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Sickle cell anaemia (SCA) results from a single mutation in the β globin gene. It is seldom symptomatic in the first semester of life. We analysed the expression pattern of 9 adhesion molecules on red blood cells, in a cohort of 54 SCA and 17 non-SCA very young infants of comparable age (median 144 days, 81–196). Haemoglobin F (HbF) level was unsurprisingly elevated in SCA infants (41.2% ± 11.2) and 2–4 fold higher than in non-SCA infants, yet SCA infants presented significantly decreased Hb level and increased reticulocytosis. Cytometry analysis evidenced a specific expression profile on reticulocytes of SCA infants, with notably an increased expression of the adhesion molecules Lu/BCAM, ICAM-4 and LFA-3, both in percentage of positive cells and in surface density. No significant difference was found on mature red cells. Our findings demonstrate the very early onset of reticulocyte membrane modifications in SCA asymptomatic infants and allow an insight into the first pathological changes with the release of stress reticulocytes expressing a distinctive profile of adhesion molecules., Highlights • Haemolytic anaemia is present in the first semester of life in asymptomatic infants with sickle cell anaemia. • Reticulocyte membrane modifications and stress reticulocytosis occur even when HbF production is still high. • Reticulocytes of SCA infants display a specific profile with notably an increased expression of the adhesion molecules Lu/BCAM, ICAM-4 and LFA-3.

Sickle cell disease results from a homozygous missense mutation in the ß-globin gene that causes polymerization of hemoglobin S. Gene therapy for patients with this disorder is complicated by the complex cellular abnormalities and challenges in achieving effective, persistent inhibition of polymerization of hemoglobin S. We describe our first patient treated with lentiviral vector-mediated addition of an antisickling ß-globin gene into autologous hematopoietic stem cells. Adverse events were consistent with busulfan conditioning. Fifteen months after treatment, the level of therapeutic antisickling ß-globin remained high (approximately 50% of ß-like-globin chains) without recurrence of sickle crises and with correction of the biologic hallmarks of the disease. (Funded by Bluebird Bio and others; HGB-205 ClinicalTrials.gov number, NCT02151526 .).

International audience; This paper presents a microfluidic system, characterizing the red blood cell (RBC) deformability through its transit time in a microchannel, which dimensions reproducing blood microcirculation capillary. Electrodes are integrated within the microchannel for electrical measurements. The transit time is measured with the electrical blockade produced when the RBC squeezes in the microchannel (d = 5 μm diameter, while the cell diameter is 8 μm). In order todetermine most suitable electrical frequency range for the voltage applied to excitation electrodes, FEM numerical simulation of the bioimpedance is achieved and presented in this paper. We investigated the frequency response of the bioimpedance taking into account the electrode polarization, the dielectric properties of the medium, a single shell model for RBC and finally the RBC position within the microchannel.

Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) characterised by the V617F activating mutation in the tyrosine kinase JAK2. PV patients exhibit increased haemoglobin levels and red cell mass because of uncontrolled proliferation of the erythroid lineage. Thrombosis and transformation to acute leukaemia are the major causes of morbidity and mortality in this disease. Increased thrombotic risk in PV patients is multifactorial and complex; it is associated with high levels of haemoglobin, impaired rheology and increased viscosity resulting from erythrocytosis. An additional parameter that might contribute to this risk was recently brought to light by work from our group showing abnormal activation of adhesion proteins in PV RBCs. In this review we provide an overview of these recent findings and discuss how the pro-adhesive features of JAK2V617F-positive red blood cells might initiate and contribute to the circulatory complications described in PV.

This review discusses the unexpected role of red blood cell (RBC) adhesiveness in the pathophysiology of two red cell diseases, hereditary spherocytosis and polycythemia vera, and two 'nonerythroid' disorders, central retinal vein occlusion and Gaucher disease. These pathologies share common clinical manifestations, that is vaso-occlusion and/or thrombotic events.Recently, the direct involvement of RBC adhesion to the vascular endothelium has been demonstrated in the occurrence of vaso-occlusive events, in particular in sickle cell disease (SCD). Several erythroid adhesion molecules and their ligands have been identified that belong to different molecular classes (integrins, Ig-like molecules, lipids...) and are activated by a variety of signaling pathways. Among these, the laminin receptor, Lutheran/basal cell adhesion molecule, which is activated by phosphorylation, appears to play a central role in several pathologies.RBC adhesiveness might be involved in complications such as the vaso-occlusive crisis in SCD, thrombosis in polycythemia vera, splenic sequestration in hereditary spherocytosis, occlusions in central retinal vein occlusion and bone infarcts in Gaucher disease. Characterization of this pathological process at the cellular and molecular levels should prove useful to develop new therapeutic approaches based on the blockade of RBC abnormal interactions with vascular endothelium and/or circulating blood cells.

Vaso-occlusive crises are the main acute complication in sickle cell disease. They are initiated by abnormal adhesion of circulating blood cells to vascular endothelium of the microcirculation. Several interactions involving an intricate network of adhesion molecules have been described between sickle red blood cells and the endothelial vascular wall. We have shown previously that young sickle reticulocytes adhere to resting endothelial cells through the interaction of α4β1 integrin with endothelial Lutheran/basal cell adhesion molecule (Lu/BCAM). In the present work, we investigated the functional impact of endothelial exposure to hydroxycarbamide (HC) on this interaction using transformed human bone marrow endothelial cells and primary human pulmonary microvascular endothelial cells. Adhesion of sickle reticulocytes to HC-treated endothelial cells was decreased despite the HC-derived increase of Lu/BCAM expression. This was associated with decreased phosphorylation of Lu/BCAM and up-regulation of the cAMP-specific phosphodiesterase 4A expression. Our study reveals a novel mechanism for HC in endothelial cells where it could modulate the function of membrane proteins through the regulation of phosphodiesterase expression and cAMP-dependent signaling pathways.

Background LentiGlobin gene therapy contains autologous CD34+ hematopoietic stem cells (HSCs) transduced with the BB305 lentiviral vector (LVV), encoding human β-globin with a T87Q substitution. This substitution confers anti-sickling properties to the gene therapy-derived hemoglobin (HbAT87Q) and allows for its quantification in transduced HSCs. The proof of concept for LentiGlobin gene therapy in patients with transfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD) was established in the recently completed HGB-205 study (NCT02151526). Herein, we provide the safety and efficacy outcomes and long-term follow-up data for all 7 treated patients, 4 with TDT and 3 with SCD. Methods Patients 5−35 years old with TDT (≥ 100 mL/kg of packed red blood cells [pRBCs]/year) or severe SCD (e.g., ≥ 2 acute chest syndromes [ACS] or ≥ 2 vaso-occlusive crises in the preceding year or the year before regular transfusions) were enrolled. CD34+ HSCs were obtained by mobilization and apheresis in patients with TDT or by bone marrow harvest in patients with SCD. Following collection, cells were transduced with the BB305 LVV. Patients underwent busulfan myeloablative conditioning and were infused with transduced cells. Patients were monitored for engraftment, adverse events (AEs), HbAT87Q levels, and other hematologic and clinical parameters. After 2 years in HGB-205, patients transitioned into the long-term follow-up study, LTF-303 (NCT02633943). Summary statistics are shown as median (min-max). Results As of June 2019, patients with TDT (n=4) and SCD (n=3) had a median follow-up of 49.6 (40.5-60.6) and 28.5 (25.5-52.5) months, respectively. Table 1 shows patient and drug product characteristics and several key efficacy outcomes. All patients achieved HSC engraftment. LentiGlobin safety profile was consistent with busulfan myeloablative conditioning and, in case of SCD, with the underlying disease state. The most common non-hematologic Grade ≥ 3 AEs post-LentiGlobin gene therapy (≥ 2 patients) for patients with TDT were stomatitis (n=4) and increased aspartate aminotransferase (n=2), and for patients with SCD were ACS (n=2) and vaso-occlusive pain (n=2). In all 4 patients with TDT, total Hb and HbAT87Q levels remained generally stable up to 5 years post-LentiGlobin infusion. Three of 4 patients achieved transfusion independence (TI; defined as weighted average Hb ≥ 9g/dL without pRBC transfusions for ≥ 12 months), for an ongoing duration of 56.3 (38.2-57.6) months. Weighted average total Hb during TI was 11.4 (10.5-13.0) g/dL. One patient has been off transfusions for 37.5 months and had total Hb of 7.7 g/dL, which was below the ≥ 9 g/dL requirement to meet the protocol definition of TI. At last visit, HbAT87Q levels in these 4 patients ranged from 6.2-11.2 g/dL, which contributed 73.8-86.8% of the total Hb. The first patient treated with LentiGlobin for SCD experienced one vaso-occlusive pain episode, which developed at 30 months after LentiGlobin gene therapy following a case of acute gastroenteritis with fever and dehydration. The second SCD patient had 2 serious AEs (SAEs) of ACS approximately 6 and 8 months after LentiGlobin gene therapy. The patient resumed chronic pRBC transfusions and hydroxyurea treatment and subsequently experienced 2 SAEs of vaso-occlusive pain; no additional SAEs of vaso-occlusive pain or ACS were reported during the last 16 months of follow-up after LentiGlobin infusion. The third SCD patient had no episodes of vaso-occlusive pain or ACS during 25.5 months of follow-up post-LentiGlobin gene therapy as of the data cut-off. Two patients with SCD who have been off chronic pRBC transfusions, showed improvement in hemolysis markers post-LentiGlobin treatment and stabilization of HbAT87Q expression at approximately 6 months post-LentiGlobin infusion. Total Hb levels for patients with SCD at last visit were 13.0 g/dL (patient 1), 9.4 g/dL (patient 2), and 9.8 g/dL (patient 3), with corresponding HbAT87Q contributions of 47.9%, 7.9%, and 25.8%, respectively. Summary With up to 5 years of follow-up, treatment with LentiGlobin gene therapy was well tolerated and resulted in improvement in hematologic parameters and disease-related symptoms. Further results from the completed study will be presented. Disclosures Hermine: Celgene: Research Funding; Novartis: Research Funding; AB science: Consultancy, Equity Ownership, Honoraria, Research Funding. Brousse:bluebird bio, Inc: Consultancy; AddMedica: Consultancy. El Nemer:Hemanext: Other: Other. Bartolucci:Novartis: Membership on an entity's Board of Directors or advisory committees; AddMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; HEMANEXT: Membership on an entity's Board of Directors or advisory committees. Asmal:bluebird bio, Inc: Employment, Equity Ownership. Whitney:bluebird bio, Inc: Employment, Equity Ownership. Gayron:bluebird bio, Inc: Employment, Equity Ownership. Huang:bluebird bio, Inc.: Employment, Equity Ownership. de Montalembert:AddMedica: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio, Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Ribeil:bluebird bio, Inc: Employment, Equity Ownership. Cavazzana:SmartImmune: Other: Founder.

