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.

Microfluidic electrical impedance assessment of red blood cell mediated microvascular occlusion

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Yuncheng Man ◽  
Debnath Maji ◽  
Ran An ◽  
Sanjay Ahuja ◽  
Jane A Little ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Red Blood Cell ◽  
Blood Cells ◽  
Electrical Impedance ◽  
Cell Disease ◽  
Blood Disorders

Alterations in the deformability of red blood cells (RBCs), occurring in hemolytic blood disorders such as sickle cell disease (SCD), contributes to vaso-occlusion and disease pathophysiology. However, there are few...

scholarly journals Whole Blood Adhesion to VCAM-1 and P-Selectin and RBC Mechanical Fragility Can be Compromised in Long COVID-19 Patients with Sickle Cell Disease

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 959-959
Author(s):  
Michael Tarasev ◽  
Marta Ferranti ◽  
Cidney Allen ◽  
Xiufeng Gao ◽  
Kayla Topping ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Sickle Cell ◽  
Whole Blood ◽  
Stock Options ◽  
Membrane Stability ◽  
Cell Disease ◽  
Adhesive Properties ◽  
Rbc Membrane ◽  
Privately Held

Abstract Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause severe vascular complications associated with endothelial dysfunction and systemic inflammation. COVID19-specific IgG are detectable within a week of infection. Long COVID-19 has been described in patients continuing to exhibit symptoms after the virus is no longer detectable in the respiratory secretions, including fatigue, dyspnea, headache, and brain fog. The recent FAIR Health study reviewed a total of 1,959,982 COVID-19 patients for the prevalence of long COVID symptoms and reported that 23.2% had at least one post-COVID symptom [1]. The underlying biologic mechanisms of long COVID remain unclear, thus treatments are limited to symptomatic relief and supportive care. Many long COVID symptoms are consistent with systemic inflammation and impaired oxygen delivery observed in individuals with sickle cell disease (SCD), in turn associated with elevated blood cell adhesion and decreased red blood cell (RBC) stability. The aim of this study was to determine if deleterious changes in in blood cell properties related to adhesion and membrane stability under stress can be associated with the symptoms of long COVID-19. In this work we evaluated 7 SCD patients that were diagnosed with SARS-Cov-2 and tracked their recovery using semiquantitative IgG and blood cell function assays. Methods: Blood samples were collected by the Foundation for Sickle Cell Disease (SCD) Research from SCD (homozygous SS, n=6) patients coming for regular or urgent clinic visit with SARS-CoV-2 serological and blood cell functions tests performed per the standard of care. Semiquantitative IgG assay was performed using DXi-80 (Beckman Coulter). Flow adhesion of whole blood to VCAM-1 (FA-WB-VCAM)and P-Selectin (FA-WB-Psel) substrates were determined by counting the cells that remain adherent in a microfluidics channel after perfusion with whole blood 1:1 diluted with HBSS buffer and washed by reversed flow at 1 dyne/cm 2. Red blood cell mechanical fragility (RBC MF) was measured as hemolysis induced by an oscillating cylindrical magnet with periodic non-invasive probing of cell-free hemoglobin fraction. Six individuals with SCD recovering from SARS-Cov-2 with biomarker data available both before and for more than 3 months after the infection (179±62 days) were included in the study. Results: IgG levels varied from less than 0.1 to 37, with positive values being defined as IgG > 1. The median estimated half-life of IgG decline was 53 days ranging from 25 to 90 days (the last, for the hospitalized patient). Averaged for IgG positive (IgG+) and IgG negative (IgG-) conditions, combining pre- and post-infection IgG- conditions, values of patient hemoglobin (Hb), FA-WB-VCAM, FA-WB-Psel, and RBC MF cell properties lacked statistical significance (under both a paired t-test and population statistics). Hb levels remained essentially unchanged regardless of the time from infection or IgG status. However, FA-WB-VCAM, FA-WB-Psel, and RBC MF were all significantly elevated after SARS-Cov-2 seroconversion and remained elevated despite declining IgG levels (e.g., Fig. 1). These increases in biomarker values were statistically significant for both FA-WB-VCAM and RBC MF, and were approaching significance for FA-WB-Psel (p<0065). These increases were highly patient-specific with potential return to pe-infection values observed in some cases at about 5-6 months after the infection. A qualitative review of the medical records indicated a new subjective report of fatigue in 5 of 6 patients. Longer observations are required to determine if abnormal blood cell adhesive properties and RBC membrane instability are mechanisms of long-COVID-19 pathophysiology. Conclusions: Whole blood adhesion to both p-selectin and VCAM-1 as well as RBC membrane stability can be significantly impaired in convalescent SARS-Cov-2 patients suggesting an association with long COVID-19. New and emerging treatments that modify whole blood adhesive properties and RBC membrane stability should be investigated for their potential to accelerated recovery from long COVID-19. Health F. A Detailed Study of Patients with Long-Haul COVID: An Analysis of Private Healthcare Claims; White Paper. June 15, 2021 Disclosures Tarasev: Functional Fluidics: Current holder of stock options in a privately-held company. Ferranti: Functional Fluidics: Current holder of stock options in a privately-held company. Allen: Functional Fluidics: Current Employment. Gao: Functional Fluidics: Current Employment. Topping: Functional Fluidics: Current Employment. Ferranti: Functional Fluidics: Current Employment. Makinde-Odesola: Functional Fluidics: Other: conduct research for academic program. Hines: Functional Fluidics: Current holder of stock options in a privately-held company.

