Search

This is a related article to: Nucleoside ENTry modulates erythropoiesis (cf ci-dessous); International audience; Abstract The tight regulation of intracellular nucleotides is critical for the self-renewal and lineage specification of hematopoietic stem cells (HSCs). Nucleosides are major metabolite precursors for nucleotide biosynthesis and their availability in HSCs is dependent on their transport through specific membrane transporters. However, the role of nucleoside transporters in the differentiation of HSCs to the erythroid lineage and in red cell biology remains to be fully defined. Here, we show that the absence of the equilibrative nucleoside transporter (ENT1) in human red blood cells with a rare Augustine-null blood type is associated with macrocytosis, anisopoikilocytosis, an abnormal nucleotide metabolome, and deregulated protein phosphorylation. A specific role for ENT1 in human erythropoiesis was demonstrated by a defective erythropoiesis of human CD34+ progenitors following short hairpin RNA-mediated knockdown of ENT1. Furthermore, genetic deletion of ENT1 in mice was associated with reduced erythroid progenitors in the bone marrow, anemia, and macrocytosis. Mechanistically, we found that ENT1-mediated adenosine transport is critical for cyclic adenosine monophosphate homeostasis and the regulation of erythroid transcription factors. Notably, genetic investigation of 2 ENT1null individuals demonstrated a compensation by a loss-of-function variant in the ABCC4 cyclic nucleotide exporter. Indeed, pharmacological inhibition of ABCC4 in Ent1−/− mice rescued erythropoiesis. Overall, our results highlight the importance of ENT1-mediated nucleotide metabolism in erythropoiesis.

Sickle cell disease (SCD) is a severe hemoglobinopathy due to abnormal hemoglobin S (HbS). Although red blood cell dysfunction is at the core of the SCD pathophysiology, several studies have highlighted the important role of inflammatory cells like neutrophils. One of the most serious complications of SCD is cerebral vasculopathy (CV), due to the occlusion of one or more intracranial or cervical arteries. In 1998, the STOP study demonstrated that monthly blood transfusions could reduce the risk of stroke by 90% in children with CV. However, there is large heterogeneity in the evolution of CV under chronic transfusion, sometimes requiring exchange transfusion (ET) program for years without succeeding in healing the CV. The aim of the study is to investigate the impact of long-term transfusion program on neutrophil dysfunction, in order to understand if persistent inflammation could contribute to the non-healing of CV despite HbS permanently below 40%. In SCD children undergoing ET program for at least 1 year, we analysed i)the phenotype of neutrophils with 8 markers of activation/adhesion/ageing, ii)the plasmatic levels of elastase, witnessing the NETose activity of neutrophils, and iii)the ex-vivo adhesion of neutrophils on activated endothelial cells. One hundred and two SCD children with an ET transfusion program for at least 6 months because of CV were included in the study. ET session, carried out every 5 weeks and most of the time by erythrapheresis, reached their biological objectives with a mean HbS rate after ET session of 14.1%, and 35.4% before the next ET session, which means that these patients globally live at an average HbS level of 24% for at least 1 year. We managed to limit iron overload with a mean ferritinemia of 207 µg/L in the whole cohort. Despite these satisfactory results in terms of HbS reduction, the efficiency in curing the CV was modest in accordance with the previously described efficiency of ET program in SCD children: after a mean ET program duration of 4.4 years only 22% of them had an improvement of their CV since the beginning of the ET program, while 60% of them had a stagnation of their CV, and 18% of them worsened their vascular lesions. Considering inflammatory parameters, the patients had persistence of high leukocytosis and high neutrophils count (respective mean of 9810 G/L and 5742 G/L), significantly not different of neutrophils count before inclusion in the ET program. In a random subgroup of 20 patients, we analysed neutrophils phenotype, NETose and endothelial adhesion and compared them to healthy controls and SCD children without ET, treated or not with Hydroxyurea (HU). Overall, we observed as expected an activated, aged and adherent profile of neutrophils from untreated SCD children compared to healthy controls, characterized by an overexpression of CD18/CD11b (p=0,03), CD18/CD11a (p=0,02), CD162 (p=0,01), CD66a (p=0,01) and the ageing markers CD184 high/CD62Llow (p=0,04) as well as a higher plasmatic level of elastase (p=0. 