Sickle cell disease (SCD) is a severe, life-threatening disorder caused by a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) β-chain that causes Hb polymerization with consequent red blood cell (RBC) rigidity, anemia, vaso-occlusive crises, organ damage and reduced life expectancy. The co-inheritance of genetic mutations causing a sustained fetal γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin, HPFH) decreases sickling and significantly reduces the clinical severity of SCD. HPFH mutations cluster at several loci in the promoters of the two γ-globin genes, HBG1 and HBG2, and, disrupt binding sites for transcriptional repressors (e.g., BCL11A and LRF), leading to elevated levels of fetal hemoglobin (HbF) representing up to 40% of total hemoglobin tetramers. In addition, SNPs at position -158 of both γ-globin promoters are associated with enhanced γ-globin expression and may identify a putative transcriptional repressor binding site. Here, we used the CRISPR/Cas9 system to mimic HPFH mutations in the HBG promoters by generating insertion and deletions (InDels) leading to disruption of known and putative binding sites for transcriptional repressors via non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) DNA repair mechanisms. Efficient editing of the LRF binding site (≥ 3 γ-globin promoters/cell in >70% of cells) in SCD patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in a robust, virtually pancellular HbF reactivation and a concomitant reduction in βS-globin levels, recapitulating the phenotype of asymptomatic SCD-HPFH patients. Of note, in RBCs derived from edited HSPCs, HbF levels exceeded 40% of total Hb tetramers, suggesting that CRISPR/Cas9 mediated disruption of the LRF binding site is even more potent than naturally occurring HPFH point mutations in reactivating HbF expression. RBCs derived from edited HSPCs displayed HbF levels sufficient to substantially correct the SCD cell phenotype (~80% of non-sickling RBCs). Although larger deletions (in part generated likely by MMEJ) disrupt more efficiently the LRF binding site, shorter InDels mainly produced by NHEJ, the most active repair mechanism in bona fide hematopoietic stem cells (HSCs), were still able to induce a potent γ-globin reactivation and correct the SCD cell phenotype. Interestingly, upon targeting of the -158 site, we observed HBG de-repression, which, however, was mild, indicating that this region contains a sequence that moderately inhibits γ-globin expression in adult cells, consistently with the mild increase in HbF associated with the -158 SNP. To evaluate the efficiency and the safety of this approach in long-term repopulating HSCs, untreated and edited HSPCs were injected into NSG immunodeficient mice. 16 weeks after transplantation, the engraftment of control and HBG-edited cells was comparable, as analyzed in bone marrow, spleen, thymus and blood. Edited HSCs were capable to differentiate into multiple hematopoietic cell types, with no skewing towards any particular lineage. Importantly, xenotransplantation of HSPCs edited using the gRNAs targeting the LRF binding site demonstrated a high editing efficiency in long-term repopulating HSCs (up to 75%) and a modest reduction in the frequency of deletions likely generated by MMEJ. Overall, this study identifies the binding site for the LRF repressor as a novel, potent and safe target for a genome editing-based treatment of SCD. Disclosures El Nemer: Hemanext: Other: Other. Cavazzana:SmartImmune: Other: Founder.

Polycythemia vera (PV) is characterized by an increased RBC mass, spontaneous erythroid colony formation, and the JAK2V617F mutation. PV is associated with a high risk of mesenteric and cerebral thrombosis. PV RBC adhesion to endothelial laminin is increased and mediated by phosphorylated erythroid Lu/BCAM. In the present work, we investigated the mechanism responsible for Lu/BCAM phosphorylation in the presence of JAK2V617F using HEL and BaF3 cell lines as well as RBCs from patients with PV. High levels of Rap1-GTP were found in HEL and BaF3 cells expressing JAK2V617F compared with BaF3 cells with wild-type JAK2. This finding was associated with increased Akt activity, Lu/BCAM phosphorylation, and cell adhesion to laminin that were inhibited by the dominant-negative Rap1S17N or by the specific Rap1 inhibitor GGTI-298. Surprisingly, knocking-down EpoR in HEL cells did not alter Akt activity or cell adhesion to laminin. Our findings reveal a novel EpoR-independent Rap1/Akt signaling pathway that is activated by JAK2V617F in circulating PV RBCs and responsible for Lu/BCAM activation. This new characteristic of JAK2V617F could play a critical role in initiating abnormal interactions among circulating and endothelial cells in patients with PV.

Vaso-occlusive crisis (VOC) is the main acute complication in sickle cell anaemia (SS) and several clinical trials are investigating different drugs to improve the clinical severity of SS patients. A phase III study is currently exploring the profit of Velopoloxamer in SS during VOCs. We analysed, in-vitro, the effect of poloxamer (P188) on red blood cell (RBC) properties by investigating haemorheology, mechanical and adhesion functions using ektacytometry, microfluidics and dynamic adhesion approaches, respectively. We show that poloxamer significantly reduces blood viscosity, RBC aggregation and adhesion to endothelial cells, supporting the beneficial use of this molecule in SS therapy.

In sickle cell anemia, reticulocytes express enhanced levels of α4β1 integrin that interact mainly with vascular cell adhesion molecule-1 and fibronectin, promoting vaso-occlusion. These interactions are known to be highly sensitive to the inflammatory chemokine IL-8. The Duffy antigen receptor for chemokines (DARC) modulates the function of inflammatory processes. However, the link between α4β1 activation by chemokines and DARC erythroid expression is not or poorly explored. Therefore, the capacity of α4β1 to mediate Duffy-negative and Duffy-positive sickle reticulocyte (SRe) adhesion to immobilized vascular cell adhesion molecule-1 and fibronectin was evaluated. Using static adhesion assays, we found that, under basal conditions, Duffy-positive SRe adhesion was 2-fold higher than that of Duffy-negative SRes. Incubating the cells with IL-8 or RANTES (regulated on activation normal T cell expressed and secreted) increased Duffy-positive SRe adhesion only, whereas Mn(2+) increased cell adhesion independently of the Duffy phenotype. Flow cytometry analyses performed with anti-β1 and anti-α4 antibodies, including a conformation-sensitive one, in the presence or absence of IL-8, revealed that Duffy-positive and Duffy-negative SRes displayed similar erythroid α4β1 expression levels, but with distinct activation states. IL-8 did not affect α4β1 affinity in Duffy-positive SRes but induced its clustering as corroborated by immunofluorescence microscopy. Our results indicate that in Duffy-negative SRes α4β1 integrin is constitutively expressed in a low affinity state, whereas in Duffy-positive SRes α4β1 is expressed in a higher chemokine-sensitive affinity state. This activation state associated with DARC RBC expression may influence the intensity of the inflammatory responses encountered in sickle cell anemia and participate in its interindividual clinical expression variability.

Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder characterized by chronic hemolytic anemia in association with acute and chronic life-threatening complications mainly related to acute vaso-occlusive events (VOCs) due to amplified inflammatory response and defective pro-resolving events. Imatinib is an oral Tyrosine (Tyr)-kinase inhibitor, developed for the treatment of chronic myeloid leukemia (CML). Few case reports on SCD patients with CML undergoing imatinib treatment highlight the beneficial impact of imatinib on severity and recurrence of acute VOCs in SCD. In red blood cells (RBCs), Imatinib has been shown to interfere with Tyr-phosphorylation state of the integral membrane protein band 3, affecting RBC microparticle formation. Here, we study the actions of Imatinib on a model of acute VOCs using humanized SCD mice (Hbatm1(HBA)TowHbbtm2(HBG1,HBB*)Tow). We treated SCD and control healthy mice (AA, Hbatm1(HBA)Tow Hbbtm3(HBG1,HBB)Tow) (n=6-7 animals in each group) with Imatinib 50 mg/Kg/d for 2 weeks before hypoxia/Reoxygenation (H/R) stress used to mimic acute VOCs. Under normoxia, we found that in SCD mice Imatinib significantly reduced (i) Tyr-phosphorylation state of sickle red cell membrane proteins; (ii) the amount of phosphatidyl-serine (PS)+ sickle RBCs; and (iii) the release of erythroid microparticles, which was associated with accumulation of hemichromes and a more efficient erythrophagocytosis compared to vehicle treated animals. In SCD mice exposed to H/R stress, imatinib significantly decreased the H/R-induced (i) increase in peripheral neutrophil count; (ii) lung inflammatory cell infiltrate; (iii) kidney inflammatory cell infiltrate. In the lung of H/R SCD mice, Imatinib inhibited the H/R induced NF-kB activation and prevented the up-regulation of (i) pro-inflammatory cytokines such as ET-1; (ii) vascular endothelial activation markers (VCAM-1, ICAM-1); (iii) pro-fibrotic markers such as PDGF-B and (iv) the activation of UPR system. In the kidney of H/R SCD mice, Imatinib significantly reduced H/R induced expression of ET-1, VCAM-1 and E-selectin, which were again associated with inhibition of NF-kB. In addition, Imatinib significantly upregulated the expression of the microRNAs miR200a/b, which has been described to reduce renal fibrosis. Finally, in isolated aorta from Imatinib treated SCD exposed to H/R stress, a significant reduction in vascular activation markers was observed compared to vehicle treated animals. Collectively, our data indicate that Imatinib acts on multiple targets, modulating signal transduction and reducing inflammatory vasculopathy and extracellular matrix remodeling process related to VOC in SCD. Thus, Imatinib might represent a new therapeutic tool in clinical management of SCD patients. Disclosures El Nemer: Imara: Research Funding.

