin gene (HbS), leading to red blood cell (RBC) sickling, vaso-occlusive episodes (VOE), and organ damage. Chronic hemolysis, inflammation, and repeated red blood cell transfusions in SCD can disrupt iron homeostasis. Patients who receive multiple blood transfusions develop iron-overload, and another subpopulation of SCD patients manifest iron deficiency. SCD mice exhibit increased iron absorption via upregulation of the intestinal transcription factor hypoxia inducible factor-2a(HIF-2a) leading to the development of iron overload. Disruption of intestinal HIF-2a or limiting iron intake by dietary iron restriction in SCD mice, significantly reduced systemic iron overload, lowered cellular sickle hemoglobin, and consequently improved hemolysis and ameliorated anemia. Our lab previously demonstrated that microbial signals from the gut also participated in initiation of VOE and organ damage in SCD mice. To elucidate connections between dietary iron, the microbiota-gut axis, and SCD pathogenesis, we hypothesize that dietary iron restriction may reduce microbiota load and gut permeability, and contributes to amelioration of acute and chronic complications in SCD mice. Methods: SCD mice were generated by transplanting bone marrow cells from Berkeley SCD (Tg[Hu-miniLCRα1GγAγδβS]Hba−/− Hbb−/−) mice into 8-week old, lethally-irradiated C57BL/6J mice. Immediately after transplantation, SCD mice were fed either a regular-iron diet (Ctrl) containing 185 parts per million (ppm) iron or an otherwise identical iron-restricted diet (IRD) containing 3 ppm iron. VOE and organ damage analysis were conducted 3-4 months after treatments. Results: IRD resulted in a reduced organ iron concentration in SCD compared to Ctrl group(0.6±0.1 v.s. 4.0±0.7ug iron/mg liver tissue; P=0.001), confirming that dietary iron restriction reverses organ iron deposition in SCD mice. SCD mice treated with IRD exhibited reduced MCHC (19.7±0.3 v.s. 20.1±0.2 pg; P=0.006) and serum indirect bilirubin (0.4±0.04 v.s. 0.7±0.07 mg/dL; P=0.001) indicating a decrease in cellular sickle hemoglobin and hemolysis. Furthermore, IRD reduced the fraction of aged neutrophils by >50% in SCD mice which corresponded with improved VOE parameters (RBC velocity: 2.0±0.2 v.s. 1.6±0.1mm/s; P=0.01; blood flow rate: 314±33 v.s. 230±13pL/s; P=0.01; leukocyte adhesion: 10±1 v.s. 14±1cells/100um vein; P=0.01; IRD v.s. Ctrl). Importantly, IRD led to prolonged survival after TNF-a challenge in SCD mice (Fig1). IRD resulted in a dramatic reduction in lung leukocyte infiltration, indicating significantly reduced lung inflammation in SCD mice (Fig2A). Compared to Ctrl liver samples, IRD resulted in significantly less liver necrosis, leukocyte infiltration and fibrosis (Fig2B). We further hypothesized that, in addition to reducing iron concentration in organs, IRD ameliorates organ damage by preventing translocation of microbial compounds. Microbiota load in IRD treated SCD mice were decreased to 25% of Ctrl level. IRD inhibited translocation of FITC-Dextran by 5-fold compared to Ctrl suggesting functionally improved gut permeability. We detected significantly decreased TLR2 ligands in serum of IRD compared to Ctrl mice (0.31±0.04 v.s. 0.46±0.05 TLR2 ligand activities O.D. 650nm; P=0.04) suggesting ligands from gram-positive bacteria (TLR2 agonists) may play a role in triggering SCD complications. Conclusion: Our study demonstrates first time that dietary iron restriction reduces VOE severity and alleviates chronic organ damage in SCD mice. These findings provide strong evidence for the therapeutic potential of manipulating iron homeostasis and the gut microbiome to ameliorate SCD pathophysiology. Many treatments under development focus on lowering the systemic iron concentration to relieve disease complications, and our data suggest that iron-induced changes in microbiota load and gut integrity are related and novel therapeutic targets. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Estimation of Malondialdehyde Levels and Determination of Total Antioxidant Capacity in Serum and Saliva of Patient Affected with Sickle Cell Anemia

Introduction: Hemoglobinopathy is an inherited blood disorder in which an individual has an abnormal form of hemoglobin (variant) or decreased production of hemoglobin. The molecular pathogenesis of sickle cell disease explains the phenomenon well when there is adeficiency of oxygen carriage by the erythrocytes. The horrendous patterns of sickling/unsickling bring about the accumulation of lipid oxidation items and reactive oxygen speciesthat gather inside sickle-shaped red blood cells (RBCs). Aims: To estimate the malondialdehyde levels and determine the total antioxidant capacity in serum and saliva of sickle cell anemic patients. Methods and Material: Fiftysubjects aged 5–40 years were selected for the study. The subjects were divided into two groups. Blood and saliva samples were collected from both groups and were subjected to the phosphomolybdenum method. Absorbance was read spectrophotometrically at 695 nm. SPSS software was used, and statistical analysis was performed using Student's unpaired ttests. Results: Oxidative stress results f an imbalance between reactive oxygen species formation and enzymatic and non-enzymatic antioxidants. Conclusion: Salivary levels of total antioxidant capacity, malondialdehyde, and electrolytes make saliva a potential prognostic and diagnostic medium as there is an alteration of oxidative stress in sickle cell anemia (SCA).