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

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.

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

Frontiers in Medicine ◽

10.3389/fmed.2021.728693 ◽

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.

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Caring for Patients with Sickle Cell Disease during a Pandemic: Continuing to Provide Automated Red Blood Cell Exchange Transfusions in Difficult Times

Blood ◽

10.1182/blood-2020-142535 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 25-26

Author(s):

Richard Curtis Godby ◽

Ashton Kornbrust ◽

Denis Noubouossie ◽

Jose Lima ◽

Marisa B. Marques ◽

...

Keyword(s):

Sickle Cell Disease ◽

Blood Cell ◽

Sickle Cell ◽

Red Blood Cell ◽

Blood Product ◽

T Test ◽

World Health ◽

Cell Disease ◽

Global Pandemic ◽

The Mean

Introduction: The World Health Organization declared COVID-19 a global pandemic on 03/11/20. Subsequent concerns around caring for patients with sickle cell disease who require automated red blood cell (RBC) exchange transfusions emerged, especially in the setting of physical distancing and national shortages in blood product supplies. In this vulnerable population at high risk of allo-immunization, ideal transfusion parameters (e.g., antigen optimization) will likely grow increasingly difficult to satisfy and require careful evaluation and strategic planning. Methods: Automated RBC exchange transfusions were performed at the University of Alabama at Birmingham (UAB) in patients with sickle cell disease for a variety of clinical indications with the primary objective of lowering the amount of Hemoglobin S (goal 15%) and replacing it with Hemoglobin A. We collected the number of weekly RBC exchange transfusions performed and then compared the frequencies between 01/05/20 and 03/14/20 (pre-pandemic) to those between 03/15/20 and 08/01/20 (intra-pandemic) using a one-tailed t-test. We also examined the number of RBC units ordered per week at UAB, in both the inpatient and outpatient settings, shortly before and after the declaration of a global pandemic using a one-tailed t-test. Results: The mean frequency of RBC exchange transfusions performed per week was 8.1 [standard deviation 2.3] pre-pandemic and 8.6 [2.3] intra-pandemic (Figure 1a). There was no statistically significant difference (p=0.27) in the frequency between these two periods. Shortly prior to the start of the pandemic (02/23/20-03/14/20), a mean of 77.3 [17.9] units/week were ordered for outpatient RBC exchange transfusions. Shortly after the start of the pandemic (03/15/20-04/26/20), a mean of 55.3 [22.8] units/week were ordered for outpatient RBC exchange transfusions, which was also not significantly different (p=0.09). During this time period, the mean number of RBC units per week ordered in the inpatient surgical setting significantly declined from 719.3 [43.1] to 390.0 [46.8] as elective procedures were delayed (p<0.005) (Figure 1b). Conclusions/Future Directions: The frequency of automated RBC exchange transfusions performed at UAB did not decrease after the onset of the pandemic. UAB was able to continue caring for patients with sickle cell disease receiving RBC exchange transfusions as the pandemic emerged and national blood product supplies declined despite a similar overall demand. Interestingly, there was also a concomitant decrease in the demand for RBCs from inpatient surgical settings as elective procedures were delayed, possibly contributing to the blood bank's ability to maintain ideal transfusion parameters and perform antigen optimization of transfused RBCs. As the COVID-19 pandemic continues, the national shortage of blood product supplies will likely worsen and necessitate multidisciplinary efforts, including intra-institutional and inter-institutional collaborations, to continue caring for patients with sickle cell disease receiving RBC exchange transfusions. Furthermore, community education, safely structured blood drives, and other efforts to encourage donations are essential to maintain the national blood product supply. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v106.11.2330.2330 ◽

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.

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Drugs for preventing red blood cell dehydration in people with sickle cell disease

10.1002/14651858.cd003426 ◽

2002 ◽

Cited By ~ 13

Author(s):

C Riddington ◽

L De Franceschi

Keyword(s):

Sickle Cell Disease ◽

Blood Cell ◽

Sickle Cell ◽

Red Blood Cell ◽

Cell Disease ◽

Cell Dehydration

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Effects of Genotypes and Treatment on Oxygenscan Parameters in Sickle Cell Disease

Cells ◽

10.3390/cells10040811 ◽

2021 ◽

Vol 10 (4) ◽

pp. 811

Author(s):

Camille Boisson ◽

Minke A. E. Rab ◽

Elie Nader ◽

Céline Renoux ◽

Celeste Kanne ◽

...

Keyword(s):

Steady State ◽

Sickle Cell Disease ◽

Blood Cell ◽

Sickle Cell ◽

Red Blood Cell ◽

Cell Disease ◽

Oxygen Gradient ◽

Acute Complication ◽

Adults And Children ◽

The Difference

(1) Background: The aim of the present study was to compare oxygen gradient ektacytometry parameters between sickle cell patients of different genotypes (SS, SC, and S/β+) or under different treatments (hydroxyurea or chronic red blood cell exchange). (2) Methods: Oxygen gradient ektacytometry was performed in 167 adults and children at steady state. In addition, five SS patients had oxygenscan measurements at steady state and during an acute complication requiring hospitalization. (3) Results: Red blood cell (RBC) deformability upon deoxygenation (EImin) and in normoxia (EImax) was increased, and the susceptibility of RBC to sickle upon deoxygenation was decreased in SC patients when compared to untreated SS patients older than 5 years old. SS patients under chronic red blood cell exchange had higher EImin and EImax and lower susceptibility of RBC to sickle upon deoxygenation compared to untreated SS patients, SS patients younger than 5 years old, and hydroxyurea-treated SS and SC patients. The susceptibility of RBC to sickle upon deoxygenation was increased in the five SS patients during acute complication compared to steady state, although the difference between steady state and acute complication was variable from one patient to another. (4) Conclusions: The present study demonstrates that oxygen gradient ektacytometry parameters are affected by sickle cell disease (SCD) genotype and treatment.

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Microfluidic electrical impedance assessment of red blood cell mediated microvascular occlusion

Lab on a Chip ◽

10.1039/d0lc01133a ◽

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...

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