Introduction: Gene therapy is a highly promising therapeutic strategy in sickle cell disease (SCD). The Phase 1/2 HGB-205 (NCT02151526) clinical study in France is evaluating the safety and efficacy of LentiGlobin gene therapy, which consists of autologous CD34+ cells transduced with a lentiviral vector encoding a human β-globin gene with a point mutation (T87Q) that confers anti-sickling properties. Data from the first successfully treated patient have been published (Ribeil et al, 2017 NEJM). In order to establish the effect of βAT87Q-globin production on red blood cell properties, we have analyzed membrane properties, hemolysis markers, morphology, hemoglobin content, and the extent of HbS polymerization. Methods: Whole blood samples were obtained from 3 patients with SCD (1204, 1207 and 1208) treated in HGB-205 during their clinical follow-up. HbS polymerization level was assessed by O2 dissociation and association curves and cell morphology. Membrane properties were evaluated by RBC density curves in phthalate gradient, deformability under increasing osmolality (LORRCA) and level of adherence to surfaces coated with thrombospondin (TSP), under increasing shear stress (from 0.5 to 5 dynes/cm²). Hemolytic level was determined by measurement of classical markers (LDH, bilirubin and haptoglobin). Hemoglobin contents of total RBCs and reticulocytes (CD71-positive cells sorted) were assessed by reverse-phase HPLC. Results were compared against untreated βSβS patients (n=11 for deformability assay, n=4 for adhesion assay) and healthy donors (n=10 for deformability assay, n=3 for adhesion assay). Results: As of May 29 2018, follow-up, total Hb and HbAT87Q contribution to total Hb for patients 1204, 1207 and 1208 were: 42, 18 and 15 months, 12.2, 8.4, and 10.4 g/dL, and 49.44, 7.77 and 26.99%, respectively. At approximately 30 months post-infusion patient 1204 developed vaso-occlusive pain following an episode of acute gastroenteritis, since then the patient has not had any vaso-occlusive episodes or acute chest syndrome (ACS). Patient 1207 had 2 episodes of ACS approximately 6 and 8 months after LentiGlobin gene therapy and has since been on chronic transfusions and hydroxyurea treatment; the patient subsequently experienced 1 vaso-occlusive pain episode. Patient 1208 has had no episodes of VOCs or ACS post LentiGlobin gene therapy. Dissociation and association of O2 curves for RBCs isolated from the 2 patients free of chronic transfusions (1204 and 1208) and performed 36 and 8 months post infusion, respectively, showed only a slight increase in P50 during re-oxygenation, indicating anti-sickling capability of transgenic HbAT87Q and low levels of HbS polymerization. Density curves showed an overall normal RBC hydration at multiple time points during follow-up, with dense cells contributing 0-4% compared to a mean (±SD) of 12.8% (±7.8) in untreated patients. The deformability of RBCs from the 2 patients (1204 and 1208) evaluated in HGB-205 study was lower than observed for healthy donors but higher than for untreated SCD patients. Under controlled shear stress, TSP adherence was consistently lower for RBCs isolated from the 2 patients (1204 and 1208) in HGB-205 compared to untreated patients with SCD. Slight intravascular hemolysis was observed for the 3 HGB-205 patients during follow-up, but the hemolytic levels improved compared to baseline. RP-HPLC analysis of total RBCs isolated at last visit showed an increase in βAT87Q and a decrease in βS in comparison to reticulocytes, indicating an improved survival of RBCs expressing more anti-sickling β-globin transgene (Table 1). Data on deformability, distribution of fetal Hb and additional adhesion markers will be presented. Conclusions: Our results suggest an improvement in RBC properties for 2 of 3 patients with SCD treated with LentiGlobin gene therapy in the HGB-205 clinical trial compared to non-treated patients with SCD, suggesting a promising potential of this treatment. Disclosures El Nemer: Imara: Research Funding. Negre:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Ribeil:Vitalaire: Research Funding; Bluebird Bio, inc.: Employment. Bartolucci:Addmedica: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees; Fondation Fabre: Research Funding; Novartis US: Membership on an entity's Board of Directors or advisory committees.

International audience; BACKGROUND: Abnormal interactions between red blood cells, leukocytes and endothelial cells play a critical role in the occurrence of the painful vaso-occlusive crises associated with sickle cell disease. We investigated the interaction between circulating leukocytes and red blood cells which could lead to aggregate formation, enhancing the incidence of vaso-occlusive crises. DESIGN AND METHODS: Blood samples from patients with sickle cell disease (n=25) and healthy subjects (n=5) were analyzed by imaging and classical flow cytometry after density gradient separation. The identity of the cells in the peripheral blood mononuclear cell layer was determined using antibodies directed specifically against white (anti-CD45) or red (anti-glycophorin A) blood cells. RESULTS: Aggregates between red blood cells and peripheral blood mononuclear cells were visualized in whole blood from patients with sickle cell disease. The aggregation rate was 10-fold higher in these patients than in control subjects. Both mature red blood cells and reticulocytes were involved in these aggregates through their interaction with mononuclear cells, mainly with monocytes. The size of the aggregates was variable, with one mononuclear cell binding to one, two or several red blood cells. Erythroid Lu/basal cell adhesion molecule and α(4)β(1) integrin were involved in aggregate formation. The aggregation rate was lower in patients treated with hydroxycarbamide than in untreated patients. CONCLUSIONS: Our study gives visual evidence of the existence of circulating red blood cell-peripheral blood mononuclear cell aggregates in patients with sickle cell disease and shows that these aggregates are decreased during hydroxycarbamide treatment. Our results strongly suggest that erythroid Lu/basal cell adhesion molecule proteins are implicated in these aggregates through their interaction with α(4)β(1) integrin on peripheral blood mononuclear cells.

Lu/BCAM, the unique erythroid receptor for laminin 511/521, interacts with the erythrocyte membrane skeleton through spectrin binding. It has been reported that Hereditary Spherocytosis red blood cells (HS RBC) exhibit increased adhesion to laminin. We investigated the role of Lu/BCAM-spectrin interaction in the RBC adhesion properties of 2 splenectomised HS patients characterized by 40% spectrin deficiency. Under physiological flow conditions, HS RBC exhibited an exaggerated adhesion to laminin that was completely abolished by soluble Lu/BCAM. Triton extraction experiments revealed that a greater fraction of Lu/BCAM was unlinked to the membrane skeleton of HS RBC, as compared to normal RBC. Disruption of the spectrin interaction site in Lu/BCAM expressed in the transfected K562 cell line resulted in a weakened interaction to the skeleton and an enhanced interaction to laminin. These results demonstrated that the adhesion of HS RBC to laminin was mediated by Lu/BCAM and that its interaction with the spectrin-based skeleton negatively regulated cell adhesion to laminin. Finally, the results of this study strongly suggest that the reinforced adhesiveness of spectrin-deficient HS RBC to laminin is partly brought about by an impaired interaction between Lu/BCAM and the membrane skeleton.

KLF1 is an essential erythroid transcription factor (EKLF) involved both in the ß-globin switch from fetal to adult and in definitive erythropoiesis. KLF1 also activates genes encoding heme biosynthetic enzymes, as well as genes involved in the cell cycle and the synthesis of membrane proteins. Heterozygosity for one or several nucleotide changes in KLF1 may be responsible for the so-called dominant Lu(a-b-) phenotype, also known as In(Lu) for "Inhibitor Lutheran" and characterized by a dramatic reduction in the expression levels of the Lutheran blood group antigens and a slight elevation in hemoglobin F (HbF). Several other blood group antigens are also under the direct control of KLF1, but their number and expression level in In(Lu) red blood cells (RBCs) remain a matter of debate. In addition, mutations in KLF1 in humans lead to anemia, acantocytosis and some of these variants, such as CDA type IV, are characterized by ineffective terminal differentiation and defects in enucleation. Given the major role of KLF1 in erythropoiesis, it would not be surprising that In(Lu) RBCs show abnormal properties. In this work, we performed proteomic studies to quantify membrane proteins in In(Lu) vs control RBCs. Results: using label-free mass spectrometry, we analyzed the expression levels of membrane proteins in 5 In(Lu) and 4 control RBCs. Hierarchical clustering allowed to identify two modulation profiles of protein expression (Figure 1). The first profile (red color) is composed of 49 proteins over-expressed in In(Lu) RBCs, with the majority of them corresponding to the "26S proteasome complex" (PSMA, PSMB, PSMD, and PSME). In addition, we confirmed the overexpression of the 26S proteasome complex in In(Lu) RBCs by western blot analyses. The second profile (green color) includes 23 membrane proteins with a lower expression level in In(Lu) RBCs; these proteins correspond to blood group antigens (e.g. Lu/BCAM, CD44), and to cytoskeletal proteins (e.g. dematin, flotillin). Five proteins carrying blood group systems show a decrease of expression in In(Lu) RBCs over 40%: Chido/Rodgers, Xg, Indian, Duffy and Lutheran: antigens. While our results indicating of decreased expression of Indian and Chido/Rodgers systems are consistent with previous reports, the Duffy expression was unexpectedly found to be decreased by 86%. Since we evidenced an important increase in the proteasome complex, and taking into account that KLF1 is involved in the regulation or erythropoiesis, we, investigated the expression profile of the 26 S proteasome expression during erythropoiesis. We observed an important decrease of all proteins of the proteasome complex during erythropoiesis. Conclusion: from our preliminary results reported in this study, we hypothesized that the increase of the 26S proteasome in the In(Lu) individuals may result from defects in organelle loss and could explain the profound dyserytropoiesis observed in the "nan" mice null for KLF1. Figure 1: Comparative proteomics of red blood cell membrane proteins in In(Lu) blood group type. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background and hypothesis: Infancy is a critical time during which the first complications of sickle cell anemia (SCA) emerge. Very early prognostic factors of severity are needed to select high-risk children to offer precisely tailored therapy. Our hypothesis was that clinical, biological or genetic parameters measurable soon after birth (3 to 6 months of age) could predict severe outcomes in the first two years of life in SCA infants. Methods: Infants with SCA were offered to participate in a prospective study (ClinicalTrials.gov: NCT01207037) that included clinical and laboratory assessment at enrollment (3-6 months) and at 12, 18 and 24 months of age. The prognostic performance of conventional and SCA-specific biomarkers (notably erythroid adhesion markers, dense cells, ektacytometry indices) on disease severity was assessed using Cox's proportional hazard regression models. The disease severity was defined as time to first occurrence of either acute splenic sequestration (ASS), vaso occlusive (VOC) event requiring hospitalization, transfusion, conditional or abnormal Trans Cranial Doppler, acute chest syndrome and death. Hazard ratios (HRs) were calculated with 95% Cis. Results: Fifty-seven infants (55 SS; 2 Sβ°; 54.4% males), diagnosed through neonatal screening, were included in the study at a mean age of 4.4 ± 1 months and longitudinally assessed with a median follow-up of 19.4 months (range 3.1-23.2). Only 1 of 57 infants had experienced a SCA-related event prior to enrolment (dactilytis). At inclusion, clinical examinations were unremarkable except for pallor found in 15 (26,3%) and none of the infants had an enlarged or palpable spleen. Growth parameters were normal. Main biological characteristics were a mild hemolytic anemia (Hb 9.3 ±1.3 g/dL; reticulocytes count: 151.2 ±76 x 109/L), increased HbF level (3.8 ±1.3 g/dL. or 41.4 ±11.7%) and presence of dense red cells (23.1 % ±10.8). Genetic analysis showed one alpha globin deletion in 18 (32.1%) and a balanced distribution of beta globin haplotypes: 16 (30.8%), 18 (34.6%) and 18 (34.6%) in the favorable (SEN/SEN, BEN/SEN, CAM/SEN or SEN/ATYP), unfavorable (CAR/CAR, CAM/CAR, CAR/ATYP or BEN/CAR) and intermediate category (other) respectively. During the 2 years duration of the study, 44 of 57 (77%) infants required 157 hospital admissions, median (range): 2 (1-12) per patient. Infection was a leading cause of hospitalization although no serious adverse event related to pneumococcal infection was noted. Eight (14%) children experienced an episode of ASS at a median (range) age of 13.4 months (7.8- 15.9) while 13 infants (22.8%) experienced at least one VOC event at a median age of 12.7 months (7-22.5), with 6 experiencing ≥ 2 episodes. Nineteen infants (33.3%) required at least one transfusion with 10 (17.6%) requiring more than 2. Altogether, 22 (38.6%) infants of this cohort experienced a SCA-related severe clinical event by 24 months of age. The Kaplan Meier estimate of the 24-month event-free rate was 54.4% (95% CI, 39.7 to 74.5%) (Figure). Univariate analysis of potential prognostic markers at inclusion showed that higher HbF % and concentration were the strongest protective parameters for ASS in particular and for all severe outcomes, except for VOC (p < 0.001). Unfavorable haplotypes were also associated with severe outcome (HR (95% CI) 4.73 (1.31-17.01), p=0.017). Protective factors for VOC were higher Hb level (threshold > 8 g/dL) and low % of circulating dense cells (p=0.04 and 0.02, respectively). In a multivariate analysis, Hb level ≥ 8 g/dL and HbF ≥ 2.8 g/dL proved to be two independent prognostic factors of a SCA-related severe event (adjusted HRs (95% CI) 0.27 (0.11-0.73) and 0.16 (0.06-0.43) respectively). Interestingly, absolute neutrophil or reticulocyte counts, level of expression of known potentially pathogenic erythroid adhesion markers (CD36, Lu/B-CAM, ICAM-4/LW), % of red dense cell, or deformability parameters failed to be prognostic factors of specific complications or overall severity in this very young cohort. Conclusion: HbF and Hb levels measured between 3 and 6 months of age in SCA infants predict the risk of subsequent severe clinical outcome in the next 2 years. These events are frequent even in a high-income setting where neonatal screening is implemented. These findings further support the early use of HbF inducers such as hydroxyurea in high-risk infants to sustain a protective HbF level. Figure 1 Figure 1. Disclosures Brousse: Add Medica: Membership on an entity's Board of Directors or advisory committees. De Montalembert: Novartis: Consultancy, Honoraria, Research Funding; Addmedica: Consultancy, Honoraria, Research Funding.

