scholarly journals Extracellular Vesicles in Sickle Cell Disease: Plasma Concentration, Blood Cell Types Origin Distribution and Biological Properties

2021 ◽  
Vol 8 ◽  
Author(s):  
Elie Nader ◽  
Yohann Garnier ◽  
Philippe Connes ◽  
Marc Romana
Keyword(s):  
Plasma Concentration ◽  
Sickle Cell Disease ◽  
Blood Cell ◽  
Extracellular Vesicles ◽  
Sickle Cell ◽  
Globin Gene ◽  
Clinical Manifestations ◽  
Cell Types ◽  
Single Mutation ◽  
Cell Disease

Prototype of monogenic disorder, sickle cell disease (SCD) is caused by a unique single mutation in the β-globin gene, leading to the production of the abnormal hemoglobin S (HbS). HbS polymerization in deoxygenated condition induces the sickling of red blood cells (RBCs), which become less deformable and more fragile, and thus prone to lysis. In addition to anemia, SCD patients may exhibit a plethora of clinical manifestations ranging from acute complications such as the frequent and debilitating painful vaso-occlusive crisis to chronic end organ damages. Several interrelated pathophysiological processes have been described, including impaired blood rheology, increased blood cell adhesion, coagulation, inflammation and enhanced oxidative stress among others. During the last two decades, it has been shown that extracellular vesicles (EVs), defined as cell-derived anucleated particles delimited by a lipid bilayer, and comprising small EVs (sEVs) and medium/large EVs (m/lEVs); are not only biomarkers but also subcellular actors in SCD pathophysiology. Plasma concentration of m/lEVs, originated mainly from RBCs and platelets (PLTs) but also from the other blood cell types, is higher in SCD patients than in healthy controls. The concentration and the density of externalized phosphatidylserine of those released from RBCs may vary according to clinical status (crisis vs. steady state) and treatment (hydroxyurea). Besides their procoagulant properties initially described, RBC-m/lEVs may promote inflammation through their effects on monocytes/macrophages and endothelial cells. Although less intensely studied, sEVs plasma concentration is increased in SCD and these EVs may cause endothelial damages. In addition, sEVs released from activated PLTs trigger PLT-neutrophil aggregation involved in lung vaso-occlusion in sickle mice. Altogether, these data clearly indicate that EVs are both biomarkers and bio-effectors in SCD, which deserve further studies.

scholarly journals Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia

2016 ◽  
Vol 113 (38) ◽  
pp. 10661-10665 ◽  
Author(s):  
Lin Ye ◽  
Jiaming Wang ◽  
Yuting Tan ◽  
Ashley I. Beyer ◽  
Fei Xie ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Genome Editing ◽  
Sickle Cell ◽  
Globin Gene ◽  
Autologous Transplantation ◽  
Clinical Manifestations ◽  
Fetal Hemoglobin ◽  
Compound Heterozygous ◽  
Cell Disease ◽  
Hematopoietic Stem

Hereditary persistence of fetal hemoglobin (HPFH) is a condition in some individuals who have a high level of fetal hemoglobin throughout life. Individuals with compound heterozygous β-thalassemia or sickle cell disease (SCD) and HPFH have milder clinical manifestations. Using RNA-guided clustered regularly interspaced short palindromic repeats-associated Cas9 (CRISPR-Cas9) genome-editing technology, we deleted, in normal hematopoietic stem and progenitor cells (HSPCs), 13 kb of the β-globin locus to mimic the naturally occurring Sicilian HPFH mutation. The efficiency of targeting deletion reached 31% in cells with the delivery of both upstream and downstream breakpoint guide RNA (gRNA)-guided Staphylococcus aureus Cas9 nuclease (SaCas9). The erythroid colonies differentiated from HSPCs with HPFH deletion showed significantly higher γ-globin gene expression compared with the colonies without deletion. By T7 endonuclease 1 assay, we did not detect any off-target effects in the colonies with deletion. We propose that this strategy of using nonhomologous end joining (NHEJ) to modify the genome may provide an efficient approach toward the development of a safe autologous transplantation for patients with homozygous β-thalassemia and SCD.

