Association of haemolysis markers, blood viscosity and microcirculation function with organ damage in sickle cell disease in sub-Saharan Africa (the BIOCADRE study).

Sickle cell anaemia (SCA) is a monogenic disease with a highly variable clinical course. We aimed to investigate associations between microvascular function, haemolysis markers, blood viscosity and various types of SCA-related organ damage in a multicentric sub-Saharan African cohort of patients with SCA. In a cross-sectional study, we selected seven groups of adult patients with SS phenotype in Dakar and Bamako based on the following complications: leg ulcer, priapism, osteonecrosis, retinopathy, high tricuspid regurgitant jet velocity (TRV), macro-albuminuria or none. Clinical assessment, echocardiography, peripheral arterial tonometry, laboratory tests and blood viscosity measurement were performed. We explored statistical associations between the biological parameters and the six studied complications. Among 235 patients, 58 had high TRV, 46 osteonecrosis, 43 priapism, 33 leg ulcers, 31 retinopathy and 22 macroalbuminuria, whereas 36 had none of these complications. Multiple correspondence analysis revealed no cluster of complications. Lactate dehydrogenase levels were associated with high TRV, and blood viscosity was associated with retinopathy and the absence of macroalbuminuria. Despite extensive phenotyping of patients, no specific pattern of SCA-related complications was identified. New biomarkers are needed to predict SCA clinical expression to adapt patient management, especially in Africa, where healthcare resources are scarce.

Erythrocyte type 1 equilibrative nucleoside transporter expression in sickle cell disease and sickle cell trait.

Hypoxia-mediated red blood cell (RBC) sickling is central to the pathophysiology of sickle cell disease (SCD). The signalling nucleoside adenosine is thought to play a significant role in this process. This study investigated expression of the erythrocyte type 1 equilibrative nucleoside transporter (ENT1), a key regulator of plasma adenosine, in adult patients with SCD and carriers of sickle cell trait (SCT). Relative quantitative expression analysis of erythrocyte ENT1 was carried out by Western blot and flow cytometry. Patients with SCD with steady state conditions, either with SS or SC genotype, untreated or under hydroxycarbamide (HC) treatment, exhibited a relatively high variability of erythrocyte ENT1, but with levels not significantly different from normal controls. Most strikingly, expression of erythrocyte ENT1 was found to be significantly decreased in patients with SCD undergoing painful vaso-occlusive episode and, unexpectedly, also in healthy SCT carriers. Promoting hypoxia-induced adenosine signalling, the reduced expression of erythrocyte ENT1 might contribute to the pathophysiology of SCD and to the susceptibility of SCT individuals to altitude hypoxia or exercise to exhaustion.

Renal allograft DARCness in subclinical acute and chronic active ABMR.

Gene expression profiling of renal allograft biopsies revealed the Duffy antigen receptor for chemokines (DARC) as being strikingly upregulated in antibody-mediated rejection (ABMR). DARC has previously been shown to be associated with endothelial injury. This study aimed at assessing the value of DARC immunohistochemistry as diagnostic marker in ABMR. The study was performed on 82 prospectively collected biopsies of a clinically well-defined population (BORTEJECT trial, NCT01873157) of DSA-positive patients with gene expression data available for all biopsies. Diagnostic histologic assessment of biopsies was performed according to the Banff diagnostic scheme. DARC expression was focally accentuated, on peritubular capillaries (PTC) mostly in areas of interstitial fibrosis and/or inflammation. DARC positivity was associated with diagnosis of ABMR and correlated with DARC gene expression levels detected by microarray analysis. Still, as previously described, a substantial number of biopsies without signs of rejection showed DARC-positive PTC. We did not observe significantly reduced graft survival in cases showing histologic signs of ABMR and being DARC-positive, as compared to DARC-negative ABMR. In summary, the upregulation of DARC, detected by immunohistochemistry, is associated with but not specific for ABMR. We did not observe reduced graft survival in DARC-positive patients.

Cell-derived microparticles and sickle cell disease chronic vasculopathy in sub-Saharan Africa: A multinational study.