scholarly journals Whole blood viscosity and red blood cell adhesion: Potential biomarkers for targeted and curative therapies in sickle cell disease

2020 ◽  
Vol 95 (11) ◽  
pp. 1246-1256 ◽  
Author(s):  
Erdem Kucukal ◽  
Yuncheng Man ◽  
Ailis Hill ◽  
Shichen Liu ◽  
Allison Bode ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Sickle Cell Disease ◽  
Blood Cell ◽  
Sickle Cell ◽  
Red Blood Cell ◽  
Blood Viscosity ◽  
Whole Blood ◽  
Cell Disease ◽  
Whole Blood Viscosity ◽  
Potential Biomarkers

Persistance of Inflammatory Phenotype of Neutrophils in Sickle Cell Disease Children Despite Chronic Exchange Transfusion Program: A Cause of the Progression of Cerebral Vasculopathy?

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 2-3
Author(s):  
Abdoul Karim Dembele ◽  
Patricia Hermand-Tournamille ◽  
Florence Missud ◽  
Emmanuelle Lesprit ◽  
Malika Benkerrou ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Whole Blood ◽  
Blood Cells ◽  
Exchange Transfusion ◽  
Integrin Expression ◽  
Activation Pattern ◽  
Cell Disease ◽  
Plasmatic Level