Hydroxyurea-Induced Changes of Components Involved In the Modulation of Adenosine Levels, In Blood Cells From Sickle Cell Disease Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2674-2674
Author(s):  
Rodrigo Alexandre Panepucci ◽  
Ana Cristina S Pinto ◽  
Carolina Dias-Carlos ◽  
Felipe Saldanha-Araujo ◽  
Patricia VB Palma ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Cell Types ◽  
Potential Effect ◽  
T Test ◽  
Lower Percentage ◽  
Cell Disease ◽  
Local Synthesis

Abstract Abstract 2674 Introduction. Recent studies have demonstrated the role of high adenosine levels in priapism episodes in a mouse model of sickle cell disease (SCD). Interestingly, adenosine signaling is related to several physiopathological processes that may relate to clinical features observed in patients with SCD. Adenosine (ADO) is a purine nucleoside that plays diverse roles in distinct physiological contexts. Extracellular ADO production occurs sequentially by the ectonucleotidases CD39 (which converts ATP and ADP to 5′-AMP) and CD73 (which convert 5′-AMP to ADO). Moreover, ADO levels are controlled by its conversion to inosine by the enzyme Adenosine Deaminase (ADA). ADA can be anchored in the cell membrane by CD26, leading to an increased localized action and consequently, to reduced local concentrations of adenosine. Hydroxyurea (HU) is the only drug approved by FDA to reduce vaso-occlusive episodes in patients with SCD, partly by the induction of fetal hemoglobin (HbF) and reduction of polymerization of HbS. However, the clinical improvement of patients is not always associated with increased HbS levels, indicating the potential effect of HU on other processes. Given the known (or proposed) contribution of distinct blood cell types in the physiopathology of SCD, in this study, we aimed to evaluate the possible modulation in the expression of CD39, CD73 and CD26 on lymphocytes and monocytes from SCD patients, in HU treated patients. Methods. The expression of CD39, CD73 and CD26 was evaluated by flow cytometry on total lymphocytes (CD3+) and monocytes (CD14+) in the peripheral blood (PB) of 12 patients treated with HU, 21 untreated and seven control healthy individuals. Results. On average, while less than 0.3% and 1.7% of monocytes of controls and untreated patients express CD26, respectively; in patients treated with HU, more than 10% of the monocytes express CD26 (p=0.0171, unpaired T-test). Additionally, in treated patients, a significantly lower percentage of lymphocytes express CD39, as compared to untreated (p=0.0431, unpaired T-test). The CD73 protein was not expressed by monocytes, and there was no modulation of its levels in lymphocytes. Conclusions. During inflammation (a processes associated with the physiopathology of SCD), the extracellular concentration of adenosine is increased and distinct blood cell types localize to the affected tissue. The results indicate a potential mechanism of action of HU in SCD patients, mediated by the increased expression of CD26 on monocytes (with subsequent co-localization of the enzyme ADA) and by the decreased expression of CD39 on lymphocytes. As a result of the observed changes, a decrease in the local synthesis of adenosine, associated with its increased conversion to inosine, would be expected. Thus, HU may drive the reduction of adenosine levels, thereby reducing the aggravating effects of this molecule in different physiopathological processes affected in patients with SCD. Supported by: FAPESP, CNPQ, FINEP and INSERM. Disclosures: No relevant conflicts of interest to declare.