Plasmodium falciparum is transmitted from humans to Anopheles mosquito vectors via the sexual erythrocytic forms termed gametocytes. Erythrocyte filtration through microsphere layers (microsphiltration) had shown that circulating gametocytes are deformable. Compounds reducing gametocyte deformability would induce their splenic clearance, thus removing them from the blood circulation and blocking malaria transmission. The hand-made, single-sample prototype for microsphiltration was miniaturized to a 96-well microtiter plate format, and gametocyte retention in the microsphere filters was quantified by high-content imaging. The stiffening activity of 40 pharmacological compounds was assessed in microtiter plates, using a small molecule (calyculin) as a positive control. The stiffening activity of calyculin was assessed in spleen-mimetic microfluidic chips and in macrophage-depleted mice. Marked mechanical retention (80% to 90%) of mature gametocytes was obtained in microplates following exposure to calyculin at concentrations with no effect on parasite viability. Of the 40 compounds tested, including 20 antimalarials, only 5 endoperoxides significantly increased gametocyte retention (1.5- to 2.5-fold; 24 h of exposure at 1 μM). Mature gametocytes exposed to calyculin accumulated in microfluidic chips and were cleared from the circulation of macrophage-depleted mice as rapidly as heat-stiffened erythrocytes, thus confirming results obtained using the microsphiltration assay. An automated miniaturized approach to select compounds for their gametocyte-stiffening effect has been established. Stiffening induces gametocyte clearance both in vitro and in vivo . Based on physiologically validated tools, this screening cascade can identify novel compounds and uncover new targets to block malaria transmission. Innovative applications in hematology are also envisioned.

The Lutheran (Lu) blood group and basal cell adhesion molecule (BCAM) antigens are both carried by 2 glycoprotein isoforms of the immunoglobulin superfamily representing receptors for the laminin alpha(5) chain. In addition to red blood cells, Lu/BCAM proteins are highly expressed in endothelial cells. Abnormal adhesion of red blood cells to the endothelium could potentially contribute to the vaso-occlusive episodes in sickle cell disease. Considering the presence of integrin consensus-binding sites in Lu/BCAM proteins, we investigated their potential interaction with integrin alpha(4)beta(1), the unique integrin expressed on immature circulating sickle red cells. Using cell adhesion assays under static and flow conditions, we demonstrated that integrin alpha(4)beta(1) expressed on transfected cells bound to chimeric Lu-Fc protein. We showed that epinephrine-stimulated sickle cells, but not control red cells, adhered to Lu-Fc via integrin alpha(4)beta(1) under flow conditions. Antibody-mediated activation of integrin alpha(4)beta(1) induced adhesion of sickle red cells to primary human umbilical vein endothelial cells; this adhesion was inhibited by soluble Lu-Fc and vascular cell adhesion molecule-1 (VCAM-1)-Fc proteins. This novel interaction between integrin alpha(4)beta(1) in sickle red cells and endothelial Lu/BCAM proteins could participate in sickle cell adhesion to endothelium and potentially play a role in vaso-occlusive episodes.

Red blood cells (RBCs) are deformable and flow through vessels narrower than their own size. Their deformability is most stringently challenged when they cross micrometer-wide slits in the spleen. In several inherited or acquired RBC disorders, blockade of small vessels by stiff RBCs can trigger organ damage, but a functional spleen is expected to clear these abnormal RBCs from the circulation before they induce such complications. We analyzed flow behavior of RBCs in a microfluidic chip that replicates the mechanical constraints imposed on RBCs as they cross the human spleen. Polymer microchannels obtained by soft lithography with a hydraulic diameter of 25 μm drove flow into mechanical filtering units where RBCs flew either slowly through 5- to 2-μm-wide slits or rapidly along 10-μm-wide channels, these parallel paths mimicking the splenic microcirculation. Stiff heated RBCs accumulated in narrow slits seven times more frequently than normal RBCs infused simultaneously. Stage-dependent retention of Plasmodium falciparum-infected RBCs was also observed in these slits. We also analyzed RBCs from patients with hereditary spherocytosis and observed retention for those having the most altered mechanical properties as determined by ektacytometry. Thus, in keeping with previous observations in vivo and ex vivo, the chip successfully discriminated poorly deformable RBCs based on their distinct mechanical properties and on the intensity of the cell alteration. Applications to the exploration of the pathogenesis of malaria, hereditary spherocytosis, sickle cell disease and other RBC disorders are envisioned.

Lutheran (Lu) blood group and basal cell adhesion molecule (B-CAM) antigens reside on two glycoprotein (gp) isoforms Lu and Lu(v13) that belong to the Ig superfamily and differ only by the size of their cytoplasmic tail. Lu/B-CAM gps have been recognized as laminin alpha5 receptors on red blood cells and epithelial cells in multiple tissues. It has been shown that sickle red cells exhibit enhanced adhesion to laminin alpha5 when intracellular cAMP is up-regulated by physiological stimuli such as epinephrine and that this signaling pathway is protein kinase A- and Lu/B-CAM-dependent. In this study, we analyzed the relationship between the phosphorylation status of Lu/B-CAM gps and their adhesion function to laminin alpha5. We showed that Lu isoform was phosphorylated in sickle red cells as well as in erythroleukemic K562 and epithelial Madin-Darby canine kidney cells and that this phosphorylation is enhanced by different stimuli of the PKA pathway. Lu gp is phosphorylated by glycogen synthase kinase 3 beta, casein kinase II, and PKA at serines 596, 598, and 621, respectively. Alanine substitutions of serines 596 and 598 abolished phosphorylation by glycogen synthase kinase 3 beta and casein kinase II, respectively, but had no effect on adhesion of K562 cells to laminin under flow conditions. Conversely, mutation of serine 621 prevented phosphorylation by PKA and dramatically reduced cell adhesion. Furthermore, stimulation of K562 cells by epinephrine increased Lu gp phosphorylation by PKA and enhanced adhesion to laminin. It is postulated that modulation of the phosphorylation state of Lu gp might be a critical factor for the sickle red cells adhesiveness to laminin alpha5 in sickle cell disease.

Lutheran (Lu) and Lu(v13), two glycoprotein (gp) isoforms belonging to the immunoglobulin superfamily, represent adhesion molecules that act as erythrocyte receptors for laminin 10/11. These two gps, which differ only by the length of their cytoplasmic tail, carry both Lu blood group and Basal Cell Adhesion Molecule (B-CAM) antigens. Here, analysis of the Triton extractability of recombinant Lu and Lu(v13) gps in K562 transfected cells showed that both gps were mainly associated with the detergent-insoluble material. Patching experiments using Cholera Toxin subunit B indicated that Lu gps were not localized in lipid rafts. Glutathione-S-transferase capture assays showed that the cytoplasmic domain of Lu and Lu(v13) bound to erythroid spectrin, present in a low ionic strength extract from red cell ghosts. Direct interaction with spectrin was confirmed by plasmon resonance assays. Site-directed mutagenesis mapped a major interaction site with spectrin to the RK573-574 motif, located on the cytoplasmic tail of Lu gp, in close vicinity to the inner leaflet of the membrane lipid bilayer. The two Lu adhesion gps represent the first example of a direct link between transmembrane proteins and spectrin in red blood cells. Since Lu gps are low abundant proteins, we speculate that their interaction with spectrin might be critical for signalling and receptor function rather than for participating in the linkage of the lipid bilayer to the red cell skeleton.