01) and higher adhesion of neutrophils to endothelial cells. All these parameters were alleviated in SCD patients treated with HU. In SCD patient undergoing ET program, we found a similar profile of activated neutrophils to that of untreated SCD patients with a similar expression of activation molecules, high level of elastase and the same increase of neutrophils adhesion to endothelial cells compared to controls, witnessing a persistence of chronic inflammation despites years of ET. Overall, our study highlights that the replacement of sickle red blood cells, even for years, is not sufficient to reverse the deleterious inflammatory phenotype of neutrophils. Given the major role of inflammation in endothelial dysfunction, these could contribute to the persistence of CV in a majority of patients despite efficient ET programs. This raises the question of systematically combining ET program with anti-inflammatory treatment such as HU or P-selectin inhibitors in children with CV. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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

Epidemiological, clinical and biological characteristics of colorectal cancer vary across continents probably because of different risk factors that are not yet fully listed in countries with limited resources. This study describes the epidemiological and clinical features of colorectal cancer at a University hospital in Bamako, Mali.A retrospective study that concerned the period from 2005 to 2011 was carried out. It included records of patients with colorectal cancer histologically documented in the service. Were analyzed epidemiological, clinical and biological data stored in files.One hundred-thirteen (113) cases of colorectal cancer were diagnosed representing 15% of all cancers documented over the 7 years. The number of cases increased year by year. The cases of colorectal cancer are not uncommon in young patients (23% under 30 years of age, 60% under 50). The male is significantly more represented in this population of colorectal carriers with a sex ratio M / F = 2. The time between first the symptom and care is long and the diagnosis is made in 85% of cases at a metastatic stage. Localization of the cancer is more frequent in the colon (56%) than in the rectum (44%). Cancer is an adenocarcinoma in 97% of cases.These epidemiological and clinical features invite practitioners to reconsider the concept of rarity of colorectal cancer in Africa and vigilance in front of a digestive disorder, even the patient is young, they also emphasize the need to conduct prospective studies to identify specific risk factors and develop appropriate strategies for the prevention and treatment of colorectal cancer in Mali.Les caractéristiques épidémiologiques, cliniques et biologiques du cancer colorectal sont variables selon les continents, probablement à cause de facteurs de risque différents et non encore complètement répertoriés dans les pays à faibles moyens. Cette étude décrit les particularités épidémiologiques et cliniques du cancer colorectal dans un service hospitalier de référence spécialisé de Bamako au Mali.L'étude rétrospective a concerné la période de 2005 à 2011. Elle a inclus les dossiers des patients atteints de cancer colorectal documenté histologiquement. Ont été analysés, les données épidémiologiques, cliniques et biologiques enregistrées dans les dossiers.Cent-treize (113) cas de cancer colorectal ont été colligés et représentaient 15% de l'ensemble des cancers diagnostiqués sur 7 ans. Ces cas étaient en augmentation d'année en année et s'observaient chez une population souvent jeune (23% avant 30 ans, 60% avant 50 ans). Le sexe masculin était significativement plus représenté, soit 2 hommes pour une femme. Le délai entre le premier symptôme et la prise en charge du malade était long avec dans 85% des cas, un diagnostic porté à un stade métastatique. La localisation était plus souvent colique (56%) que rectale (44%). Le cancer était un adénocarcinome dans 97% des cas.Ces particularités épidémiologiques et cliniques invitent à reconsidérer le concept de rareté du cancer colorectal en Afrique et à la vigilance de la part des praticiens devant un trouble digestif même chez un sujet jeune, ils soulignent aussi la nécessité de conduire des études prospectives pour identifier des facteurs de risque particuliers et élaborer les stratégies les mieux adaptées pour la prévention et la prise en charge du cancer colorectal au Mali.