Contributions of Immune Cells to Vascular Occlusion in Sickle Cell Disease.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. sci-44-sci-44
Author(s):  
Paul S. Frenette ◽  
Andres Hidalgo ◽  
Jungshan Chang ◽  
Anna Peired
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Blood Cells ◽  
High Speed ◽  
Globin Gene ◽  
Clinical Manifestations ◽  
Leading Edge ◽  
Cell Disease ◽  
Humanized Mouse Model

Abstract Sickle cell disease (SCD) results from a single missense mutation in the β-globin gene, making the gene product susceptible to polymerize in conditions of low O2 tension. The resulting polymers can disrupt the normal architecture of sickle red blood cells (sRBC), altering their membrane and promoting adherence. sRBC adhesion events within the vasculature produces the painful vaso-occlusive episodes that account for most of the morbidity and mortality of this disease.1 The mechanisms mediating vaso-occlusion (VOC) in SCD are not well understood. Several studies have shown that sRBC, most notably the immature reticulocytes, can bind to endothelial cells (EC) though multiple overlapping adhesion mechanisms. However, whether these interactions are directly involved in VOC is controversial. sRBC can clearly interact with other blood cells inside the vasculature. For example, in a humanized mouse model of SCD, sRBCs interact prominently with leukocytes that are adherent in inflamed venules.2 A role for leukocytes in VOC is consistent with clinical data that have linked leukocyte counts, but not reticulocyte counts, with clinical manifestations of SCD. High-speed multichannel fluorescence intravital microscopy (MFIM) using low doses of lineage-specific antibodies has shown that sRBCs interact specifically with adherent neutrophils.3 Most adherent neutrophils are not immobile but rather crawl along inflamed venules, and virtually all of them exhibit a polarized appearance in vivo with clustered L-selectin at the uropod. Inhibition of or deficiency in the endothelial selectins (E- and P-selectins) protects SCD mice from VOC. Recent studies into the individual role of each selectin have revealed, unexpectedly, a major function for E-selectin. Whereas E-selectin inhibition or deficiency does not significantly affect neutrophil adhesion in inflamed venules, it dramatically blocks the interactions of RBC with adherent neutrophils, suggesting that E-selectin signaling into an adherent leukocyte may induce RBC capture. We have found that RBCs carrying normal hemoglobin (nRBCs) also interact with adherent neutrophils in inflamed venules of wildtype mice. In the mouse, three glycoproteins, P-selectin glycoprotein ligand-1 (PSGL-1, encoded by the Selplg gene), CD44 (encoded by Cd44), and E-selectin ligand-1 (ESL-1, encoded by Glg1) mediate all E-selectin binding activity of leukocytes.4 Detailed analyses of Selplg−/− mice, Cd44−/− mice, or chimeric mice, in which Glg1 is knocked-down by RNA interference, revealed that E-selectin-induced signals are mediated specifically by Glg1. Using MFIM analyses, we have mapped the location of RBC captures on the leading edge of polarized neutrophils. Further investigations have revealed that RBC capture was mediated by the β2 integrin Mac-1 (αMβ2). We have developed a new method to assay Mac-1 activation on adherent leukocytes in live mice. These analyses have shown that Glg1-mediated signals activate αMβ2 regionally at the leading edge, allowing RBC capture. These results suggest a new pathway for the development of targeted therapies for VOC. The fact that genetically normal RBCs are captured by neutrophils through mechanisms similar to sRBCs also suggests broad functions for this paradigm in other thrombo-inflammatory diseases.

Aggregates of Reticulocytes, Monocytes and Platelets Exist in Whole Blood of Sickle Cell Patients: Roles for Plasma Fibronectin, α4 Integrins and PSGL-1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2330-2330
Author(s):  
Julia E. Brittain ◽  
Shantres C. Clark ◽  
Kenneth I. Ataga ◽  
Eugene P. Orringer ◽  
Leslie V. Parise
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Sickle Cell ◽  
Whole Blood ◽  
Blood Cells ◽  
Cell Types ◽  
Potential Contribution ◽  
Plasma Fibronectin ◽  
Cell Disease ◽  
Color Flow