Sickle cell disease (SCD) is a severe hemoglobinopathy due to the production of abnormal hemoglobin S (HbS). Although red blood cell (RBC) dysfunction is the major contributor to disease, several studies highlighted the important role of polymorphonuclear neutrophils (PMNs), both during acute and chronic complications. One of the most severe complication of SCD is ischemic stroke due to large cerebral artery occlusion. In 1998, the Stroke Prevention (STOP) trial demonstrated that monthly blood transfusions could reduce the risk of stroke by 90% in SCD children with cerebral vasculopathy (CV). However, there is a wide heterogeneity in the course of CV in patients receiving chronic transfusions, since only about half of them improved their CV under transfusion program, while 25% are only stabilized and 29% continue to get worse despite a percentage of HbS permanently below 30%. The aim of our study is to investigate the impact of transfusion programs on neutrophils activation and ageing, in order to identify if inflammation could contribute to the persistence of SCD complications despite red cell transfusion. We performed a prospective study including 58 homozygous SCD children and 10 healthy donors. Of these, 12 had no specific treatment, 11 were on Hydroxyurea (HU) treatment, 21 were on an exchange transfusion program, and 14 were on both an exchange transfusion program and HU treatment for an average of 4.9 years due to persistent CV. Monthly exchange transfusion are carried out either by erythrapheresis or by manual exchanges, consisting of the continuous bleeding of whole blood compensated by simultaneous transfusion of packed red blood cells. Neutrophils were isolated from fresh blood samples before exchange transfusion session and labelled with 8 markers specific of adhesion, activation and ageing. We quantified by flow cytometry the expression of 3 integrins (CD18, CD11a, CD11b), 3 ageing markers (CD182, CD184, CD62L) and 2 adhesion molecules (CD162 and CD66a). We also measured the plasmatic level of elastase, which reflects the NETose activity of PMNs As previously reported, we observed a high leukocytosis and an activated profile of PMNs in the 12 non-transfused SCD patients compared to healthy controls (Figure 1), characterized by an overexpression of the integrin CD18/CD11b (p=0,03) and CD18/CD11a (p=0,02), a higher level of circulating aged PMNs CD184 high/CD62Llow (p=0,04), a higher expression of CD162 (p=0,01) and CD66a (0,01) as well as a higher plasmatic level of elastase (p=0.01). Interestingly, in the PMNs of the 21 patients receiving monthly exchange transfusion, we found an identical expression pattern of integrins, selectins, ageing markers and elastase level compared to those of the PMNs from non-transfused patients. Furthermore, we also observed a persistence of high neutrophilic leukocytosis. This activation pattern was the same for patients on manual exchange or erythrapheresis, even with a tendency towards a more inflammatory profile in patients on erythrapheresis (Figure 1). In the PMNs from the 11 patients receiving HU compared to untreated SCD patients, we found an expected decrease in high leukocytosis and membrane integrin expression CD18/CD11b and CD18/CD11a. The addition of HU therapy in 14 patients in exchange transfusion program allows to alleviate neutrophilic leukocytosis and membrane integrin expression. Our study shows for the first time that replacing sickle RBCs with healthy RBCs is not sufficient to reverse the pathological phenotype of PMNs in SCD. A persistence of the PMNs activation pattern is observed both despite erythrapheresis, where plasma and white blood cells go back to the patient, and in manual exchanges, where the patient is bled from a large volume of whole blood. Given the major role of inflammation in endothelial damage and vasculopathy in SCD, our data could explain the incomplete efficacy of transfusion exchange programs to treat CV. This raises the question to systematically combine anti-inflammatory and anti-white blood cell adhesion treatments such as Hydroxyurea or P-Selectin inhibitors for these patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Rheological Effects of L-Glutamine in Patients with Sickle Cell Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3567-3567
Author(s):  
Celeste K. Kanne ◽  
Varun Reddy ◽  
Vivien A. Sheehan
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Blood Viscosity ◽  
Whole Blood ◽  
Blood Cells ◽  
Cell Disease ◽  
Phase 3 ◽  
Whole Blood Viscosity