Although most individuals with sickle cell disease (SCD) live in sub-Saharan Africa, the natural history of the disease on this continent remains largely unknown. Intravascular haemolysis results in activation of circulating blood cells and release of microparticles (MPs) that exert pro-inflammatory effects and contribute to vascular damage. We designed a case-control study nested in the CADRE cohort (Coeur-Artère-DRÉpanocytose, clinical trials.gov identifier NCTO3114137) and based on extreme phenotypes, to analyse blood cell-derived MPs in 232 adult SS patients at steady state in Bamako and Dakar. Thirty-six healthy adult controls matched by age and sex were recruited in Bamako. The MPs concentrations were higher in SS patients compared to AA controls with a predominance of erythrocyte- and reticulocyte-derived MPs. These erythroid-derived MPs were significantly lower in patients with retinopathy (P = 0·022). Reticulocyte-derived MPs were significantly negatively and positively associated with a history of priapism (P = 0·020) and leg ulcers (P = 0·041) respectively. We describe for the first time the comparative patterns of plasma MPs in healthy subjects and patients with SCD living in sub-Saharan Africa and exhibiting various complications. Because our present results show no clear pattern of correlation between erythroid MPs and the classical hyper-haemolytic complications, we hypothesise a weak relevance of the hyper-haemolysis versus hyper-viscous paradigm in Africa.

Prognostic factors of disease severity in infants with sickle cell anemia: A comprehensive longitudinal cohort study.

In order to identify very early prognostic factors that can provide insights into subsequent clinical complications, we performed a comprehensive longitudinal multi-center cohort study on 57 infants with sickle cell anemia (55 SS; 2 Sß°) during the first 2 years of life (ClinicalTrials.gov: NCT01207037). Time to first occurrence of a severe clinical event-acute splenic sequestration (ASS), vaso-occlusive (VOC) event requiring hospitalization, transfusion requirement, conditional/ abnormal cerebral velocities, or death-was used as a composite endpoint. Infants were recruited at a mean age of 4.4 ±1 months. Median follow-up was 19.4 months. During the study period, 38.6% of infants experienced ≥1 severe event: 14% ASS, 22.8% ≥ 1 VOC (median age: 13.4 and 12.8 months, respectively) and 33.3% required transfusion. Of note, 77% of the cohort was hospitalized, with febrile illness being the leading cause for admission. Univariate analysis of various biomarkers measured at enrollment showed that fetal hemoglobin (HbF) was the strongest prognostic factor of subsequent severe outcome. Other biomarkers measured at enrolment including absolute neutrophil or reticulocyte counts, expression of erythroid adhesion markers, % of dense red cells, cellular deformability or ϒ-globin genetic variants, failed to be associated with severe clinical outcome. Multivariate analysis demonstrated that higher Hb concentration and HbF level are two independent protective factors (adjusted HRs (95% CI) 0.27 (0.11-0.73) and 0.16 (0.06-0.43), respectively). These findings imply that early measurement of HbF and Hb levels can identify infants at high risk for subsequent severe complications, who might maximally benefit from early disease modifying treatments.

Involvement of hepcidin in iron metabolism dysregulation in Gaucher disease.

Gaucher disease (GD) is an inherited deficiency of glucocerebrosidase leading to accumulation of glucosylceramide in tissues such as the spleen, liver, and bone marrow. The resulting lipid-laden macrophages lead to the appearance of "Gaucher cells". Anemia associated with an unexplained hyperferritinemia is a frequent finding in GD, but whether this pathogenesis is related to an iron metabolism disorder has remained unclear. To investigate this issue, we explored the iron status of a large cohort of 90 type I GD patients, including 66 patients treated with enzyme replacement therapy. Ten of the patients treated with enzyme replacement were followed up before and during treatment. Serum levels of hepcidin, the iron regulatory peptide, remained within the physiological range, while the transferrin saturation was slightly decreased in children. Inflammation-independent hyperferritinemia was found in 65% of the patients, and Perl's staining of the spleen and marrow smear revealed iron accumulation in Gaucher cells. Treated patients exhibited reduced hyperferritinemia, increased transferrin saturation and transiently increased systemic hepcidin. In addition, the hepcidin and ferritin correlation was markedly improved, and, in most patients, the hemoglobin level was normalized. To further explore eventual iron sequestration in macrophages, we produce a Gaucher cells model by treating the J774 macrophage cell line with a glucocerebrosidase inhibitor and showed induced local hepcidin and membrane retrieval of the iron exporter, ferroportin. These data reveal the involvement of Gaucher cells in abnormal iron sequestration, which may explain the mechanism of hyperferritinemia in GD patients. Local hepcidin-ferroportin interaction was involved in this pathogenesis.

Unexpected macrophage-independent dyserythropoiesis in Gaucher disease.

