Pathogenesis Of Sickle Cell Disease Research Articles

Association between eNOS Gene Polymorphism (T786C and VNTR) and Sickle Cell Disease Patients in Ghana.

Endothelial nitric oxide synthase (eNOS) variants have been found to be associated with several vascular disorders as well as the pathogenesis of sickle cell disease (SCD) complications such as vaso-occlusive crises (VOC). Studies on eNOS gene variants among SCD patients are rare in Ghana and several other African countries. The current study aimed to determine a possible association between variants of the eNOS gene (variable number of tandem repeats in intron 4 and T786C) in SCD complications among Ghanaian patients. This was a cross-sectional study involving 89 HbSS patients with complications and 46 HbSS patients without complications. Genomic DNA was extracted from leukocytes in the buffy coat and separated from collected whole blood samples of the study participants. PCR amplification, followed by restriction fragment length polymorphism (RFLP) was used to genotype T786C (rs2070744) variants. Variable number of tandem repeats (VNTR) in intron 4 was genotyped by PCR and direct electrophoresis. There was a significant difference in the genotype frequency of the T786C variant between HbSS patients with complications and those without complications (p = 0.0165). However, there was no significant difference in the VNTR intron 4 variant of the eNOS gene between patients with complications and those without complications (p > 0.05). The study shows an association between the eNOS gene variant (T786C) and complications in SCD.

Generation of integration-free iPS cell lines from three sickle cell disease patients from the state of Bahia, Brazil

Sickle cell disease (SCD) is one of the most prevalent and severe monogenetic disorders, affecting several million people around the world. Clinical manifestations and complications of the disease include sickle cell pain crisis, silent cerebral infarct, stroke, nephropathy and early death. In this study, we generated induced pluripotent stem cell (iPSC) lines from three homozygous SCD patients from the state of Bahia, Brazil, where SCD is highly prevalent. Peripheral blood mononuclear cells were collected and erythroblasts were expanded for cell reprogramming with the use of non-integrative episomal vectors. The generated iPSC lines expressed high levels of pluripotency markers, presented a normal karyotype and were able to differentiate into the three germ layers in embryoid body spontaneous differentiation assays. Moreover, the expression of the episomal vectors was lost in all iPSC lines after 15 passages. These iPSC lines may help increasing the knowledge about SCD pathogenesis and can be a useful tool for drug testing and gene editing studies.

GBT440 reverses sickling of sickled red blood cells under hypoxic conditions in vitro.

Sickle cell disease is characterized by hemolytic anemia, vasoocclusion and early mortality. Polymerization of hemoglobin S followed by red blood cell sickling and subsequent vascular injury are key events in the pathogenesis of sickle cell disease. Sickled red blood cells are major contributors to the abnormal blood rheology, poor microvascular blood flow and endothelial injury in sickle cell disease. Therefore, an agent that can prevent and or reverse sickling of red blood cells, may provide therapeutic benefit for the treatment of sickle cell disease. We report here that GBT440, an anti-polymerization agent being developed for the chronic treatment of sickle cell disease, increases hemoglobin oxygen affinity and reverses in vitro sickling of previously sickled red blood cells under hypoxic conditions. Our results suggest that besides preventing sickling of red blood cells, GBT440 may mitigate vasoocclusion and microvascular dysfunction by reversing sickling of circulating sickled red blood cells in vivo.

The Association of Serum 25-Hydroxyvitamin D With Biomarkers of Hemolysis in Pediatric Patients With Sickle Cell Disease.

Although vitamin D deficiency (VDD) has been linked to anemia among sickle cell disease (SCD), its relationship with hemolysis is unclear. Serum 25-hydroxyvitamin D and biomarkers of hemolysis (hemoglobin [Hb]/hematocrit, reticulocyte percentage, absolute reticulocyte, and lactate dehydrogenase [LDH] levels) in 36 hydroxyurea-naive SCD children were quantified. Correlations were significantly positive with Hb/hematocrit (r=0.40, P=0.017; r=0.45, P=0.006, respectively); inverse with reticulocyte percentage, absolute reticulocyte, and LDH (r=-0.44, P=0.008; r=-0.47, P=0.007; r=-0.45, P=0.007, respectively). In VDD groups, Hb was lower (P=0.014), reticulocyte counts and LDH were higher (P=0.047 and 0.003, respectively). Serum 25-hydroxyvitamin D correlated with biomarkers of hemolysis in SCD and VDD may play a role in SCD pathogenesis.

The effect of xanthine oxidase and hypoxanthine on the permeability of red cells from patients with sickle cell anemia.