Abstract Leukocyte and platelets are understudied contributors to the overall pathology of sickle cell disease (SCD). Elevated leukocyte counts are common in these patients and correlate inversely with patient lifespan and overall disease severity. For example, a drop in neutrophil count typically predicts a patient’s response to hydroxyurea, while increased monocyte counts correlate directly with increased reporting of pain crises. Moreover, both RBCs and WBCs have been detected as components in vaso-occlusive blockages in mouse models, where adhesive RBCs appear to interact directly with WBCs at the vaso-occlusive site. Platelets are activated in SCD and are thought to promote the hypercoagulability in these patients. Despite the potential contribution of all blood cells to the pathology of sickle cell disease, neither a mechanism of adhesion between the WBC and RBC nor a role for soluble matrix proteins in this interaction has been elucidated in humans. To detect potential adhesive interactions between the blood cells in SCD, we collected whole blood into anticoagulants that spare divalent cations (PPACK or factor Xa inhibitor) and assayed for heterotypic cell associations by two and three color flow cytometry. Our results indicate that RBCs, WBCs and platelets exist in heterotypic, multi-cellular aggregates in blood from SCD patients but not unaffected (AA) individuals. By detecting monocyte specific markers, we determined that the primary WBC component of these aggregates was the monocyte, and the primary RBC was the young SS “stress” reticulocyte. Using both in vitro RBC/monocyte adhesion studies and whole blood samples, we demonstrate that α4-containing integrins on both SS RBCs and WBCs mediate this interaction by interacting directly with endogenous plasma fibronectin. Furthermore, we show that the α4 integrin on SS RBCs binds to the RGDS site in fibronectin, whereas the α4 integrin on monocytes binds to the CS-1 site in the molecule, suggesting a novel mechanism of interaction between SS RBCs and monocytes via a fibronectin bridge. Antibodies against the CS-1 binding site in fibronectin substantially disrupt the monocyte/RBC interaction in whole blood, further underscoring the role of fibronectin as a linker between the two cell types. However, platelet incorporation in the aggregate was insensitive to inhibition of the α4 integrin, but was sensitive to inhibition of PSGL-1, suggesting that platelet inclusion likely occurs via a P-selectin/PSGL-1-mediated interaction between the platelet and the monocyte. Interestingly, similar aggregates were also detected in two patients with chronic hemolysis and brisk reticulocytosis, potentially extending the relevance of such aggregates beyond SCD. Taken together our results suggest a new adhesive paradigm for SS RBCs and monocytes as central components of heterotypic blood cell aggregates that include platelets and that are present in whole blood of patients with SCD. Our data therefore illustrate a potentially pathological interaction of all major blood cell types in SCD patients that may impact vaso-occlusion and contribute to other erythrocyte disorders.

scholarly journals The Alberta Newborn Screening Approach for Sickle Cell Disease: The Advantages of Molecular Testing

2021 ◽  
Vol 7 (4) ◽  
pp. 78
Author(s):  
Janet R. Zhou ◽  
Ross Ridsdale ◽  
Lauren MacNeil ◽  
Margaret Lilley ◽  
Stephanie Hoang ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Newborn Screening ◽  
Sickle Cell ◽  
Red Blood Cell ◽  
Globin Gene ◽  
Adverse Outcomes ◽  
Red Blood Cell Transfusion ◽  
Molecular Testing ◽  
Cell Disease

Sickle cell disease (SCD), a group of inherited red blood cell (RBC) disorders caused by pathogenic variants in the beta-globin gene (HBB), can cause lifelong disabilities and/or early mortality. If diagnosed early, preventative measures significantly reduce adverse outcomes related to SCD. In Alberta, Canada, SCD was added to the newborn screening (NBS) panel in April 2019. The primary conditions screened for are sickle cell anemia (HbS/S), HbS/C disease, and HbS/β thalassemia. In this study, we retrospectively analyzed the first 19 months of SCD screening performance, as well as described our approach for screening of infants that have received a red blood cell transfusion prior to collection of NBS specimen. Hemoglobins eluted from dried blood spots were analyzed using the Bio-Rad™ VARIANT nbs analyzer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Targeted sequencing of HBB was performed concurrently in samples from all transfused infants. During the period of this study, 43 of 80,314 screened infants received a positive NBS result for SCD, and of these, 34 were confirmed by diagnostic testing, suggesting a local SCD incidence of 1:2400 births. There were 608 infants with sickle cell trait, resulting in a carrier frequency of 1:130. Over 98% of non-transfused infants received their NBS results within 10 days of age. Most of the 188 transfused infants and 2 infants who received intrauterine transfusions received their final SCD screen results within 21 ± 10 d of birth. Our SCD screening algorithm enables detection of affected newborns on the initial NBS specimen, independent of the reported blood transfusion status.