Gaucher disease is a rare inherited disease caused by a deficiency in glucocerebrosidase leading to lipid accumulation in cells of mononuclear-macrophage lineage known as Gaucher cells. Visceral enlargement, bone involvement, mild anemia and thrombocytopenia are the major manifestations of Gaucher disease. We have previously demonstrated that the red blood cells from patients exhibit abnormal properties, which indicates a new role in Gaucher disease pathophysiology. To investigate whether erythroid progenitors are affected, we examined the in vitro erythropoiesis from the peripheral CD34+ cells of patients and controls. CD34- cells were differentiated into macrophages and co-cultivated with erythroblasts. We showed an accelerated differentiation of erythroid progenitors without maturation arrest from patients compared to controls. This abnormal differentiation persisted in the patients when the same experiments were performed without macrophages, which strongly suggested that dyserythropoiesis in Gaucher disease is secondary to an inherent defect in the erythroid progenitors. The accelerated differentiation was associated with reduced cell proliferation. As a result, less mature erythroid cells were generated in vitro in the Gaucher disease cultures compared to the control. We then compared the biological characteristics of untreated patients according to their anemic status. Compared to the non-anemic group, the anemic patients exhibit higher plasma levels of growth differentiation factor-15, a marker of ineffective erythropoiesis, but they had no indicators of hemolysis and similar reticulocyte counts. Taken together, these results demonstrated an unsuspected dyserythropoiesis that was independent of the macrophages and could participate, at least in part, to the basis of anemia in Gaucher disease.

αII-spectrin regulates invadosome stability and extracellular matrix degradation.

Invadosomes are actin-rich adhesion structures involved in tissue invasion and extracellular matrix (ECM) remodelling. αII-Spectrin, an ubiquitous scaffolding component of the membrane skeleton and a partner of actin regulators (ABI1, VASP and WASL), accumulates highly and specifically in the invadosomes of multiple cell types, such as mouse embryonic fibroblasts (MEFs) expressing SrcY527F, the constitutively active form of Src or activated HMEC-1 endothelial cells. FRAP and live-imaging analysis revealed that αII-spectrin is a highly dynamic component of invadosomes as actin present in the structures core. Knockdown of αII-spectrin expression destabilizes invadosomes and reduces the ability of the remaining invadosomes to digest the ECM and to promote invasion. The ECM degradation defect observed in spectrin-depleted-cells is associated with highly dynamic and unstable invadosome rings. Moreover, FRAP measurement showed the specific involvement of αII-spectrin in the regulation of the mobile/immobile ß3-integrin ratio in invadosomes. Our findings suggest that spectrin could regulate invadosome function and maturation by modulating integrin mobility in the membrane, allowing the normal processes of adhesion, invasion and matrix degradation. Altogether, these data highlight a new function for spectrins in the stability of invadosomes and the coupling between actin regulation and ECM degradation.

Studies of a murine monoclonal antibody directed against DARC: reappraisal of its specificity.

Duffy Antigen Receptor for Chemokines (DARC) plays multiple roles in human health as a blood group antigen, a receptor for chemokines and the only known receptor for Plasmodium vivax merozoites. It is the target of the murine anti-Fy6 monoclonal antibody 2C3 which binds to the first extracellular domain (ECD1), but exact nature of the recognized epitope was a subject of contradictory reports. Here, using a set of complex experiments which include expression of DARC with amino acid substitutions within the Fy6 epitope in E. coli and K562 cells, ELISA, surface plasmon resonance (SPR) and flow cytometry, we have resolved discrepancies between previously published reports and show that the basic epitope recognized by 2C3 antibody is 22FEDVW26, with 22F and 26W being the most important residues. In addition, we demonstrated that 30Y plays an auxiliary role in binding, particularly when the residue is sulfated. The STD-NMR studies performed using 2C3-derived Fab and synthetic peptide corroborated most of these results, and together with the molecular modelling suggested that 25V is not involved in direct interactions with the antibody, but determines folding of the epitope backbone.

Energetic and molecular water permeation mechanisms of the human red blood cell urea transporter B.

Urea transporter B (UT-B) is a passive membrane channel that facilitates highly efficient permeation of urea. In red blood cells (RBC), while the major function of UT-B is to transport urea, it is assumed that this protein is able to conduct water. Here, we have revisited this last issue by studying RBCs and ghosts from human variants with defects of aquaporin 1 (AQP1) or UT-B. We found that UT-B's osmotic water unit permeability (pfunit) is similar to that of AQP1. The determination of diffusional permeability coefficient (Pd) allowed the calculation of the Pf/Pd ratio, which is consistent with a single-file water transport. Molecular dynamic simulations of water conduction through human UT-B confirmed the experimental finding. From these results, we propose an atomistic description of water-protein interactions involved in this permeation. Inside the UT-B pore, five water molecules were found to form a single-file and move rapidly along a channel by hydrogen bond exchange involving two critical threonines. We further show that the energy barrier for water located in the central region coincides with a water dipole reorientation, which can be related to the proton exclusion observed experimentally. In conclusion, our results indicate that UT-B should be considered as a new member of the water channel family.