Red cells from patients with sickle cell anemia (SCA) are under greater oxidative challenge than those from normal individuals. We postulated that oxidants generated by xanthine oxidase (XO) and hypoxanthine (HO) contribute to the pathogenesis of SCA through altering solute permeability. Sickling, activities of the main red cell dehydration pathways (Psickle, Gardos channel, and KCl cotransporter [KCC]), and cell volume were measured at 100, 30, and 0 mmHg O2, together with deoxygenation‐induced nonelectrolyte hemolysis. Unexpectedly, XO/HO mixtures had mainly inhibitory effects on sickling, Psickle, and Gardos channel activities, while KCC activity and nonelectrolyte hemolysis were increased. Gardos channel activity was significantly elevated in red cells pharmacologically loaded with Ca2+ using the ionophore A23187, consistent with an effect on the transport system per se as well as via Ca2+ entry likely via the Psickle pathway. KCC activity is controlled by several pairs of conjugate protein kinases and phosphatases. Its activity, however, was also stimulated by XO/HO mixtures in red cells pretreated with N‐ethylmaleimide (NEM), which is thought to prevent regulation via changes in protein phosphorylation, suggesting that the oxidants formed could also have direct effects on this transporter. In the presence of XO/HO, red cell volume was better maintained in deoxygenated red cells. Overall, the most notable effect of XO/HO mixtures was an increase in red cell fragility. These findings increase our understanding of the effects of oxidative challenge in SCA patients and are relevant to the behavior of red cells in vivo.

Assessment of some indicators of oxidative stress in nigerian sickle cell anemic patients.

Background:Oxidative stress, the imbalance between the productive of reactive species and antioxidant defences notably plays an important role in the pathogenesis of sickle cell disease. Generating the necessary data about oxidative stress in patients with sickle cell anemia (SCA) would help in developing cost-effective and accessible treatment interventions of SCA in resource-poor countries.Aim:The aim of this study is to evaluate the levels plasma lipid peroxidation product, malondialdehyde (MDA), serum nitric oxide (NO), and total antioxidant activity (TAOA) as indicators of oxidative stress in sickle cell individuals.Materials and Methods:In this pilot study, 52 patients; homozygous (Hb SS) sickle cell patients confirmed by cellulose electrophoresis and 20 age- and sex-matched healthy (Hb AA) controls were subjected to analysis of NO, MDA, and TAOA. The plasma MDA was measured by a thiobarbituric reaction, (NO) was evaluated by the method described by Guevara et al. using Griess reagent and the TAOA of serum was determined by the method of Koracevic et al.Results:The results showed a statistically significant decrease and increase in baseline levels of NO and MDA, respectively, in Hb SS group when compared with the control Hb AA (P = 0.000 and 95% confidence interval [CI] of 0.10–0.18 and −9.67–−5.57, respectively). A slight decrease in the TAOA level between the groups was observed although not statistically significant (P = 0.15 95% CI was −0.28–2.90).Conclusion:The results showed of imbalance between oxidant and antioxidant status in patients with SCA. Antioxidant supplementation may be a cheap assessable intervention for in sickle cell individuals (in the steady state or in crisis) to prevent further oxidative damage to the erythrocytes.

Relief of Acute Chest Syndrome of Sickle Cell Anaemia by Nitric Oxide Inhalation - A Report of 2 Cases

Depletion of nitric oxide (NO) in the microcirculation plays a central role in the pathogenesis of sickle cell disease (SCD) and acute chest syndrome (ACS). Various other mechanisms which are responsible for the sickle-cell vasoocclusive crisis are polymerization of sickle haemoglobin, platelet activation, endothelial damage and leucocyte adhesion. During the sickle cell crisis, the high rate of red blood cell (RBC) destruction leads to release of erythrocyte arginase (into the plasma), which thereby destroys arginine. Arginine, the substrate for NO synthase, once depleted, results in lower levels of NO in vessels, leading to vasoconstriction, pulmonary hypertension and ventilation perfusion mismatch. Increasing the availability of NO therefore has a central role in SCD, especially in cases of ACS. With the prevention of vasoconstriction by NO inhalation therapy in ACS, pain relief can be provided to the patients in sickle cell crisis. We present a report of 2 cases of sickle cell disease with ACS (Acute Chest Syndrome) in whom inhalation of NO through non-invasive ventilation (NIV) brought about pain relief and recovery from ACS.