scholarly journals Sickle Cell Anemia: A review on the most severe form of Sickle Cell Disease

Bionatura ◽  
2019 ◽  
Vol 02 (Bionatura Conference Serie) ◽  
Author(s):  
María Belén Paredes ◽  
María Eugenia Sulen
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Globin Gene ◽  
Single Point ◽  
Clinical Manifestations ◽  
Severe Form ◽  
Single Point Mutation ◽  
Cell Disease ◽  
Hemoglobin S

Sickle cell disease (SCD) is a group of hereditary disorders caused by a single point mutation in the β-globin gene. This mutation results in the formation of a mutated hemoglobin S (HbS) and the consequent sickle phenotype of erythrocytes. SCD is common in regions of malaria endemicity. However, changes in population dynamics enabled the movement of the mutated gene to other areas such as North America and Europe. Sickle cell anemia (SCA) is the most severe form of SCD and affects millions of people around the globe. The clinical manifestations of SCA arise primarily from the polymerization of deoxygenated hemoglobin S (deoxyHbS) leading to vascular occlusion and hemolytic anemia. Clinical complications of the disease are derived from deoxyHbS polymerization, but there are several therapeutic strategies to reduce the severity of the symptoms. Gene therapy has arisen as a new therapeutic approach aimed to cure rather than to treat the symptomatology of SCA by targeting the altered β-globin gene for gene correction.

Clinical and Genetic Variability of Red Blood Cell Hemolysis in Sickle Cell Anemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1077-1077
Author(s):  
Jacqueline N Milton ◽  
Paola Sebastiani ◽  
Yingze Zhang ◽  
Mehdi Nouraie ◽  
Janet Lee ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Sickle Cell ◽  
Red Blood Cell ◽  
Globin Gene ◽  
Red Cell ◽  
Cell Disease ◽  
Intravascular Hemolysis ◽  
Plasma Hemoglobin ◽  
Cell Storage