Resume La vasculopathie drepanocytaire expose a un risque eleve d’accident vasculaire cerebral (AVC) pouvant entrainer le deces ou un handicap important. Le doppler transcrânien (DTC) permet d’identifier les drepanocytaires a risque de cette complication majeure de la maladie, afin de reduire par un traitement approprie, la morbidite et la mortalite qu’elle peut engendrer. De 2008 a 2013, nous avons effectue un depistage systematique de la vasculopathie cerebrale par DTC chez 572 drepanocytaires, âges de 1 a 17 ans et repartis entre 375 sujets SS, 144 SC, 26 S/β 0 et 27 S/β + thalassemiques. Apres exclusion de 30 cas de DTC incomplets et d’un cas de DTC pathologique observe dans un contexte de defaillance multiviscerale, nous avons constate un resultat pathologique ou limite chez 18 % des 541 enfants examines a l’etat basal. Les prevalences de DTC pathologique les plus elevees ont ete observees chez les homozygotes SS (8,1 %). Aucun cas de DTC anormal n’a ete observe chez les enfants S/β + thalassemiques. Les taux d’hemoglobine (Hb) etaient significativement plus bas en cas de resultat au DTC limite ou pathologique. Parmi 68 enfants dont les resultats etaient limites, 9 (13 %) ont presente des resultats pathologiques au bout d’un an de suivi. Dans ce groupe, une diminution ou une augmentation du taux d’Hb de base etait predictive d’un resultat limite ou pathologique au controle. Les enfants dont le resultat etait pathologique ont beneficie, autant que possible, de transfusions ou d’echanges transfusionnels mensuels. Un cas d’AVC transitoire sans signe neurologique, dans un contexte de deglobulisation massive par paludisme et un deces ont ete observes. Ces resultats soulignent la necessite de la pratique systematique du DTC au cours du suivi de l’enfant drepanocytaire et l’urgence de la mise en œuvre de programmes de transfusions chroniques adaptes aux contextes de difficultes d’acces a des transfusions regulieres et securisees.

Although most individuals with sickle cell disease (SCD) live in sub-Saharan Africa, the history of the disease in this continent remains largely unknown. SCD is characterized by chronic hemolytic anemia, acute-vaso occlusive events and progressive vascular organ damage. The CADRE study is a large cohort of SCD patients in five countries of West and Central Africa aiming at studying SCD-related vascular complications. The inclusion data of this study did not match the hyper-hemolysis paradigm (Dubert et al. Blood 2018), but several methodological limitations were raised, including probable mortality bias and questionable reliability of classical hemolysis markers in Africa. For the 5-year follow-up of the CADRE study, we designed a case-control study nested in the cohort, based on extreme phenotypes, to look for new markers of vasculopathy, including new markers of hemolysis. Patients and Methods SS adult patients of the CADRE cohort were selected in the centres of Dakar (Senegal) and Bamako (Mali), depending on the presence of none or at least one of the following complications at inclusion: tricuspid regurgitant jet velocity (TRJV)>2,5 m/s (which may indicate pulmonary hypertension), albuminuria/creatininuria>100 mg/g, leg ulcer, priapism, aseptic osteonecrosis and retinopathy. We chose the youngest patients with a vascular complication and the oldest without any complication. Overall, 6 groups of 40 SS patients with extreme phenotypes were constituted (20 in each centre). Patients were called for a specific visit and investigated at steady state. Besides clinical examination, usual laboratory blood tests and albuminuria measure, additional plasma and saliva samples were collected. A trained investigator isolated microparticles immediately after blood sampling by successive centrifugations, measured blood and plasma viscosities and assessed microcirculation function using peripheral arterial tonometry. A cardio-echography was performed by a trained cardiologist. Plasma samples were stored at -80 °C and shipped to Paris. High technology tests were performed in Paris, including blood cell derived microparticles, free hemoglobin, inflammatory cytokines, neutrophile extracellular trap (NETs). Using saliva DNA, we also genotyped the known SCD genetic modifiers and new candidate genes implicated in the catabolism of hemoglobin. Potential associations between those markers, usual hematological parameters, and the vascular complications were assessed statistically . Results We recalled 240 selected patients 5 years after their first visit: 38 could not be retrieved, 21 had deceased, 62 had at least one new complication, and only 15 still had no complication. Therefore, we selected 56 more patients to obtain at least 30 patients in each group. 237 SS adults were eventually investigated and the plasma samples of 232 SS patients were analyzable in Paris (106 from Bamako and 126 from Dakar). In these patients, at a mean age of 29 years (+/- 11), high TRJV was present in 58, macroalbuminuria in 33, leg ulcers in 36, priapism in 43, aseptic osteonecrosis in 45 and retinopathy in 31 whereas 28 had no vascular complication. A principal component analysis found no cluster of complications. Among patients with one "hyper-viscous" complication (retinopathy or osteonecrosis) or more, 78% also had at least one "hyper-haemolytic" complication (high TRJV, albuminuria, leg ulcer or priapism) and 49% of patients with a "hyper-haemolytic" complication also had a "hyper-viscous" complication. The microvascular function was not associated with any of the complications, whereas higher blood viscosity was associated with retinopathy. The results of the associations between the vascular complications and specific biological tests are presented in other publications. Conclusion This study illustrates the feasibility of high-technology experiments in SCD patients living in sub-Saharan Africa, but was particularly challenging because of the difficulty to prepare, store and transport frozen plasma samples. Moreover, in agreement with previously published data from the CADRE study, we found that the dichotomization of vascular complications into hyperhemolytic and hyperviscous subgroups is not clinically relevant in Africa. Other simple predictive markers of vascular complication are needed to optimize the follow-up of African patients with SCD. Disclosures No relevant conflicts of interest to declare.

Introduction Although most individuals with sickle cell disease (SCD) live in sub-Saharan Africa, the history of the disease on this continent remains largely unknown. SCD is characterized by the association of chronic hemolytic anemia with episodes of acute vaso-occlusive events and progressive vascular organ damage. Several pathophysiological pathways in SCD result in the activation of circulating blood cells and the release of microparticles (MPs). In the present study, we investigated cell-derived MPs in patients with SCD living in Africa and analyzed their relationship with clinical complications. Patients and Methods This cross-sectional case-control study is nested in the CADRE cohort (clinical trials.gov identifier NCTO3114137). We included 232 SS adults in two African centers: Bamako (Mali) and Dakar (Senegal). Patients were chosen depending on the absence or the presence of at least one of the following complications: tricuspid regurgitant jet velocity (TRJV) >3 m/s (which may indicate pulmonary hypertension), macroalbuminuria, leg ulcer, priapism, aseptic osteonecrosis, and retinopathy. Overall, 7 groups of 40 SS patients were constituted (20 in each center). Patients were investigated at steady state (i.e.,at least 15 days after a vaso-occlusive crisis, 8 days after fever or infectious disease, and 3 months after a transfusion). MPs were isolated in the African centers immediately after blood sampling by successive centrifugations at increasing speed: 2,500g x2 and 21,000g x2. MPs pellets were stored at -80 °C. The cellular origin of the MPs, erythrocyte, reticulocyte, endothelial, platelet, and leucocyte, was determined using antibodies directed against CD235a, CD71, CD106, CD41, and CD45, respectively, at the National Institute of Blood TransfusioninParis. To maintain the background at an acceptable level, events of 0.16 µm size were excluded (Fig 1A). Only MPs positive for Annexin V and the cell-type-specific labelling were retained. Potential associations between cell-derived MPs, hematological parameters, and vascular complications were assessed using logistic regression with adjustment for age, sex and country. Results The MP pellets of 106 SS patients from Bamako and 126 from Dakar were analyzed in Paris. In these patients, at a mean age of 29 years (+/- 11), high TRJV was present in 64, microalbuminuria in 84, leg ulcers in 33, priapism in 43, aseptic osteonecrosis in 45, and retinopathy in 31 patients whereas 28 patients had no complication at the time of sampling. As a typical result, Fig 1B shows erythrocyte-derived MPs labelled by Annexin V and CD235a (quarter Q2). The MPs distribution was as follows: erythroid 52% [reticulocytes (CD235a+CD71+) 14%, erythrocytes (CD235a+ CD71-) 38%], leukocyte 18%, platelet 21%, and endothelial 9%. Neither erythrocyte- nor reticulocyte-derived MPs significantly correlated with hemolysis markers (LDH, unconjugated bilirubin or reticulocytes) or hemoglobin levels. Erythrocyte- and reticulocyte-derived MPs were significantly lower in patients with retinopathy (OR=0.48, p=0.003 and OR=0.68, p=0.005, respectively). Reticulocyte-derived MPs were negatively associated with a history of priapism (OR=0.76, p=0.020) and positively associated with a history of leg ulcers (OR=1.23, p=0.040). No correlation was found between MPs of other cellular origin and chronic complications, except for a negative association between endothelial-derived MPs and priapism (OR=0.76, p=0.036). Conclusion In our African patients with SCD, erythroid-derived MPs, although recognized cellular products of hemolysis, were not associated with other markers of hemolysis. We hypothesize that erythroid MPs are not only derived from hemolysis but also probably from the sickling process in this population. They were negatively associated with retinopathy and priapism and positively associated with leg ulcers, but not with the other complications classically associated with the hyperhemolytic sub-phenotype. The search for pertinent biomarkers of SCD complications in Africa is an essential challenge. To our best knowledge, this report is the first illustrating the feasibility of high-technology experiments in an African context. Disclosures No relevant conflicts of interest to declare.

Human parvovirus B19 (HP-19) is the only Parvoviridae known to be pathogenic in human. Studies of HP-19 infection and its associated life-threatening complications in sickle cell anemia patients have been reported in Europe and the US. These results justify the development of HP-B19 prevention and strategies to reduce the incidence of severe and life-threatening complications associated with the infection in patients with sickle cell anemia, particularly in sub-Saharan Africa where the sickle cell anemia burden is high. In light of these considerations, we conducted a case-control study including 163 patients with sickle cell anemia and 163 controls. HP-B19 diagnosis was based on the detection of IgG and IgM antibodies specific for HP-B19 using commercially available enzyme immunoassays. Anti-human parvovirus B19 IgG antibodies were found in 105 of 193 (64.8%) patients vs 79 of 193 controls (48.4%). IgM antibodies were found at a higher frequency in sickle cell anemia patients than in controls. This higher frequency was found to be age-dependent. However, the reticulocyte count showed no significant decrease in Malian patients with sickle cell anemia. Further studies are needed to better characterize the implication of HP-B19 infection in sickle cell anemia mortality and morbidity and to develop preventive strategies and efficient management of the resulting complications.

Introduction Growth failure has been a well-known complication of sickle cell disease (SCD) since the 70s. More recent studies show that the proportion of underweight children with SCD has decreased significantly, thanks to modern treatments (in particular hydroxyurea and iterative transfusions), with a tendency towards overweight and even obesity. However, most studies have been carried out in high-income countries, while 80% of the affected children are born in sub-Saharan Africa, where the environment and the medical care are entirely different. We carried out a case-control study nested in a multinational African cohort to study the growth of sickle cell children and the possible factors influencing growth failure. Methods We performed a case-control transversal study nested in the CADRE cohort that includes SCD patients from five African countries: Cameroon, Gabon, Côte d'Ivoire, Mali and Senegal. All children aged 5 to 21 years-old from this cohort were included in our study. Healthy controls were recruited among the patients' siblings or the children of health workers from each center. The main parameters studied were: medical history, height, weight, blood pressure; hemoglobin phenotype (SS, Sβ0, SC or Sβ +); complete blood count; hemolysis markers (LDH and bilirubin); microalbuminuria; echocardiographic parameters. The primary endpoint was growth failure, defined as a weight, height or BMI below the 5th percentile of the WHO growth charts. We described the frequency of growth failure according to hemoglobin phenotype, age and sex. Then we assessed by multivariate logistic regression in two SCD phenotypic groups (SS or Sβ0 and SC or Sβ +) the association between growth failure and the biological characteristics or the history of SCD-related complications. Results 2296 patients (1799 SS, 114 Sβ0, 287 SC, 96 Sβ+ patients and 287 controls were enrolled in Cameroon (n=735), Ivory Coast (n=380), Gabon (n=298), Mali (n=589) and Senegal (n=581). Overall, 48% of the patients were male and their median [interquartile range] age was 12 [8-16] years-old. Growth failure was diagnosed in 51% of SS, 58% of Sβ0, 44% of SC, 38% of Sβ+patients and 32% of controls, with deeper underweight than linear growth retardation. Beyond the age of 18, the mean BMI of SCD patients was again similar to that of controls in girls, but remained lower in boys, whereas the mean height was similar to that of controls regardless of sex (Figures a to d). Growth failure was more frequent in boys than in girls and maximal between 13 and 16 years-old (Figures e and f). In univariate analysis, the prevalence of growth retardation was associated with the country (highest in Senegal, lowest in Cameroon), age, male sex, hemoglobin phenotype, levels of anemia and hemolysis (p Conclusion In sub-Saharan Africa, growth failure occurs in more than half of the SCD children and concerns weight more than height. Its prevalence is particularly high in SS or Sβ0 patients and during adolescence, due to pubertal delay. Growth failure in SCD children is associated with male sex, anemia and high hemolysis markers independently of the hemoglobin phenotype, but is not independently associated with the occurrence of acute or chronic vascular complications of sickle cell disease, apart from microalbuminuria. However, the long-term consequences of growth failure in sickle cell disease should be evaluated in a longitudinal study. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.