PADI4 Gene Polymorphism As a Risk Factor for Acute Chest Syndrome in Sickle Cell Anemia Patients

Introduction: Sickle cell anemia (SCA) is a chronic inflammatory disease with heterogeneous clinical features and the reasons for the heterogeneity of the clinical manifestations has not been fully elucidated. New mediators of the pathogenesis of SCA described recently include the formation of Neutrophil Extracellular Traps (NETs), which may contribute to the amplification of inflammation via the production of pro-inflammatory mediators. Peptidylarginine Deiminase 4 (PADI4) is a critical regulator of NETosis by mediating histone citrullination, an essential step for the generation of NETs. There appears to be a relationship between PADI4 gene polymorphisms and the pathophysiology of other inflammatory diseases in which NETosis seems to be relevant. Our aim was to investigate the association of PADI4 gene polymorphisms [rs874881(G>C), rs1748033(T>C), rs11203366(G>A), rs11203367 (T >C), rs2240340 (T>C)], which have been previously associated with increased PADI4 mRNA stability and with some clinical manifestations in cohorts of SCA patients. Methods: The study included 194 SCA patients (93 males and 101 females with mean age 33.29 ± 9.54 years) being followed up at the Hematology and Hemotherapy Foundation of Pernambuco (HEMOPE), Recife, Brazil. PADI4 gene polymorphisms were performed by Polymerase Chain Reaction (PCR) and their products were sequenced using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit v3.1 (Applied Biosystems, CA, USA). These results obtained were compared with the clinical data obtained from the patients' records. Ethical approval was obtained from Ethics Committee of HEMOPE and all patients gave informed consent. Results: The frequencies of the genotypes found were as follows: rs874881 (22.2% GG, 50.5% GC, 27.3% CC); rs1748033 (17% TT, 42.3% TC, 40.7% CC); rs11203366 (21.1% GG, 50% GA, 28.9% AA); rs11203367 (20.6% TT, 49.5% TC, 29.9% CC); rs2240340 (24.7% TT, 46.4% TC, 28.9% CC). The distribution of the genotypes was in accordance with the Hardy-Weinberg equilibrium (p > 0.05). Twenty-four patients (12.4%) presented with a history of acute chest syndrome (ACS), 27 (13.9%) with stroke, 168 (86.6%) with vaso-occlusive crisis (VOC), and 81 (47.8%) with leg ulcers (LU). No association was observed between the polymorphisms studied and the history of LU, VOC and stroke in the patients (p > 0.05). However, for rs 874881 and 1748033 with the G and T alleles, respectively, were associated with a higher risk of ACS (OR: 2.96, p = 0.02 and OR: 4.75, p = 0.01, respectively). Conclusion: In the present study, we found an association between the wild type alleles (rs874881G and rs1748033T) and a history of ACS in our cohort of SCA patients. There is need for future studies on these polymorphisms in larger cohorts of SCA patients to affirm this association. DisclosuresNo relevant conflicts of interest to declare.

Cardiorespiratory pathogenesis of sickle cell disease in a mouse model

The nature and development of cardiorespiratory impairments associated with sickle cell disease are poorly understood. Given that the mechanisms of these impairments cannot be addressed adequately in clinical studies, we characterized cardiorespiratory pathophysiology from birth to maturity in the sickle cell disease SAD mouse model. We identified two critical phases of respiratory dysfunction in SAD mice; the first prior to weaning and the second in adulthood. At postnatal day 3, 43% of SAD mice showed marked apneas, anemia, and pulmonary vascular congestion typical of acute chest syndrome; none of these mice survived to maturity. The remaining SAD mice had mild lung histological changes in room air with an altered respiratory pattern, seizures, and a high rate of death in response to hypoxia. Approximately half the SAD mice that survived to adulthood had an identifiable respiratory phenotype including baseline tachypnea at 7–8 months of age, restrictive lung disease, pulmonary hypertension, cardiac enlargement, lower total lung capacity, and pulmonary vascular congestion. All adult SAD mice demonstrated impairments in exercise capacity and response to hypoxia, with a more severe phenotype in the tachypneic mice. The model revealed distinguishable subgroups of SAD mice with cardiorespiratory pathophysiology mimicking the complications of human sickle cell disease.

The super sickling haemoglobin HbS‐Oman: a study of red cell sickling, K+ permeability and associations with disease severity in patients heterozygous for HbA and HbS‐Oman (HbA/S‐Oman genotype)

Studying different sickle cell genotypes may throw light on the pathogenesis of sickle cell disease (SCD). Here, the clinical profile, red cell sickling and K+ permeability in 29 SCD patients (15 patients with severe disease and 14 with a milder form) of HbA/S-Oman genotype were analysed. The super sickling nature of this Hb variant was confirmed. The red cell membrane permeability to K+ was markedly abnormal with elevated activities of Psickle , Gardos channel and KCl cotransporter (KCC). Results were consistent with Ca2+ entry and Mg2+ loss via Psickle stimulating Gardos channel and KCC activities. The abnormal red cell behaviour was similar to that in the commonest genotype of SCD, HbSS, in which the level of mutated Hb is considerably higher. Although activities of all three K+ transporters also correlated with the level of HbS-Oman, there was no association between transport phenotype and disease severity. The super sickling behaviour of HbS-Oman may obviate the need for solute loss and red cell dehydration to encourage Hb polymerisation, required in other SCD genotypes. Disease severity was reduced by concurrent α thalassaemia, as observed in other SCD genotypes, and represents an obvious genetic marker for prognostic tests of severity in young SCD patients of the HbA/S-Oman genotype.

Hemoglobin inhibits albumin uptake by proximal tubule cells: implications for sickle cell disease.

Proximal tubule (PT) dysfunction, including tubular proteinuria, is a significant complication in young sickle cell disease (SCD) that can eventually lead to chronic kidney disease. Hemoglobin (Hb) dimers released from red blood cells upon hemolysis are filtered into the kidney and internalized by megalin/cubilin receptors into PT cells. The PT is especially sensitive to heme toxicity, and tubular dysfunction in SCD is thought to result from prolonged exposure to filtered Hb. Here we show that concentrations of Hb predicted to enter the tubule lumen during hemolytic crisis competitively inhibit the uptake of another megalin/cubilin ligand (albumin) by PT cells. These effects were independent of heme reduction state. The Glu7Val mutant of Hb that causes SCD was equally effective at inhibiting albumin uptake compared with wild-type Hb. Addition of the Hb scavenger haptoglobin (Hpt) restored albumin uptake in the presence of Hb, suggesting that Hpt binding to the Hb αβ dimer-dimer interface interferes with Hb binding to megalin/cubilin. BLAST searches and structural modeling analyses revealed regions of similarity between Hb and albumin that map to this region and may represent sites of Hb interaction with megalin/cubilin. Our studies suggest that impaired endocytosis of megalin/cubilin ligands, rather than heme toxicity, may be the cause of tubular proteinuria in SCD patients. Additionally, loss of these filtered proteins into the urine may contribute to the extra-renal pathogenesis of SCD.

Hydroxycarbamine: from an Old Drug Used in Malignant Hemopathies to a Current Standard in Sickle Cell Disease.

While hydroxycarbamide (hydroxyurea, HU) has less and fewer indications in malignant hemopathies, it represents the only widely used drug which modifies sickle cell disease pathogenesis. Clinical experience with HU for patients with sickle cell disease has been accumulated over the past 25 years in Western countries. The review of the literature provides increasing support for safety and efficacy in both children and adults for reducing acute vaso-occlusive events including pain episodes and acute chest syndrome. No increased incidence of leukemia and teratogenicity was demonstrated. HU has become the standard-of-care for sickle cell anemia but remains underused. Barriers to its use should be identified and overcome.

Role of Circulating Endothelial Progenitor Cells in Micro VascularDamage in Sickle Cell Patients

The vascular wall participates in the pathogenesis of sickle cell disease (SCD). Circulating Endothelial progenitor cells (cEPCs) also play a key role in the vascular pathology of SCD, including the painful crisis. In previous investigations, reduced levels of cEPCs were found in conditions in which vascular injury is implicated such as myocardial infarction. The aim of this research is to study the role of cEPCs in the vascular pathology of SCD. cEPCs were enumerated using flow cytometry with the Flow Activated Cell Sorting machine (FACS). To differentiate the various genotypes-SS, SC, AS, AC and AA, Hb Electrophoresis was employed. Results showed that the median % cEPCs (CD34+/VEGF-2+) was lower in patients with SCD [0.555(0.4, 0.765)] than in healthy controls [(1.08(0.87, 1.39) (p=0.001)]. Patients in crisis had a higher cEPCs (0.65+0.39) than those in steady state (0.59+0.28) (p=0.522). SS group recorded the highest mean GGT (73.66+73.35). Only total cholesterol demonstrated a positive correlation (r=0.378, p=0.00814) with cEPCs in subjects; a trend unseen in healthy controls. Patients’ WBC, Hb and Liver enzymes- ALT, GGT, ALP showed no correlation with cEPCs. In healthy controls though, WBCs showed an inverse correlation with cEPCs (r=-0.6293, p=0.0003). SCD is as much a disease of endothelial dysfunction as it is a hemoglobinopathy that triggers erythrocyte polymerization: cEPC is a surrogate bio-marker for vascular function in SCD patients. The results suggest that SCD patients have depleted cEPCs compared with healthy controls. Oxidative stress, Nitric oxide activity, loss of CD133 during homing could influence progenitor cell populations. Total cholesterol positively correlates with cEPCs. High total cholesterol could spell the onset of painful crisis. Liver enzymes are not related to cEPCs correlatively even though the liver is involved in endothelial injury in SCD patients. SS individuals tend to have a high GGT. Overall, we have shown the direct correlation between total cholesterol and cEPCs in sickle cell patients.