Abstract Abstract 1077 Intravascular hemolysis is an important pathological mechanism underlying some complications of sickle cell disease and other hemolytic anemias. Hemolysis contributes to endothelial dysfunction, pulmonary and systemic vasculopathy, and platelet and hemostatic activation via nitric oxide catabolism by plasma hemoglobin and arginine catabolism by red blood cell arginase. Little is known about the molecular mechanisms of hemolysis and how the propensity of erythrocytes to hemolyze is modulated. Hemoglobin F concentration and the presence of ∝ thalassemia affect the level of hemolysis but it is likely that other genes and their products are also important. We hypothesize that genetic variation, much of which is outside the β-globin gene-like cluster, underlies the susceptibility of erythrocytes to hemolyze in response to diverse disease stressors. We first characterized hemolysis by creating a principal component analysis (PCA) of age-adjusted values for LDH, AST, reticulocyte count and total bilirubin, but not hemoglobin concentration, to develop a hemolytic component that reflects shared variability among markers. The development of such a component helps to resolve the problem of dealing with correlated predictors in multivariate analyses and confounding variables such as site, and it permits for adjustment for the degree of anemia. To validate the PCA, we measured the plasma hemoglobin levels and red cell microparticle levels in the first and fourth quartile of PCA intensity of hemolysis in 118 HbS-only patients without detectable HbA, from the Walk-PHASST cohort We observed a highly significant increase in plasma hemoglobin (p<0.0001) and red cell microparticles (p=0.0004) based on PCA quartile. Despite the small sample size of this validating cohort we reproduced significant associations between high hemolytic rate and the subphentypes of low arterial oxygen saturation, high pulse pressure, leg ulcers, TRV, high NT-proBNP levels, and low 6-minute walk test distance. More patients with ∝ thalassemia and more females were present in the lower hemolytic index quartile (p=0.006). The hemolytic index and its individual components were then used as phenotypes in genome-wide association studies (GWAS) in the CSSCD (Cooperative Study of Sickle Cell Disease) and walk-PHaSST cohorts to discover novel genes that might be associated with hemolysis. As further validation of our approach using PCA stratification, patients in the quartile with the lowest hemolytic index from the CSSCD also had a much higher prevalence of ∝ thalassemia than patients within the highest quartile of hemolytic index (p=2.2E-16). We first examined 1117 cases from the CSSCD and found 303 SNPs, 265 with a MAF >0.05, that reached a threshold of significance of p<5E-4. For replication, we examined these SNPs in the Walk-PHASST cohort. Eight SNPs replicated with the same effects in a GWAS in 449 subjects from Walk-PHAAST and p-value<0.01. Of the 8 SNPs that replicated, 4 SNPs were in olfactory receptor (OR) genes on chromosome (chr) 11p; OR51L2 (rs7948471, rs7938426. rs1391617), and OR51L1 (rs2445284). Several of these SNPs were also associated with HbF in previous GWAS analyses. Polymorphisms in the OR gene cluster upstream of HBG might modulate HbF levels by altering chromatin structure within the HBB globin gene-like cluster. One SNP in an intron of NPRL3 (rs7203566) on chr16p is ∼34 kb upstream from a SNP causing ∝ thalassemia. In CSSCD cases there was an association of SNPs in NPRL3 with reticulocytes (p=5.1E-0006) and LDH (p=0.0003). In silico analysis did not predict any function for this SNP. Genetic studies to discover new biologic modifiers of hemolysis will help to identify critical molecular determinants of hemolysis for functional studies, to develop new disease severity biomarkers, and to suggest candidate therapies for some common human diseases with intravascular hemolysis. We anticipate that our studies will identify genetic variants enriched in the African-American population primarily determined by the evolved human response to endemic malaria infection. These studies are expected to broadly impact many human diseases and blood banking by providing genomic markers of susceptibility to hemolytic anemia, red cell storage stability and transfusion risk, and insights into novel strategies to reduce anemia and to enhance red blood cell storage and post-transfusion erythrocyte recovery. Disclosures: Gladwin: Patents filed related to treating hemolysis.: Patents & Royalties.

scholarly journals Sickle Cell Disease in the Post Genomic Era: A Monogenic Disease with a Polygenic Phenotype

2009 ◽  
Vol 2 ◽  
pp. GEI.S2626 ◽  
Author(s):  
A. Driss ◽  
K.O. Asare ◽  
J.M. Hibbert ◽  
B.E. Gee ◽  
T.V. Adamkiewicz ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Cell Interaction ◽  
Monogenic Disease ◽  
Acute Chest Syndrome ◽  
Single Mutation ◽  
Beta Globin ◽  
Genetic Modifiers ◽  
Cell Disease

More than half a century after the discovery of the molecular basis of Sickle Cell Disease (SCD), the causes of the phenotypic heterogeneity of the disease remain unclear. This heterogeneity manifests with different clinical outcomes such as stroke, vaso-occlusive episodes, acute chest syndrome, avascular necrosis, leg ulcers, priapism and retinopathy. These outcomes cannot be explained by the single mutation in the beta-globin gene alone but may be attributed to genetic modifiers and environmental effects. Recent advances in the post human genome sequence era have opened the door for the identification of novel genetic modifiers in SCD. Studies are showing that phenotypes of SCD seem to be modulated by polymorphisms in genes that are involved in inflammation, cell–cell interaction and modulators of oxidant injury and nitric oxide biology. The discovery of genes implicated in different phenotypes will help understanding of the physiopathology of the disease and aid in establishing targeted cures. However, caution is needed in asserting that genetic modifiers are the cause of all SCD phenotypes, because there are other factors such as genetic background of the population, environmental components, socio-economics and psychology that can play significant roles in the clinical heterogeneity.

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