Background: ENDARITM (oral pharmaceutical L-glutamine powder) received FDA approval in 2017 as a treatment for sickle cell disease (SCD). A pivotal phase 3 clinical study conducted by Emmaus Medical, Inc. showed that L-glutamine resulted in a lower incidence of vaso-occlusive crises (VOC) as well as a lower rate of hospitalizations and shorter hospital stays. No changes in standard clinical laboratory values were noted. The clinical improvements associated with sickle cell complications are believed to be due to an increase in the proportion of the reduced form of nicotinamide adenine dinucleotides in the red blood cells (RBC) of patients with SCD, reducing the oxidative stress. While the endpoints in the phase 3 study are clinically important, it is essential that we identify biomarkers or measurable laboratory changes that can serve as endpoints for future clinical trials assessing dose optimization and the efficacy and safety of L-glutamine in SCD individuals, including those with hepatic and renal dysfunction. RBC rheology is markedly abnormal in SCD; blood is more viscous for a given hematocrit than normal individuals, dense red blood cells (DRBC) are packed with HbS, potentiating sickling, and RBCs are less deformable than those of HbAA or HbAS individuals. High whole blood viscosity, high DRBCs, and poor RBC deformability are associated with higher rates of VOC. Given the demonstrated reduction in pain events, we hypothesized that L-glutamine might improve RBC rheology and sought to test this in vitro and in vivo using a battery of rheological tests. Methods: For the in vitro study, 6 mL of whole blood was drawn into an EDTA vacutainer from ten pediatric patients with sickle cell anemia (HbSS or HbSβ0) during routine clinical checkups under an IRB approved protocol. The cohort included 3 female and 7 male patients, ages 2-19 years old. All patients were on a steady dose of hydroxyurea and did not receive a transfusion within the 3 months prior to sample collection. A 200 mM stock solution of L-glutamine and water was mixed and filtered under light-protected conditions. Aliquots were stored at -20°C to avoid multiple freeze/thaw cycles. L-glutamine was added to 3 mL of whole blood for a final concentration of 1 mM (average in vivo L-glutamine plasma concentration in patients with SCD treated with L-glutamine); 3 mL of the same patient sample with water added served as a control. After a 24-hour incubation period at 4°C, whole blood viscosity was measured using a cone and plate viscometer at 37°C (DV3T Rheometer, AMETEK Brookfield, USA), %DRBCs were measured on an ADVIA 120 Hematology System (Siemens Healthcare Diagnostics, Inc., USA), and deformability measured using a Laser Optical Rotational Red Cell Analyzer (Lorrca®) (RR Mechatronics, the Netherlands) with the Oxygenscan module. The Oxygenscan measures RBC deformability at normoxia (Elmax), deformability upon deoxygenation (EImin), and point of sickling (PoS), the oxygen tension at which deformability begins to decline, reflecting the patient-specific pO2 at which sickling begins. Paired samples (with and without added L-glutamine) were analyzed using Student's t-test. For the in vivo study, rheological tests were performed on peripheral blood from one patient (18-year-old male on hydroxyurea) at baseline and treated with L-glutamine as part of his routine clinical care. Results and conclusions: Addition of L-glutamine in vitro significantly reduced the PoS, meaning RBCs incubated with L-glutamine could tolerate a lower pO2 before sickling compared to the control. RBCs incubated with L-glutamine also had significantly higher EImin, meaning deoxygenated RBCs were more flexible and deformable. Whole blood viscosity at 45s-1 and 225s-1 did not change significantly following incubation with L-glutamine; %DRBCs also did not change significantly (Table 1). The in vivo patient sample tested exhibited a similar improvement in PoS and EImin (Figure 1). We therefore propose to further test the performance of the PoS and EImin as possible biomarkers of response to L-glutamine in vivo. If validated, these biomarkers may also help further elucidate the mechanisms of action of L-glutamine in SCD. Disclosures No relevant conflicts of interest to declare.

scholarly journals Red blood cells modulate structure and dynamics of venous clot formation in sickle cell disease

Blood ◽  
2019 ◽  
Vol 133 (23) ◽  
pp. 2529-2541 ◽  
Author(s):  
Camille Faes ◽  
Anton Ilich ◽  
Amandine Sotiaux ◽  
Erica M. Sparkenbaugh ◽  
Michael W. Henderson ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Sickle Cell Disease ◽  
Plasminogen Activator ◽  
Sickle Cell ◽  
Whole Blood ◽  
Blood Cells ◽  
Cell Disease ◽  
Increased Risk ◽  
Blood Clots ◽  
Tissue Plasminogen

Abstract Sickle cell disease (SCD) is associated with chronic activation of coagulation and an increased risk of venous thromboembolism. Erythrocyte sickling, the primary pathologic event in SCD, results in dramatic morphological changes in red blood cells (RBCs) because of polymerization of the abnormal hemoglobin. We used a mouse model of SCD and blood samples from sickle patients to determine if these changes affect the structure, properties, and dynamics of sickle clot formation. Sickling of RBCs and a significant increase in fibrin deposition were observed in venous thrombi formed in sickle mice. During ex vivo clot contraction, the number of RBCs extruded from sickle whole blood clots was significantly reduced compared with the number released from sickle cell trait and nonsickle clots in both mice and humans. Entrapment of sickled RBCs was largely factor XIIIa–independent and entirely mediated by the platelet-free cellular fraction of sickle blood. Inhibition of phosphatidylserine, but not administration of antisickling compounds, increased the number of RBCs released from sickle clots. Interestingly, whole blood, but not plasma clots from SCD patients, was more resistant to fibrinolysis, indicating that the cellular fraction of blood mediates resistance to tissue plasminogen activator. Sickle trait whole blood clots demonstrated an intermediate phenotype in response to tissue plasminogen activator. RBC exchange in SCD patients had a long-lasting effect on normalizing whole blood clot contraction. Furthermore, RBC exchange transiently reversed resistance of whole blood sickle clots to fibrinolysis, in part by decreasing platelet-derived PAI-1. These properties of sickle clots may explain the increased risk of venous thromboembolism observed in SCD.