The linkage between blood group-related cell surface proteins and the detergent-insoluble material (DIM) was estimated by flow cytometry using a panel of specific monoclonal antibodies (mAbs) as a comparison of the antibody-binding capacity of intact and Triton-X100-treated cells. Studies were performed with K562 cells expressing endogenous or recombinant proteins and with human erythroid progenitors during their proliferation and differentiation in vitro. Glycophorin C (GPC) was found to be Triton-insoluble in both cellular models. When expressed (erythroid progenitors), Band 3 remained Triton-insoluble. Glycophorin A (GPA), however, behaved as Triton-soluble or insoluble according to the absence (K562) or the presence (erythroid progenitors) of Band 3 respectively. Comparison of the cellular models regarding the proteins that compose the Rh complex also indicated that Rh(D), RhAG and CD47 were resistant to Triton extraction in cells lacking Band 3. Similarly, RhAG and CD47 remained predominantly Triton-insoluble in K562 cells and early progenitors before Rh and Band 3 expression. Further analysis showed that the Kell protein was DIM-associated. In contrast, CD99 and DARC (Fy) proteins were not, or were very poorly, DIM-associated. Additionally, the adhesion molecules CD44 and Lu were completely or partially resistant to detergent extraction respectively. Deletion of the Lu cytoplasmic tail or its replacement by the cytoplasmic domain of GPC resulted in significant increase or decrease of the Triton solubility of the transfected proteins respectively. These data suggest that Triton insolubility of Lu results in part from direct attachment of its cytoplasmic tail with the cytoskeleton. We assume that this method should provide a useful tool to map interaction sites localized in the cytoplasmic domain of recombinant transmembrane proteins.

Lutheran (Lu) blood group antigens and the basal cell adhesion molecule antigen reside on two glycoproteins that belong to the Ig superfamily (IgSF) and carry five Ig-like extracellular domains. These glycoproteins act as widely expressed adhesion molecules and represent the unique receptors for laminin-10/11 in erythroid cells. Here, we report the mapping of IgSF domains responsible for binding to laminin. In plasmonic resonance surface experiments, only recombinant Lu proteins containing the N-terminal IgSF domains 1-3 were able to bind laminin-10/11 and to inhibit binding of laminin to Lu-expressing K562 cells. Mutant recombinant proteins containing only IgSF domain 1, domains 1 + 2, domains 1 + 3, domains 2 + 3, domain 3, domain 4, domain 5, and domains 4 + 5 failed to bind laminin as well as a construct containing all of the extracellular domains except domain 3. Altogether, these results indicate that IgSF domains 1-3 are involved in laminin binding and that a specific spatial arrangement of these three first domains is most probably necessary for interaction. Neither the RGD nor the N-glycosylation motifs present in IgSF domain 3 were involved in laminin binding.

Introduction: Although Sickle Cell Disease (SCD) is due to abnormal hemoglobin, many cell types, including endothelial cells and polymorphonuclear neutrophils (PMNs), play a key role in the pathophysiology of the disease, particularly in the vaso-occlusive events. Adhesion of PMNs to activated endothelium is critical in SCD and might contribute to vaso-occlusion; thus targeting PMN interactions with the endothelium may represent a good opportunity for new therapeutics. We looked for candidate mediators that may be involved in PMN activation and recruitment in tissues. We focused on endothelin-1 (ET-1) since (i) high levels of cytokines have been reported in SCD patients; (ii) we previously demonstrated that ET-1 receptors blockade in the SAD mouse model of SCD leads to reversal or prevention of vaso-occlusive events, nitrative stress, kidney and lung damage, and even death. While demonstrating that ET receptor (ETR) antagonism inhibited a tonic ET-1-dependent vasoconstriction during experimental vaso-occlusive crisis, we observed unexpected inhibition of PMN recruitment in lungs and kidneys. We postulated that activation of ETRs might stimulate a pathogenic proinflammatory role for PMNs in SCD. Methods: In the present study, we combinedintravital videomicroscopy of the cremaster muscle microcirculation in SAD mice and quantitative microfluidic fluorescence microscopy of human blood to investigate the involvement of the ETRs in the interaction between neutrophils and endothelial cells. Results: Experiments performed both on SAD mice and SCD patients indicate that blocking ETRs hinders PMN recruitment to endothelial cells in inflammatory conditions (Fig.1). In SAD mice, we showed that the ETRs are involved at several steps of PMN microvascular recruitment. Rolling adhesion involves the ETB receptor only; firm adhesion and post-adhesive dynamic events with transmigration involve both the ETA and the ETB receptors. Inhibition experiments performed with the highly selective BQ788 antagonist (specific for ETB receptor) provide potent anti-inflammatory action in SAD mice. In SCD patients and healthy volunteers, we evidenced that human PMNs display functional ETB receptors that trigger a downstream calcium signaling leading to enhanced adhesion to endothelial cells. Furthermore, we investigated the expression of preproET-1 mRNA and the secretion of ET-1 in human PMNs from SCD patients and healthy controls and we highlighted the ability of PMNs to produce and secrete ET-1. We also found an unexpected stimulatory role for the ETB receptor in PMN adhesion to endothelial cells in laminar flow conditions. Last, we showed that this abnormal adhesion involves ETB receptors on both endothelial cells and PMNs. Conclusion: SAD mice and SCD patients both provide consistent evidence for a powerful anti-adhesion role of ETB blockade. We emphasize that our work is the first to unravel the impact of the ET receptors on the different phases of PMN-endothelial interaction, further complementing early evidence that endothelins are chemoattractants for neutrophils in vitro. Our findings involving a potent contribution of the ETB receptor to vascular inflammation are novel as well as the fact that this pathogenic phenomenon is found in sickle but not in normal mice. Overall, the aforementioned findings indicated that human neutrophils display functional ETB receptors with calcium signaling capability, and we confirmed that human PMNs synthesize ET-1 that may be involved in autocrine and paracrine pathophysiological actions. Thus, the ET-1- ETB axis should be considered a cytokine-like potent proinflammatory pathway in SCD. If endothelin receptors antagonists prove safe and effective for preventing or treating acute vasoocclusive events in the clinical setting, they should include anti- ETB potency and may provide major benefits for lung and renal integrity, quality of life, and survival of SCD patients. Figure 1 Effect of BQ123 and BQ788 on neutrophils recruitment. (A) Representative images of cremasteric venules from SAD mice after local TNFa stimulation associated or not with specific blocking of ETA (BQ123) or ETB (BQ788) receptors. (B) Representative images of labeled PMNs (in green) that have gradually adhered to the endothelial cells. The three microfluidic channels were infused with the same batch of whole blood from a SCD patient without treatment (NT) or preincubated with BQ123 or BQ788 Figure 1. Effect of BQ123 and BQ788 on neutrophils recruitment. (A) Representative images of cremasteric venules from SAD mice after local TNFa stimulation associated or not with specific blocking of ETA (BQ123) or ETB (BQ788) receptors. (B) Representative images of labeled PMNs (in green) that have gradually adhered to the endothelial cells. The three microfluidic channels were infused with the same batch of whole blood from a SCD patient without treatment (NT) or preincubated with BQ123 or BQ788 Disclosures No relevant conflicts of interest to declare.

Introduction: β-globin gene transfer into hematopoietic stem cells (HSCs) has the potential to reduce or eliminate the symptoms of severe sickle cell disease (SCD) and reduce or eliminate transfusion requirements in transfusion-dependent β-thalassemia (TDT). LentiGlobin Drug Product (DP) contains autologous CD34+ cells transduced with the BB305 lentiviral vector, which encodes a human β-globin gene containing a single point mutation (AT87Q) designed to confer anti-sickling properties similar to those observed with γ-globin. We previously reported proof of concept for LentiGlobin DP treatment in severe SCD and early data from 4 treated patients with TDT. We now report 18 months of follow-up for the patient with SCD and between 9 and 30 months of follow-up for the 4 patients with TDT. Patients (5-35 years of age) with severe SCD (e.g. ≥2 acute chest syndrome episodes or ≥2 vaso-occlusive crises [VOC] in preceding year/in year prior to regular transfusions) or TDT (≥100mL/kg of packed red blood cells [RBCs] per year) were enrolled. Following mobilization and apheresis (for TDT) or bone marrow harvest (for SCD), autologous CD34+ cells were transduced with the BB305 lentiviral vector. Patients underwent myeloablative conditioning with busulfan prior to infusion of the transduced cells. After infusion, patients were monitored for hematologic engraftment, vector copy number (VCN), and HbAT87Q expression. Disease-specific assessments included transfusion requirements for TDT, or VOCs and hospitalizations for SCD. Safety assessments included adverse events (AEs) and integration site analysis. Results: As of July 2016, 1 patient with severe SCD (male; 13 years old) and 4 patients with TDT (2 male, 2 female; 16-19 years old) have received LentiGlobin DP in Study HGB-205. The median LentiGlobin DP cell dose was 8.9 (range 5.6-13.6) x 106 CD34+ cells/kg with a DP VCN of 1.2 (range: 0.8-2.0) vector copies/diploid genome. Median post-infusion follow-up as of July 6, 2016 is 20.8 months (range 9.5-31.3). All subjects successfully engrafted after receiving LentiGlobin DP, with a median time to neutrophil engraftment of 17 days (range 14-38 days). VCN in peripheral blood has remained generally consistent from Month 3 in all patients with a range of 0.2 to 3.4 at last measurement. The toxicity profile observed from start of conditioning to latest follow-up remains consistent with myeloablative conditioning with single-agent busulfan, with no DP-related ≥Grade 3 AEs or serious AEs and no evidence of clonal dominance reported to date. Three patients with TDT have β0/βE genotypes and 1 is homozygous for the severe β+ mutation IVS1 nt 110 G>A. The 2 patients who have completed the 2-year primary follow-up period (both β0/βE) have not required RBC transfusions for 31 and 28 months, with total Hb of 10.9 and 13.5 g/dL, and HbAT87Q expression of 7.7 and 10.1 g/dL, respectively, at most recent study visit. Iron chelation has been discontinued and phlebotomy initiated for 1 of the patients. The remaining patient with β0/βE genotype has 9 months of follow-up and has not required transfusions since 4 days post-LentiGlobin DP infusion, achieving a total Hb of 11.3 g/dL at last study visit. The patient with the severe IVS1 genotype has 12 months of follow-up and has been free of transfusions for 9 months, with a total Hb of 8.3 g/dL at last study visit. The patient with severe SCD, who prior to study enrollment received regular RBC transfusions, has experienced no clinical symptoms or complications of SCD in the 18 months since treatment, despite discontinuing transfusions 3 months after LentiGlobin DP infusion. Total Hb in this patient was 12.5 g/dL, with 6.6 g/dL HbAT87Q (53%) and 5.7 g/dL HbS (45%) at last study visit. Compared with values at screening, unconjugated bilirubin had dropped 78% (50 to 11 μmol/L), lactate dehydrogenase had dropped 54% (626 to 287 U/L), and reticulocyte count had dropped 45% (238x109/L to 132x109/L) by Month 18. Conclusions: Data from this ongoing Phase 1/2 clinical study suggest that treatment with LentiGlobin DP can result in sustained production of therapeutic HbAT87Q, which ameliorates the clinical and biochemical effects of severe SCD and TDT, with an acceptable safety profile. Gene therapy presents a potentially promising therapy for patients with severe hemoglobinopathies. Further follow-up and additional data from patients are needed to confirm the encouraging results seen to date in this study. Disclosures Ribeil: Bluebirdbio: Consultancy; Addmedica: Research Funding. Payen:bluebird bio: Patents & Royalties. Hermine:Alexion: Research Funding; Celgene: Research Funding; Novartis: Research Funding; AB science: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau. Asmal:bluebird bio: Employment, Equity Ownership. Joseney-Antoine:bluebird bio: Employment, Equity Ownership. De Montalembert:Addmedica: Research Funding; Novartis: Research Funding, Speakers Bureau. Leboulch:bluebird bio, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