Regulation of Elastase By SerpinA3N Contributes to Pain in Sickle Cell Disease

SerpinA3N, a serine protease inhibitor, has been shown to ameliorate neuropathic pain by inhibiting leukocyte elastase activity (Vicuña et al., Nature Med 2015, 21:518-523). Patients with sickle cell disease (SCD) have activated leukocytes and experience neuropathic pain. We observed that SerpinA3N protein expression is significantly reduced in the dorsal root ganglion (DRG) of 5 month old homozygous HbSS-BERK sickle mice, compared to age-matched HbAA-BERK control mice expressing normal human hemoglobin A (p < 0.05). Since HbSS-BERK sickle mice demonstrate tonic hyperalgesia (Kohli et al., Blood 2010, 116:456-465), we hypothesize that decreased expression of SerpinA3N contributes to hyperalgesia by increasing elastase activity in these mice. We examined our hypothesis by using both genders of HbSS-BERK sickle and HbAA-BERK control mice, treated intravenously with and without sivelestat, a small molecule inhibitor of elastase. Mechanical, thermal and musculoskeletal/deep tissue hyperalgesia were analyzed before (considered baseline, BL) and after treatments. Elastase activity was determined using a fluorescent elastase substrate MeOSuc-AAPV-AMC (EMD Milipore).We observed that elastase activity is significantly increased in the DRG and lungs of male and female sickle mice compared to gender and age-matched control mice (p < 0.05 and < 0.005 for female and male DRG, respectively; p < 0.001 and < 0.05 for female and male lungs, respectively). Thus, decreased SerpinA3N expression perhaps contributes to increased elastase activity in sickle mice. Treatment of sickle mice with a single dose of 2 mg/Kg sivelestat led to a significant decrease in mechanical, heat and cold hyperalgesia for up to 24 hours compared to BL (p < 0.05), but no effect was noted in deep tissue hyperalgesia. Increasing the single dose to 4.5 mg/Kg led to a significant decrease in mechanical and thermal as well as deep tissue hyperalgesia for up to 24 hours in both genders compared to BL or saline treated mice (p < 0.05). Continuation of treatment with sivelestat 4.5 mg/Kg/day for 3 days led to significantly reduced hyperalgesia for up to 6 days in both genders of mice. These findings suggest that elastase activity contributes to nociceptive mechanisms, since hyperalgesia remained reduced for up to 3 days even after discontinuation of sivelestat. Reduced hyperalgesia following 3 days of sivelestat (4.5 mg/Kg/day) treatment was accompanied by a significant decrease in elastase activity in the DRG (p < 0.005) and lungs (p < 0.05) of both genders of sickle mice compared to saline treatment, but no histopathological changes were observed in lungs, kidney, liver, spleen and heart. Activating transcription factor 3 (ATF3), a marker of neuropathic pain, is elevated in the DRG of sickle as compared to control mice (Vincent et al., Blood 2013, 122:1853-1862). Sivelestat treatment significantly reduced ATF3-immunoreactivity compared to saline treatment in the DRG of sickle mice (p < 0.05), suggesting a decrease in neuronal injury which underlies neuropathic pain. None of the treatments had any effect on control mice. Sivelestat treatment showed no significant effect on serum tryptase or cutaneous mast cell degranulation in sickle or control mice. Thus, sivelestat specifically inhibits elastase activity. However, both elastase and tryptase activate protease-activated receptor-2 (PAR2) in the peripheral nervous system, stimulating the release of pro-nociceptive neuropeptides and activation of transient receptor potential vanilloid (TRPV) channels, leading to hyperalgesia. Since PAR-2 and TRPV channels are activated in sickle mice, it is likely that sivelestat treatment has an inhibitory effect on these mechanisms.It has been suggested that leukocytes, including activated neutrophils, contribute to acute lung injury (a common complication of SCD) and the pathogenesis of SCD (Zhang et al., Blood 2016, 127:801-809). Our observations that decreased SerpinA3N protein expression leads to increased elastase activity in DRG and lung in sickle mice suggest that this mechanism may contribute to sickle pathobiology and pain. In Japan, sivelestat is already approved for treatment of acute lung injury. Therefore, pharmacological inhibition of elastase may offer the advantage of treating chronic pain while concomitantly ameliorating one of the pathogenic mechanisms of SCD. DisclosuresNo relevant conflicts of interest to declare.

Influence of Genetic Modifiers on Levels of Invariant Natural Killer T-Cells in Patients with Sickle Cell Anemia

Although patients homozygous for the sickle cell disease (SCD) mutation have an identical genotype, the severity of the disease can be extremely variable. The hemoglobin (Hb) S mutation has been associated with five different beta S-globin gene cluster haplotypes (βS-haplotypes) that show different clinical expression. Because genetic modifiers can modulate treatment response, we hypothesized that βS-haplotypes can affect levels of invariant natural killer T (iNKT) cells, which are known to play a key role in the pathogenesis of SCD, and are being considered as a potential target to treat acute crises in SCD patients. Herein we evaluated the impact of βS-haplotypes and HbF concentration on iNKT cell and dendritic cell (DC) subsets in a well-defined group of patients with SCD in a steady state. Sickle cell anemia patients were selected based on the history of patients, clinical examination and hematological findings. The βS-haplotypes were identified by polymerase chain reaction-restriction fragment length polymorphism analysis for seven restriction sites. The iNKT cell subsets were characterized by the positive-staining of Vα24Jα18 T cell receptor alpha chain, along with CD3, CD4 and CD8 surface markers and the intra-cellular cytokine production of interferon-gamma (Th1-like), interleukin-4 (Th2-like) and interleukin-17 (Th17-like) cells using flow cytometry and cell culture. The myeloid DC (mDC) and plasmacytoid DC (pDC) cells were identified by the expression of HLA-DR, CD123, CD11c, Lin and CD1d, a non-classical molecule that induces the activation of iNKT cells by flow cytometry. Comparisons among βS-haplotypes were performed using ANOVA and unpaired t test, while the Spearman’s correlation was used to assess associations. Among the 125 sickle cell anemia patients studied, Benin haplotype (40%) was the most common followed by Bantu (21%), Arab/Indian (18%), Atypical (12%) and Benin/Bantu or Benin/Arab-Indian or Bantu/Arab-Indian (9%). The majority of subjects with Benin/Benin βS-haplotype had a severe to moderate clinical profile similar to Bantu/Bantu or Arab/Indian βS-haplotype groups (P=0.23). A trend toward increased levels of CD3iNKT, CD4iNKT and CD8iNKT cell subsets was observed in the subjects with the Benin/Benin βS-haplotype, when compared to other βS-haplotype groups (P=0.06). Interestingly, subjects with the Benin/Benin βS-haplotype exhibited slightly higher levels of Th1-like cells, but not Th2-like and Th17-like cells, when compared to subjects with the Bantu/Bantu or Arab/Indian βS-haplotype groups (P=0.047). Comparisons between the levels of mDC and pDC cell subsets, as well as the expression of CD1d on these DC cells, showed no statistically significant differences among the βS-haplotype groups (P=0.42). Similarly, levels of iNKT cell subsets, Th1-like, Th2-like, and Th17-like cells as well as DC subsets were similar among patients who received or not hydroxyurea therapy, independently of βS-haplotype groups (P= 0.09). Likewise, coexistence of high HbF and βS-haplotypes did not show any significant association with iNKT cell subsets. Collectively, our findings suggest that neither the βS-haplotypes or HbF levels nor the hydroxyurea therapy or clinical severity appeared to be associated with iNKT cell or DC cell subsets in patients with SCD. The effects of other genetic modifiers such as alpha/beta-thalassemia and G6PD deficiency on iNKT cell subsets need to be evaluated in future studies. DisclosuresNo relevant conflicts of interest to declare.

Efficacy and Safety of Long-Term Treatment with the LSD1 Inhibitor RN-1 to Increase HbF in Normal, Non-Anemic Baboons

Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease. New and more effective strategies to increase HbF are needed because current therapies are ineffective in a significant proportion of patients. The lysine-specific demethylase, LSD1, an enzyme that demethylates Histone H3 mono- and dimethyl- K4 and K9 residues, is a component of the DRED co-repressor complex that represses the γ-globin gene (Cui et al, Mol Cell Biol 31:3298, 2011). Our laboratory has recently shown that RN-1, a pharmacological inhibitor of LSD1, increases γ-globin expression in sickle cell disease (SCD) transgenic mice (Rivers et al, Exp Hematol 43:546, 2015; Cui et al, Blood 126:386, 2015) and in baboons (Rivers et al, Haematol 101:688, 2016), widely acknowledge as the best animal model to test the activity of HbF-inducing drugs. To evaluate the safety and efficacy of RN-1, two normal, non-anemic baboons were treated for an extended period with RN-1 (PA8695, 264d; PA8698, 278d; 0.25mg/kg/d; 5d/wk, sc) and the effect on HbF, F cells, and hematological parameters evaluated weekly. Both animals exhibited weight gain (PA8695: 14.4%; PA8698: 20%) during the course of the study. F retics were increased 8-10 fold by the second week (d9) and were maintained at high levels throughout the course of the study (PA8695: 55.7±17.7% (mean±SD), median (M)=59.0%; PA8698: 56.4±14.5%, M=56.9%). F cell levels increased until d169 and were maintained thereafter at levels 18-25 times greater than pre-treatment (PA8695: 54.5±3.3%, M=54.2%; PA8698: 51.7±3.2%, M=51.4%). HbF levels also increased until d169 and were also maintained thereafter at levels 10-12 times greater than pre-treatment (PA8695: 12.8±1.7%, M=12.1%; PA8698: 11.4±1.4%, M=11.5%). Elevated levels of γ-globin chain synthesis were observed in the peripheral blood on multiple days (d162, d190, d267) of treatment (PA8695, 0.28±0.08 γ/γ+β;PA8698, 0.26±0.08 γ/γ+β). RT-PCR analysis showed that γ-globin mRNA levels were increased 9-22 fold (p<0.01) in FACS-purified CD105+ CD117+/- glyA+ BM erythroid precursor subpopulations of RN-1 treated baboons compared to normal baboons. Absolute neutrophil counts showed no overall decline compared to pre-treatment (PA8695: pre-treatment=2110/μL, post-treatment=3118±1452/μL, M=2765/μL; PA8698: pre-treatment=2690/μL, post-treatment=2936±696/μL, M=2460/μL) although variations were observed. In PA8695 the ANC declined below 1500/μL twice (1490, 1160) and five times (1250, 1410, 1420, 1330, 1000) in PA8595. Monocytes increased 2-3 fold in each animal (PA8695: pre-treatment=100/μL, post-treatment=252±97/μL, M=246/μL; PA8698: pre-treatment=161/μL, post-treatment=425±319/μL, M=347/μL). Hemoglobin (Hb), RBC, and hematocrit (HCT) levels exhibited small overall decreases during the course of treatment but remained within the normal range. Decreases in RBC, Hb, and HCT were associated with blood loss during menstruation (PA8695) and following an accidental laceration of the perineal swelling (PA8698). Rapid recovery was observed in both animals. Significant changes in MCV, MCH, and MCHC during treatment were not observed. Platelet levels decreased approximately 40% in each animal but nevertheless were maintained within the normal range (PA8695: pre-treatment=351 X 103/μL, post-treatment=236±64 X 103/μL, M=219 X 103/μL; PA8698: pre-treatment=224 X 103/μL, post-treatment=143±78 X 103/μL, M=119 X 103/μL). No significant differences in either PT or aPTT assays were observed compared to normal baboons. In vitro platelet activation assays performed to investigate effects on platelet function showed that the fraction of platelets expressing P-selectin (CD62P) on the surface following addition of thrombin was reduced 14% (p<0.02) and the level of CD62P expression reduced 46% (p<0.0001) in RN-1 treated baboons compared to controls. Normal platelet function was restored 10d post-drug. Elevated platelet activation is associated with the pathogenesis of sickle cell disease and inhibitors of platelet activation are currently in clinical trials. We conclude that long-term treatment with RN-1 increases and maintains elevated levels of HbF and F cells in normal baboons and is well-tolerated. Pharmacological inhibitors of LSD1 are therefore likely to be beneficial in patients with SCD. DisclosuresRivers:Acetylon: Research Funding. DeSimone:EpiDestiny: Consultancy, Other: patents around decitabine and tetrahydrouridine. Lavelle:Acetylon: Research Funding.

Red Cell Transport Abnormalities As Predictors of the Development of Renal Disease in Children with Sickle Cell Disease

Background:Patients with sickle cell disease (SCD) have the abnormal haemoglobin (Hb) HbS in their red blood cells (RBCs). The complications of SCD may be extensive but are markedly variable between patients. Renal damage (sometimes called sickle cell nephropathy, SCN) is a common finding in about a third of children with SCD, a significant number of whom will proceed to renal failure. As yet, it is not possible to predict which children will develop SCN and who would therefore benefit from earlier and aggressive management. Various biomarkers have been investigated but none have proved efficacious. Changes in RBC membrane permeability are closely linked with polymerisation of HbS and, as such, may represent an important early event in pathogenesis.We postulated that certain RBC characteristics (sickling, K+ transport, PS exposure), which may be inherited independently of the HbS mutation, may correlate with renal pathology, but more importantly, may be identifiable in advance of damage, thereby providing prognostic markers to inform patient management.Participants and Methods:Children (>4 years old) with sickle cell anaemia (SCA) were recruited from the paediatric haematology clinic at King’s College Hospital in South London, UK. Patients who had received blood transfusion in the preceding four months or who were on medications known to directly alter RBC cation transport (e.g. dipyridamole and Ca2+ channel blockers) were excluded. Patients on hydroxyurea were included. All patients were in steady state, with no acute symptoms requiring treatment for at least seven days. Clinical and laboratory parameters were collected prospectively at routine clinical attendance. Details of enuresis were collected using specifically developed proforma and patients were divided into two groups: those who stopped wetting their bed before the age of five years and those who were still enuretic after five years of age. RBC permeability was assessed using 86Rb+ as a congener for K+ to measure the activity of main cation transport systems involved in RBC dehydration. The activity of RBC transport pathways and percentage of sickling was measured at 100mmHg, 35mmHg, 15mmHg and 0mmHg. Statistical analysis was conducted using IBM-SPSS, New York.Results: 112 patients of HbSS genotype (SCA) consented to take part in the study. Their clinical profile is summarised Table 1. The percentage of children with enuresis, hypertension and significant albuminuria is shown in Table 2. There was significant correlation between activity of Gardos channel at O2 tensions of 35mmHg, 15mmHg and 0mmHg and enuresis after the age of five years. Psickle activity also showed significant correlation but only in fully deoxygenated RBCs (O2 of 0mmHg.) Table 3.ConclusionIn conclusion, the present findings emphasise the significance of a thorough appreciation of RBC permeability to the pathogenesis of SCD. Whilst preliminary, these findings are exciting because they show that the activity of a particular transport pathways, abnormally elevated in SCA patients, are associated with enuresis. These findings suggest that the early identification of increased Gardos and Psickle activity may identify children who would benefit from early treatment for nocturnal enuresis. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Biomechanics and biorheology of red blood cells in sickle cell anemia

Sickle cell anemia (SCA) is an inherited blood disorder that causes painful crises due to vaso-occlusion of small blood vessels. The primary cause of the clinical phenotype of SCA is the intracellular polymerization of sickle hemoglobin resulting in sickling of red blood cells (RBCs) in deoxygenated conditions. In this review, we discuss the biomechanical and biorheological characteristics of sickle RBCs and sickle blood as well as their implications toward a better understanding of the pathophysiology and pathogenesis of SCA. Additionally, we highlight the adhesive heterogeneity of RBCs in SCA and their specific contribution to vaso-occlusive crisis.

Rein et hémoglobinopathies

Sickle-cell disease (SCD), one of the most common severe monogenic disorders into the world, is associated with an increased frequency of chronic kidney disease. SCD is caused by a point mutation in the gene encoding β globin gene which leads to the formation of hemoglobin S that polymerises after deoxygenation. HbS polymerisation is associated with erythrocyte rigidity and vaso-occlusive episodes that play a central role into SCD pathogenesis. The spectrum of renal diseases during SCD is broad and includes various renal manifestations which become more apparent with increasing age. Underlying pathophysiological processes involved in sickle cell nephropathy are multifactorial but endothelial dysfunction related to chronic hemolysis is a key factor contributing to renal involvement. Our review focuses on the pathogenesis and on the spectrum of renal manifestations occurring in SCD patients.

Understanding the roles of intrinsic disorder in subunits of hemoglobin and the disease process of sickle cell anemia.

One of the common genetic disorders is sickle cell anemia, in which 2 recessive alleles must meet to allow for destruction and alteration in the morphology of red blood cells. This usually leads to loss of proper binding of oxygen to hemoglobin and curved, sickle-shaped erythrocytes. The mutation causing this disease occurs in the 6th codon of the HBB gene encoding the hemoglobin subunit β (β-globin), a protein, serving as an integral part of the adult hemoglobin A (HbA), which is a heterotetramer of 2 α chains and 2 β chains that is responsible for binding to the oxygen in the blood. This mutation changes a charged glutamic acid to a hydrophobic valine residue and disrupts the tertiary structure and stability of the hemoglobin molecule. Since in the field of protein intrinsic disorder, charged and polar residues are typically considered as disorder promoting, in opposite to the order-promoting non-polar hydrophobic residues, in this study we attempted to answer a question if intrinsic disorder might have a role in the pathogenesis of sickle cell anemia. To this end, several disorder predictors were utilized to evaluate the presence of intrinsically disordered regions in all subunits of human hemoglobin: α, β, δ, ε, ζ, γ1, and γ2. Then, structural analysis was completed by using the SWISS-MODEL Repository to visualize the outputs of the disorder predictors. Finally, Uniprot STRING and D2P2 were used to determine biochemical interactome and protein partners for each hemoglobin subunit along with analyzing their posttranslational modifications. All these properties were used to determine any differences between the 6 different types of subunits of hemoglobin and to correlate the mutation leading to sickle cell anemia with intrinsic disorder propensity.