Clinical Effects of Chronic Red Blood Cell Exchange and Different Types of Red Cell Concentrates in Patients with Sickle Cell Disease

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4823-4823
Author(s):  
Sergio Cabibbo ◽  
Agostino Antolino ◽  
Giovanni Garozzo ◽  
Carmelo Fidone ◽  
Pietro Bonomo
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Iron Overload ◽  
Sickle Cell ◽  
Red Blood Cell ◽  
Whole Blood ◽  
Clinical Effects ◽  
Red Cell ◽  
Blood Components ◽  
Cell Disease

Abstract For patients with severe SCD not eligible for hydroxyurea, two major therapeutic options are currently available: blood transfusion, and bone marrow transplantation. Either urgent or chronic red blood cell transfusion therapy, is widely used in the management of SCD but determines a progressive increase of ferritin level and is also limited by the development of antibodies to red cell antigens. The introduction of chronic red blood cell exchange and prestorage filtration to remove leucocytes and the use of techniques for multicomponent donation could be a good solutions. Thus, the aims of our studies were to evaluate the clinical effects of the different blood components in terms of annual transfusion needs and the intervals between transfusion, moreover we evaluated the efficacy of chronic red blood cell exchange (manual or automatic with cell separator) in preventing SCD complications and limiting iron overload. In our center we follow 78 patients affected by Sickle Cell Disease. We selected 36 patients occasionally treated with urgent red blood cell exchange because they had less than 2 complications/Year, and 42 patients regularly treated with chronic red blood cell exchange because they had more than 2 complications/Year with Hospital Admission. Moreover among these we selected 10 patients for fulfilling the criteria of continuous treatment at the Centre for at least 48 months with no interruptions, even sporadic and absolute transfusion dependency. All 10 patients were evaluated for a period of 4 years, during which two different systems of producing RCC were used. In the second two the patients were transfused with RCC obtained from filtering whole blood prestorage or with RCC from apheresis filtered prestorage. These products differed from those used in the preceding two years, during which the leucodepletion was obtained by bed-side filtration For all the patients we performed 782 automatic red blood cell exchanges and 4421 units of RCC were transfused. The exchange procedures were extremely well-tolerated by the patients and adverse effects were limited to symptoms of hypocalcaemia during automatic red blood cell exchange with cell separator. After every red blood cell exchange we obtained HbS level < 30%. The10 patients selected received respectively a mean of 6.9 and 6.1 units of RBCs exchanged per automatic procedure, in the first two years and in the second two years. Alloantibody developed in 14 patients but only 2 clinically significant and about the observed frequency of transfusion reactions it was very low. All patients treated with chronic red blood cell exchange had an improvement of the quality of life with a reduced number of complications/year (<2/year) and good compliance and moreover patients had limited iron overload making chelating therapy easier. In conclusion this study was focused on the most suitable characteristics of blood components for use in sickle cell disease patients and the choice of systematically adopting prestorage filtration of whole blood, enabled us to have RCC with a higher Hb concentration than standard. Moreover chronic manual or automatic red blood cell exchange as an alternative approach to simple long-term RBC transfusions give many advantages by being more rapid and tolerable as well as clinically safe and effective and minimize the development of iron overload especially when procedure was carried out with an automatic apparatus. To note that the clinical advantages for patients derived from good selection of the donor and good practices in the production of the blood components

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.

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 Differential Efficacy of Anti-Sickling and Anti-Inflammatory Mechanisms in a Fluorescent Intravital Microscopy Dorsal Skinfold Vaso-Occlusion Model in Sickle Cell Disease Townes Mice

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2264-2264
Author(s):  
Sarah Sturtevant ◽  
Alejandra Macias-Garcia ◽  
Sriram Krishnamoorthy ◽  
Arjan van der Flier ◽  
Alexandra Hicks ◽  
...  
Keyword(s):  
Blood Flow ◽  
Sickle Cell Disease ◽  
Blood Cell ◽  
Sickle Cell ◽  
Blood Cells ◽  
Ex Vivo ◽  
Endothelial Activation ◽  
Cell Disease ◽  
Occlusion Model ◽  
Hypoxia Reoxygenation

Sickle cell disease (SCD) is characterized by acute and repetitive vaso-occlusive crises (VOC). These crises have been hypothesized to occur when blood flow is reduced following obstruction of sickle-shaped red blood cells in the vasculature. However, it is now well established that inflammation, oxidative stress, endothelial activation and pro-coagulation in sickle cell disease patients also contribute to the formation of heterocellular aggregates that can lead to VOC (Vercellotti and Belcher, 2014). Transgenic SCD mice recapitulate the pathology of human disease in response to stimuli such as heme injection and hypoxia/reoxygenation. SCD SS Townes mice, which express human α and sickle γAβS globins, AA Townes mice expressing normal human α and normal γAβA globins and heterozygous AS mice which express only one allele of the γAβS sickle gene were used. To characterize vaso-occlusion in these mice and evaluate the efficacy of different pharmacological mechanisms, we modified the skinfold vaso-occlusion model (Kalambur et al, 2004) using fluorescent intravital microscopy to visualize blood flow occlusion following hemin injection or hypoxia/reoxygenation challenge. Dorsal skinfold chambers were implanted and24 hours post-surgery mice were injected with FITC-dextran for visualization of flowing blood vessels. Skinfold bearing mice were then subjected to hemin treatment (16 μmoles/kg) or hypoxia (7%; 1 hour)/reoxygenation (1 hour) followed by the injection of Alexa fluor 647-labeled albumin to allow quantification of occluded vessels through dual fluorescent image analysis. Following hemin injection, SS mice showed significant ~30% vaso-occlusion in comparison to AA mice with ~8%, whereas the AS mice showed an intermediate phenotype with ~20% vaso-occlusion. Hypoxia/reoxygenation challenge also resulted in significant vaso-occlusion for SS mice (~25%) whereas only 5% was observed in AA mice. Interestingly, AS mice also showed a significant amount of vaso-occlusion (~25%) similar to SS mice when challenged with hypoxia/reoxygenation. Although no sickling can be observed in an ex vivo sickling assay using AS red blood cells, an intermediate amount of free Hemoglobin (Hb) can be detected in the plasma of these mice and rolling can be observed. This suggests that these vaso-occlusive models relate more on the inflammatory and endothelial activation state and are independent of the sickling potential of the red blood cell. We then used our model with hypoxia/reoxygenation challenge to evaluate the effects of dimethyl fumarate (DMF, 15 mpk BID), an anti-P-Selectin antibody (150ug/mouse) and the covalent hemoglobin oxygen affinity modulator GBT-440 (300 mpk). As anti-inflammatory agents, DMF and Anti-P-Selectin significantly reduced vaso-occlusion in SS mice by ~60% compared to the vehicle treated mice, but GBT-440 did not inhibit vaso-occlusion at a dose where a significant reduction in p50 was observed. In conclusion, our data have shown that obstruction of blood flow in the skinfold vaso-occlusion model in SCD Townes mice reflects the vascular inflammatory state of the disease and is independent of the ex vivo capacity of red blood cell to sickle. Disclosures Sturtevant: Sanofi: Employment. Macias-Garcia:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment. van der Flier:Sanofi: Employment. Hicks:Sanofi: Employment. Demers:Sanofi: Employment.

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