The McLeod syndrome is a rare X-linked recessive disorder characterized by blood group, neuromuscular and haematopoietic abnormalities. It is caused by XK gene defects and may include large deletions in the Xp21 region. Analysis of three unrelated McLeod patients for the presence of the XK, DMD, CYBB, ETX1, RPGR and OTC loci, as well as for the DXS709 marker, revealed deletions from the 39th exon of DMD to the ETX1 locus (patient Be), from the XK to RPGR loci (patient Bi) and from the XK to CYBB loci (patient Lh). All three patients normally expressed the Lutheran (Lu) red cell antigens, thus excluding the interval between the RPGR and DMD genes as site of the XS locus, previously mapped to the Xp21.2-Xq21.1 region and thought to regulate the expression of the LU blood group gene on chromosome 19.

Lu and Lu(v13) are two glycoprotein (gp) isoforms that belong to the immunoglobulin superfamily and carry both the Lutheran (Lu) blood group antigens and the basal cell adhesion molecule epithelial cancer antigen. Lu (85 kDa) and Lu(v13) (78 kDa) gps, which differ only in the length of their cytoplasmic domain, are adhesion molecules that bind laminin. In nonerythroid tissues, the Lu/basal cell adhesion molecule antigens are predominantly expressed in the endothelium of blood vessel walls and in the basement membrane region of normal epithelial cells, whereas they exhibit a nonpolarized expression in some epithelial cancers. Here, we analyzed the polarization of Lu and Lu(v13) gps in epithelial cells by confocal microscopy and domain-selective biotinylation assays. Differentiated human colon carcinoma Caco-2 cells exhibited a polarized expression of endogenous Lu antigens associated with a predominant expression of the Lu isoform at the basolateral domain of the plasma membrane and a very low expression of the Lu(v13) isoform at both the apical and basolateral domains. Analysis of transfected Madin-Darby canine kidney cells revealed a basolateral expression of Lu gp and a nonpolarized expression of Lu(v13) gp. Delivery of Lu(v13) to both apical and basolateral surfaces showed similar kinetics, indicating that this isoform is directly transported to each surface domain. A dileucine motif at position 608–609, specific to the Lu isoform, was characterized as a dominant basolateral sorting signal that prevents Lu gp from taking the apical delivery pathway.

The Lutheran (Lu) blood group antigens and the B-cell adhesion molecule (B-CAM) epithelial cancer antigen are carried by recently cloned integral glycoproteins that belong to the Ig superfamily. We have previously shown that the Lu and B-CAM antigens are encoded by the same gene, LU, and that alternative splicing of the primary transcript most likely accounts for the presence of both antigens on two isoforms that differ by the length of their cytoplasmic tails. In the present report, we isolated the human LU gene by cloning a 20-kb HindIII fragment from Lu(a − b+) genomic DNA. The LU gene is organized into 15 exons distributed over 12.5 kb. Alternative splicing of intron 13 generates the 2.5- and 4.0-kb transcript spliceoforms encoding the long tail and the short tail Lu polypeptides, respectively. Sequencing of the major mRNA species (2.5 kb) amplified from human bone marrow, kidney, placenta, and skeletal muscle did not suggest the presence of tissue-specific Lu glycoprotein isoforms. The same transcription initiation point, located 22 bp upstream from the initiation codon, was characterized in several tissues. In agreement with the wide tissue distribution of the Lu messengers, the GC-rich proximal 5′ flanking region of the LU gene does not contain TATA or CAAT boxes, but includes several potential binding sites for the ubiquitous Sp1 transcription factor. In addition, the distal 5′ region, encompassing nucleotides −673 to −764, contains clustered binding sequences for the GATA, CACCC, and Ets transcription factors. Analysis of the coding sequences amplified from genomic DNA of Lu(a + b−) or Lu(a − b+) donors showed a single nucleotide change in exon 3 (A229G) that correlates with an Aci I restriction site polymorphism and results in a His77Arg amino-acid substitution. Polymerase chain reaction/restriction fragment length polymorphism analysis indicated that the A229G mutation is associated with the Lua/Lub blood group polymorphism. When expressed in Chinese hamster ovary (CHO) cells, Lu cDNAs carrying the A229 or the G229 produced cell surface proteins that reacted with anti-Lua or anti-Lub antibodies, respectively, showing that these nucleotides specify the Lua and Lub alleles of the Lutheran blood group locus. CHO cells expressing recombinant short-tail or long-tail Lu glycoproteins reacted as well with anti-Lu as with anti–B-CAM antibodies, providing the definitive proof that the Lu blood group and B-CAM antigens are carried by the same molecules.

International audience; Background Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors. Design and Methods We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathological induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation. Results We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch from fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathological induced pluripotent stem cells into mice. Conclusions These results demonstrate that human induced pluripotent stem cells i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation; and ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment. © 2012 Ferrata Storti Foundation.

Introduction: Sickle cell anemia (SCA) is a severe monogenic hereditary disorder characterized by chronic hemolytic anemia and the occurrence of frequent painful vaso-occlusive crises (VOC). SCA classical physiological scheme involves hemoglobin S polymerization under hypoxic conditions, which triggers red blood cells (RBCs) sickling. Recent studies demonstrated that the degree of hemorheological alterations, such as blood hyper-viscosity, determines the risk for VOC. Moreover, sickle RBCs abnormally adhere to the vascular endothelium, triggering microvascular occlusions. Despite extensive research very few drugs are available to efficiently treat VOCs or VOC-like events. A first clinical trial was performed to test the efficacy of poloxamer 188 (P188) in a large sickle cell cohort of adults and children (Orringer et al., 2001). This study demonstrated a significant reduction of pain duration in the children treated with P188. Recently, a new phase III multi-center trial has been started to test the efficacy of this drug during acute VOC in children (Humphries et al., 2015). We conducted in vitro experiments using the commercial formulation named Kolliphor P188 (Sigma-Aldrich) to test the effects of this drug on RBCs biophysical properties of SCA patients. Methods: To measure deformability and mechanical properties of RBCs, we used ektacytometry and microfluidic device mimicking the diameters of the micro vessels. RBCs adhesion assays were performed on HMEC-1 (Human Microvascular Endothelial Cell line) using dynamic flow adhesion platform. RBCs from healthy (AA) and SCA individuals were used for the different experiments. Results: While P188 did not significantly affect blood viscosity in AA, P188 treatment decreased blood viscosity at the lowest shear rates in SCA (Fig 1A). When measured in plasma, RBC aggregation decreased with P188 in SCA patients but not in AA (Fig 1B). RBC deformability assessed by both ektacytometry (not shown) and microfluidic device (Fig 2) was not affected by P188. This is in agreement with the mode of action of P188 suggesting that it binds to hydrophobic surfaces and lowers surface tension without any changes in the organization of the cytoskeleton. We examined the effect of P188 treatment on SCA-RBCs adhesion to the endothelial HMEC-1 cell line. We observed a mean adhesion of 40 cells/ mm2 for the untreated SCA-RBCs versus 20 cells/mm2 in the case of P188 treated RBCs, i.e. a 50% decrease upon P188 treatment (Fig 3). As for RBC aggregation, our findings suggest that the binding of P188 to SCA-RBCs membranes prevent the interaction with endothelial cells. This is of particular importance in the context of SCA since increased RBC adhesiveness has been demonstrated to trigger VOC. Conclusion: In parallel of the phase III clinical trial studying the profit of P188 for sickle cell patients during VOCs, our results bring clarifications regarding its mode of action on RBCs. We show that P188 directly reduces blood viscosity, RBC aggregation and adhesiveness to endothelial cells, making this drug as a potential beneficial therapy in SCA. References: Orringer, E.P., et al; (2001). JAMA, 286, 2099-2106. Humphries, J.D., et al; (2015). Trends Cell Biol, 25, 388-397. Figure 1. P188 treatment decreases blood viscosity (A) and RBC aggregation (B) in SS patients but not in AA controls. Figure 1. P188 treatment decreases blood viscosity (A) and RBC aggregation (B) in SS patients but not in AA controls. Figure 2. P188 treatment does not change SCA-RBC deformability. (A) Design of the microfluidic chip containing eight filtering units organized in parallel. Each filtering unit has a height of 5 µm and pillars are organized to allow an escape route of 20 µm around the unit to avoid occlusion. The surrounding pillars line has 5 µm slits. (B) Retention percentage of untreated and P188-treated SCA-RBC in 5 µm slits. Histograms represent mean of 5 µm slits from the 8 filtering units in one chip expressed in percent of total trapped RBCs for three patients. Figure 2. P188 treatment does not change SCA-RBC deformability. (A) Design of the microfluidic chip containing eight filtering units organized in parallel. Each filtering unit has a height of 5 µm and pillars are organized to allow an escape route of 20 µm around the unit to avoid occlusion. The surrounding pillars line has 5 µm slits. (B) Retention percentage of untreated and P188-treated SCA-RBC in 5 µm slits. Histograms represent mean of 5 µm slits from the 8 filtering units in one chip expressed in percent of total trapped RBCs for three patients. Figure 3. Graph representing adherent cells per mm2 at a flow rate of 1 dyne/mm2. The mean of the 5 patients is expressed as the average number of adherent cells/mm2 ± SD. Paired t test, P < 0.05. Figure 3. Graph representing adherent cells per mm2 at a flow rate of 1 dyne/mm2. The mean of the 5 patients is expressed as the average number of adherent cells/mm2 ± SD. Paired t test, P < 0.05. Disclosures No relevant conflicts of interest to declare.

Lu/BCAM (Lutheran/basal cell-adhesion molecule) is a laminin 511/521 receptor expressed in erythroid and endothelial cells, and in epithelial tissues. The RK573–574 (Arg573-Lys574) motif of the Lu/BCAM cytoplasmic domain interacts with αI-spectrin, the main component of the membrane skeleton in red blood cells. In the present paper we report that Lu/BCAM binds to the non-erythroid αII-spectrin via the RK573–574 motif. Alanine substitution of this motif abolished the Lu/BCAM–spectrin interaction, enhanced the half-life of Lu/BCAM at the MDCK (Madin–Darby canine kidney) cell surface, and increased Lu/BCAM-mediated cell adhesion and spreading on laminin 511/521. We have shown that the Lu/BCAM–spectrin interaction mediated actin reorganization during cell adhesion and spreading on laminin 511/521. This interaction was involved in a laminin 511/521-to-actin signalling pathway leading to stress fibre formation. This skeletal rearrangement was associated with an activation of the small GTP-binding protein RhoA, which depended on the integrity of the Lu/BCAM laminin 511/521-binding site. It also required a Lu/BCAM–αII-spectrin interaction, since its disruption decreased stress fibre formation and RhoA activation. We conclude that the Lu/BCAM–spectrin interaction is required for stress fibre formation during cell spreading on laminin 511/521, and that spectrin acts as a signal relay between laminin 511/521 and actin that is involved in actin dynamics.

In this issue of Blood, Garcia-Santos et al advance the field of heme regulation, a highly complex process involving iron and globin metabolism. They focus on a key enzyme involved in heme catabolism, heme oxygenase 1 (HO-1), which, ironically, has been poorly investigated in erythroid cells, the largest pool of heme-containing cells.

Sickle cell disease is characterized by painful vaso-occlusive crises during which abnormal interactions between erythroid adhesion molecules and vessel-wall proteins are thought to play a critical role. Hydroxyurea, the only drug with proven benefit in sickle cell disease, diminishes these interactions, but its mechanism of action is not fully understood. We report that, under hydroxyurea, expression of the unique erythroid laminin receptor Lu/BCAM was increased, but red blood cell adhesion to laminin decreased. Because Lu/BCAM phosphorylation is known to activate cell adhesion to laminin, it was evaluated and found to be dramatically lower in hydroxyurea-treated patients. Analysis of the protein kinase A pathway showed decreased intracellular levels of the upstream effector cyclic adenosine monophosphate during hydroxyurea treatment. Using a cellular model expressing recombinant Lu/BCAM, we showed that hydroxyurea led to decreased intracellular cyclic adenosine monophosphate levels and diminished Lu/BCAM phosphorylation and cell adhesion. We provide evidence that hydroxyurea could reduce abnormal sickle red blood cell adhesion to the vascular wall by regulating the activation state of adhesion molecules independently of their expression level.

Lutheran blood group and basal cell adhesion molecule (Lu/BCAM) has been recognized as a unique receptor for laminin α5chain in human red blood cells and as a coreceptor in epithelial, endothelial, and smooth muscle cells. Because limited information is available regarding the function of this adhesion glycoprotein in vivo, we generated Lu/BCAM-null mice and looked for abnormalities in red blood cells as well as in kidney and intestine, two tissues showing alteration in laminin α5chain-deficient mice. We first showed that, in contrast to humans, wild-type murine red blood cells failed to express Lu/BCAM. Lu/BCAM-null mice were healthy and developed normally. However, although no alteration of the renal function was evidenced, up to 90% of the glomeruli from mutant kidneys exhibited abnormalities characterized by a reduced number of visible capillary lumens and irregular thickening of the glomerular basement membrane. Similarly, intestine analysis of mutant mice revealed smooth muscle coat thickening and disorganization. Because glomerular basement membrane and smooth muscle coat express laminin α5chain and are in contact with cell types expressing Lu/BCAM in wild-type mice, these results provide evidence that Lu/BCAM, as a laminin receptor, is involved in vivo in the maintenance of normal basement membrane organization in the kidney and intestine.

Patients with polycythemia vera (PV) have a JAK2 (a cytosolic tyrosine kinase) mutation and an increased risk of vascular thrombosis related to red blood cell (RBC) mass and platelet activation. We investigated functional RBC abnormalities that could be involved in thrombosis. RBC adhesion to human umbilical vein endothelial cells (HUVECs) was measured by a radiometric technique and in a flow system by video microscopy, and adhesion molecule expression was determined using specific antibodies (against CD36, CD49d, ICAM-4, Lu/BCAM, CD147, and CD47) and flow cytometry in a group of 38 patients with PV and a group of 36 healthy volunteers. Adhesion of PV RBCs was 3.7-fold higher than that of normal RBCs (P < .001). Adhesion was inhibited when PV RBCs were incubated with anti-Lutheran blood group/basal cell adhesion molecule (Lu/BCAM) or when HUVECs were treated with anti-laminin α5 and to a lesser extent with anti-α3 integrin. Lu/BCAM was constitutively phosphorylated in PV RBCs. Transfection of K562 cells with JAK2 617V>F resulted in increased expression and phosphorylation of Lu/BCAM. Phosphorylation of Lu/BCAM increases RBC adhesion. Our results indicate that JAK2 mutation might be linked to Lu/BCAM modification and increased RBC adhesiveness, which may be a factor favoring thrombosis in PV.

Lu (Lutheran) blood group and BCAM (basal cell adhesion molecule) antigens both reside on two gp (glycoprotein) isoforms, Lu and Lu(v13), that differ by the size of their cytoplasmic tail. They are receptors of laminin-10/11 and are expressed in RBCs (red blood cells), epithelial cells of multiple tissues and vascular endothelial cells. To gain more insights into the biological function of Lu/BCAM gps, we looked for potential partners of their cytoplasmic tail. We isolated Ubc9 (ubiquitin-conjugating enzyme 9) protein by screening a human kidney library using the yeast two-hybrid system. Lu/Ubc9 interaction was validated by GST (glutathione S-transferase) pull-down and co-immunoprecipitation experiments. Endogenous Ubc9 formed a complex with endogenous or recombinant Lu gp in A498 and MDCK (Madin–Darby canine kidney) epithelial cells respectively. Replacement of Lys585 by alanine in the Lu gp abolished in vitro and ex vivo interactions of Lu gp with Ubc9 protein. Lu K585A mutant transfected in MDCK cells exhibited a normal basolateral membrane expression but was overexpressed at the surface of polarized MDCK cells as compared with wild-type Lu. Pulse–chase experiments showed extended half-life of Lu K585A gp at the plasma membrane, suggesting an impaired endocytosis of this mutant leading to protein accumulation at the membrane. Furthermore, we showed that the ability of MDCK-Lu K585A cells to spread on immobilized laminin was dramatically decreased. Our results support a physiological role for the direct interaction between Lu gp and Ubc9 protein and reveal a role for this enzyme in regulating the stability of Lu gp at the cell membrane.

BACKGROUND: Monoclonal antibodies (MoAbs) are gradually replacing human polyclonal sera as typing reagents. Many blood group specificities, however, are not amenable to classic hybridoma technology. The phage display technology, aimed at isolating peptides or antibody fragments, offers an alternative strategy. Recombinant antibodies derived from this technology would greatly facilitate phenotyping and decrease analysis cost. STUDY DESIGN AND METHODS: A human single-chain Fv (scFv) phage-displayed library was panned on red blood cells (RBCs) in an attempt to isolate clones recognizing human RBC specificities. Three rounds of biopanning were performed. Enrichment was monitored by phage titration, and selected phage populations were analyzed further. RESULTS: Three major clones were identified by clone diversity analysis. One of them showed a specificity for Lua. This scFv was reconstructed into a human IgG1 by recombinant DNA methods. The reactivity of the reconstructed human IgG1 toward Lua is indistinguishable from its parent scFv. Moreover, the specificity of the antibody was confirmed by serologic assays, flow cytometry, and biochemical analysis with RBCs of different Lu phenotypes and a recombinant cell line expressing Lu glycoproteins. CONCLUSION: With phage display and standard recombinant DNA methods, isolation of a scFv of Lua specificity was successful, from which a complete human IgG1 MoAb of equivalent reactivity was reconstructed. To our knowledge, this is the first MoAb specific for Lua.

International audience; Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. In cancer, clinical and experimental studies have reported a link between tetraspanin expression levels and metastasis. Tetraspanins play a role as organizers of a molecular network of interactions, the "tetraspanin web". Here, we have performed a proteomic characterization of the tetraspanin web using a model of human colon cancer consisting of two cell lines derived from primary tumor and metastasis from the same patient. The tetraspanin complexes were isolated after immunoaffinity purification and the proteins were identified by MS using LC-ESI-MS/MS and MALDI-FTICR. The high resolution and mass accuracy of FTICR MS allowed reliable identification using mass finger printing with only two peptides. Thus, it could be used to resolve the composition of complex peptide mixtures from membrane proteins. Different types of membrane proteins were identified, including adhesion molecules (integrins, Lu/B-CAM, GA733 proteins), receptors and signaling molecules (BAI2, PKC, G proteins), proteases (ADAM10, TADG15), and membrane fusion proteins (syntaxins) as well as poorly characterized proteins (CDCP1, HEM-1, CTL1, and CTL2). Some components were differentially detected in the tetraspanin web of the two cell lines. These differences may be relevant for tumor progression and metastasis.

Introduction: In normal conditions, circulating red blood cells (RBCs) are not supposed to adhere. Some erythroid adhesion molecules expressed on young reticulocytes are therefore lost upon maturation. Conversely, abnormal RBC adhesiveness in sickle cell anemia (SCA) through activation, sustained or increased expression of adhesion molecules are considered central in vaso occlusive crisis (VOC), the hallmark of SCA. SCA is seldom symptomatic in the first six months of life. One main explanation lies in the sustained level of fetal hemoglobin (HbF) and F cells during this period preventing HbS polymerization. However, infra clinical vaso occlusion, particularly in the spleen, has been demonstrated to be present and the absolute reticulocyte count is paradoxically elevated in the first semester of life, evidencing the very early onset of hemolysis. Our objectives were to analyze the profile of erythroid adhesion molecules in SCA and non-SCA infants in combination with HbF level, in order to evidence significant differences specifically attributable to SCA, informative on very early pathophysiology and disease severity. Patients and methods: Infants were enrolled in a longitudinal multi center prospective study on prognostic factors in SCA between September 2010 and March 2013. Blood sampling was performed at enrolment (3-6 months) at steady state, in asymptomatic infants. In parallel, blood samples from infants with no hemoglobinopathy were collected. Flow cytometer analysis (BD FACSCanto II) was performed using murine monoclonal antibodies against CD36, CD44, CD47, CD49d, CD58, CD99, CD147, CD239 and CD242. Statistical analysis was performed with Graphpad software, Prism 6. Differences in the percentage of positive cells and the level of expression of molecules between groups were calculated with Mann Whitney test. Results were considered statistically significant at an a-risk level of 5%. Results: Fifty-four SCA infants were included and compared to 17 non-SCA infants. Median age in the two groups was not statistically different (144 days, range 81-196 versus 128, range 68-621, p=0,84). Mean HbF level was, as previously described, statistically increased in SCA compared to non-SCA infants (41.2% +/-11.2 versus 10,4 % +/- 1,8; p Reticulocytes from SCA infants display a distinct surface molecule expression profile, with a statistically increased percentage of reticulocytes expressing the following adhesion molecules: CD239 (Lu/BCAM), CD242 (ICAM-4), CD47, CD99, CD58, CD147 and CD44 (Figure 1). Furthermore the mean intensity of fluorescence is statistically increased concerning CD239, CD58 and CD242 (data not shown). Surprisingly, known adhesion markers demonstrated to play an important role in SCA pathogeny i.e. CD36 and CD49d (α4β1/Very Late Antigen-4) are not significantly increased in SCA infants (Figure 1) No significant difference of expression profile was found on mature or total RBCs between SCA and non-SCA infants (data not shown). Figure 1 Figure 1. Discussion: Recent publications have pointed to the significance of reticulocytosis associated with an increased risk of death or stroke during childhood. Our results further demonstrate that this early rise of reticulocyte count in SCA infants, despite elevated HbF level, include reticulocytes with a specific adhesion molecule expression profile. This numerically small subpopulation of red cells may play in fact a major role in this complex disease. An interesting hypothesis may be that the spleen, as long as its function is preserved, prevents VOC by trapping this subpopulation in very young infants. As spleen function decreases, proadhesive reticulocytes remain in the circulation, which may, in combination with the decline of HbF, subsequently favor extra splenic VOC. Ongoing analysis will confirm if this idiosyncratic erythroid adhesion molecule profile in infants indeed correlates with HbF level and, importantly, correlates with functional activation, in order to serve as a readily available biomarker of disease severity. Disclosures De Montalembert: Novartis: Speakers Bureau. Le Van Kim:SHIRE: Research Funding.

Lutheran (Lu) and Lu(v13), two glycoprotein (gp) isoforms belonging to the immunoglobulin superfamily, represent adhesion molecules that act as erythrocyte receptors for laminin 10/11. These two gps, which differ only by the length of their cytoplasmic tail, carry both Lu blood group and Basal Cell Adhesion Molecule (B-CAM) antigens. Here, analysis of the Triton extractability of recombinant Lu and Lu(v13) gps in K562 transfected cells showed that both gps were mainly associated with the detergent-insoluble material. Patching experiments using Cholera Toxin subunit B indicated that Lu gps were not localized in lipid rafts. Glutathione-S-transferase capture assays showed that the cytoplasmic domain of Lu and Lu(v13) bound to erythroid spectrin, present in a low ionic strength extract from red cell ghosts. Direct interaction with spectrin was confirmed by plasmon resonance assays. Site-directed mutagenesis mapped a major interaction site with spectrin to the RK573-574 motif, located on the cytoplasmic tail of Lu gp, in close vicinity to the inner leaflet of the membrane lipid bilayer. The two Lu adhesion gps represent the first example of a direct link between transmembrane proteins and spectrin in red blood cells. Since Lu gps are low abundant proteins, we speculate that their interaction with spectrin might be critical for signalling and receptor function rather than for participating in the linkage of the lipid bilayer to the red cell skeleton.

In this issue of Blood, Almeida et al report immediate benefits of hydroxyurea (HU) acute administration in diminishing vaso-occlusive processes in sickle cell disease (SCD) mice.(1)

The Lutheran antigens are recently characterized glycoproteins in which the extracellular region contains five immunoglobulin like domains, suggesting some recognition function. A recent abstract suggests that the Lutheran glycoproteins (Lu gps) act as erythrocyte receptors for soluble laminin (Udani, M., Jefferson, S., Daymont, C., Zen, Q., and Telen, M. J. (1996) Blood 88, Suppl. 1, 6 (abstr.)). In the present report, we provided the definitive proof of the laminin receptor function of the Lu gps by demonstrating that stably transfected cells (murine L929 and human K562 cell lines) expressing the Lu gps bound laminin in solution and acquired adhesive properties to laminin-coated plastic dishes but not to fibronectin, vitronectin, transferrin, fibrinogen, or fibrin. Furthermore, expression of either the long-tail (85 kDa) or the short-tail (78 kDa) Lu gps, which differ by the presence or the absence of the last 40 amino acids of the cytoplasmic domain, respectively, conferred to transfected cells the same laminin binding capacity. We also confirmed by flow cytometry analysis that the level of laminin binding to red cells is correlated with the level of Lu antigen expression. Indeed, Lunull cells did not bind to laminin, whereas sickle cells from most patients homozygous for hemoglobin S overexpressed Lu antigens and exhibited an increased binding to laminin, as compared with normal red cells. Laminin binding to normal and sickle red cells as well as to Lu transfected cells was totally inhibited by a soluble Lu-Fc chimeric fragment containing the extracellular domain of the Lu gps. During in vitro erythropoiesis performed by two-phase liquid cultures of human peripheral blood, the appearance of Lu antigens in late erythroid differentiation was concomitant with the laminin binding capacity of the cultured erythroblasts. Altogether, our results demonstrated that long-tail and short-tail Lu gps are adhesion molecules that bind equally well laminin and strongly suggested that these glycoproteins are the unique receptors for laminin in normal and sickle mature red cells as well as in erythroid progenitors.

International audience; This paper discusses the electrical sensing of red blood cell (RBC) rigidity due to Hemoglobin S (HbS) polymerization in sickle cell disease. For this purpose sickle RBCs subpopulations, differentiated by cell density, were flown through a microfluidic device equipped with integrated microelectrodes. Differences in transit time and electrical blockade were observed and analyzed.

Background: Clinical complications of BCR-ABL1-negative Myeloproliferative Neoplasms (MPNs) include mainly vascular events and on longer term transformation to Myelofibrosis (MF) or acute leukemia. MPNs are also associated with neo-angiogenesis leading to extramedullary hematopoiesis and increased vascularity in the spleen and bone marrow. The most frequent molecular alteration present in hematopoietic cells is the JAK2V617F mutation, which has also been detected in hepatic Endothelial Cells (ECs) of MPN patients with Budd-Chiari syndrome (BCS) and in ECs derived from patients' splenic capillaries. The consequences of the presence of the JAK2V617F mutation into the endothelial lineage are not known although these cells play an important role in thrombosis. Methods: Isogenic JAK2 wild-type (WT) and JAK2V617F mutant induced pluripotent stem cells (iPSC) were derived from an MPN patient. The reprogrammed clonal cells were induced to differentiate toward mesoderm for 3 days and then driven toward the endothelial lineage using Stem-Pro medium, Vascular Endothelial Growth Factor (VEGF) and Forskolin. Enriched CD34+/CD144+ progenitors were then matured in VEGF-supplemented Stem-Pro medium for 5 days before being cultured in the EGM-2 specific EC medium in the presence of the TGF-β inhibitor SB-431542. Cells were harvested and tested for specific surface markers by flow cytometry before expanding them; passages 3 to 6 were used for further characterization and functional studies. Transcriptomic analysis was performed in triplicate from two independent endothelial differentiation experiments using Affymetrix HTA 2.0 gene expression arrays. Results: Phenotypically, the endothelial-like cells obtained after differentiation from both mutant and WT JAK2 iPSC displayed a classical EC morphology and were positive for CD34 and EC markers (CD144, CD309, CD31 and ICAM-1). Such characteristics were conserved for at least 6 rounds of cell passaging. TNF-α treatment increased ICAM-1 cell surface levels and induced the expression of VCAM-1, as observed in other EC models. Our iPS-derived cells were also responsive to VEGF, which induced cellular proliferation and angiogenesis demonstrated by tube formation in Matrigel with both JAK2V617F and WT ECs. As expected, JAK2V617F ECs exhibited higher levels of phosphorylated forms of JAK2, STAT3 and Akt in a serum and growth factor-reduced medium, showing that, as in hematopoietic cells, the presence of the mutation increases basal intracellular signaling. Interestingly, Weibel-Palade bodies detection by confocal microscopy revealed a more important amount of Von Willebrand factor (vWF) in JAK2V617F ECs (2-fold increase), suggesting a higher pro-thrombotic potential for these cells. Basal surface expression of the adhesion molecule ICAM-1 was also clearly increased (1.8-fold) compared to WT ECs suggesting that JAK2V617F ECs may be more prone to recruit activated leukocytes expressing LFA-1, the ICAM-1 receptor. Gene expression microarrays analysis confirmed a significantly higher expression of cell adhesion molecules involved in leukocyte or platelet adhesion in JAK2V617F vs. WT ECs (ICAM-1, VCAM-1, or P-selectin), together with several inflammatory chemokines (IL8, CXCL1, or CXCL5), chemokine receptors (CXCR4 or IL7R) and extracellular matrix modulators (Periostin, Tenascin C, Lysyl Oxidase, or MMP-10) . Finally, Gene Set Enrichment Analysis pointed out gene sets induced in situations of inflammation, response to cytokines and hypoxia. These results could be in agreement with an activated endothelium, leukocyte recruitment and vascular remodeling. Conclusion: Previous discovery of JAK2V617F-positive endothelial cells in the splanchnic vasculature of patients with BCS or MF was of particular interest because of the well-known association between thrombosis and MPN. In this work we first demonstrate that the presence of JAK2V617F doesn't impair endothelial differentiation from stem/progenitor cells. Moreover, JAK2V617F-mutant ECs display features of pro-inflammatory and pro-thrombotic EC activation, which we are currently further characterizing with additional functional studies. Finally, this iPSC model may represent an effective screening tool for molecules antagonizing the pro-thrombotic events in a JAK2V617F context. Disclosures Kiladjian: Novartis: Honoraria, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding.