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Chronic kidney disease (CKD) is a global health condition characterized by a progressive deterioration of kidney function. It is associated with high serum levels of uremic toxins (UT), such as Indoxyl Sulfate (IS), which may participate in the genesis of several uremic complications. Anemia is one of the major complications in CKD patients that contribute to cardiovascular disease, increase morbi-mortality, and is associated with a deterioration of kidney failure in these patients. Our study aimed to characterize the impact of IS on CKD-related erythropoiesis. Using cellular and pre-clinical models, we studied cellular and molecular effects of IS on the growth and differentiation of erythroid cells. First, we examined the effect of clinically relevant concentrations of IS (up to 250 μM) in the UT7/EPO cell line. IS at 250 μM increased apoptosis of UT7/EPO cells at 48 h compared to the control condition. We confirmed this apoptotic effect of IS in erythropoiesis in human primary CD34

International audience; Acute lymphoblastic leukemias (ALL) are characterized by a large number of cytogenetic abnormalities of clinical interest that require the use of several complementary techniques. Optical genome mapping (OGM) is based on analysis of ultra-high molecular weight DNA molecules that provides a high-resolution genome-wide analysis highlighting copy number and structural anomalies, including balanced translocations. We compared OGM to standard techniques (karyotyping, fluorescent in situ hybridization, single nucleotide polymorphism-array and reverse transcription multiplex ligation-dependent probe amplification) in 10 selected B or T-ALL. Eighty abnormalities were found using standard techniques of which 72 (90%) were correctly detected using OGM. Eight discrepancies were identified, while 12 additional anomalies were found by OGM. Among the discrepancies, four were detected in raw data but not retained because of filtering issues. However, four were truly missed, either because of a low variant allele frequency or because of a low coverage of some regions. Of the additional anomalies revealed by OGM, seven were confirmed by another technique, some of which are recurrent in ALL such as LMO2-TRA and MYC-TRB fusions. Despite false positive anomalies due to background noise and a case of inter-sample contamination secondarily identified, the OGM technology was relatively simple to use with little practice. Thus, OGM represents a promising alternative to cytogenetic techniques currently performed for ALL characterization. It enables a time and cost effective analysis allowing identification of complex cytogenetic events, including those currently inaccessible to standard techniques.

Among histone deacetylases, HDAC6 is unusual in its cytoplasmic localization. Its inhibition leads to hyperacetylation of non-histone proteins, inhibiting cell cycle, proliferation and apoptosis. Ricolinostat (ACY-1215) is a selective inhibitor of the histone deacetylase HDAC6 with proven efficacy in the treatment of malignant diseases, but anaemia is one of the most frequent side effects. We investigated here the underlying mechanisms of this erythroid toxicity. We first confirmed that HDAC6 was strongly expressed at both RNA and protein levels in CD34

Numerous studies have highlighted the role of post-translational modifications in the regulation of cell proliferation, differentiation and death. Among these modifications, acetylation modifies the physicochemical properties of proteins and modulates their activity, stability, localization and affinity for partner proteins. Through the deacetylation of a wide variety of functional and structural, nuclear and cytoplasmic proteins, histone deacetylases (HDACs) modulate important cellular processes, including hematopoiesis, during which different HDACs, by controlling gene expression or by regulating non-histone protein functions, act sequentially to provide a fine regulation of the differentiation process both in early hematopoietic stem cells and in more mature progenitors. Considering that HDAC inhibitors represent promising targets in cancer treatment, it is necessary to decipher the role of HDACs during hematopoiesis which could be impacted by these therapies. This review will highlight the main mechanisms by which HDACs control the hematopoietic stem cell fate, particularly in the erythroid lineage.

The Gluthation peroxidase (GPX) enzymes are part of the protective system against lipid peroxydation that includes prevention of oxydation and reduction of already oxidized lipid through enzymatic reactions catalyzed by GSH. GPX4 is one of the five GPX able to incorporate selenium. It is also the only GPX able to directly reduce in the membrane the oxidized fatty acids and cholesterol. Recent reports identified GPX4 as the central inhibitor of ferroptosis, a process during which iron-induced peroxydation of membrane lipids causes a specific cell death that can be reverted by lipophilic antioxydants or by iron chelators. GPX4 has recently been involved during mice erythropoiesis: GPX4-/-mice present a hemolytic anemia and a high apoptotic rate in spleen erythroid progenitors. Although transcriptomics and proteomics found it expressed in human erythroid precursors, its role during human erythropoiesis has not been described. Using an in-vitroerythroid differentiation protocol from CD34+cells obtained from apheresis, we confirmed that GPX4 expression was induced at RNA and protein level during differentiation. RSL3, a specific GPX4 inhibitor, didn't affect early steps of erythropoiesis (i.eclonogenic potential and progenitor amplification) nor the early maturation of erythroid precursors (assessed by sequential CD49d/CD235/CD71 staining) but led to a significant decrease in the enucleation rate as assessed by Hoechst staining using flow cytometry (74%±9 DMSO versus 35%±6 RSL3, p Using Western Blot, we observed that RSL3 exposure induced a strong GPX4 depletion in erythroid progenitors while it didn't affect GPX1, another member of the GPX family expressed in erythroid cells. In order to confirm that enucleation defect was related to GPX4 knockdown, we used an Sh-RNA strategy that allowed a 62%±6 GPX4 decrease at RNA level and a 46%±5 at protein level. We observed a significant defect in terminal enucleation in the cells transduced with shGPX4-lentiviruses in comparison with sh-Scramble (59%±5 Sh-Scr versus 39%±6 Sh-Gpx4, p We investigated then whether GPX4-knock-down affected quantitatively or qualitatively the membrane lipid content, which was shown to be involved in the enucleation process. Addition of Cholesterol to RSL3 in the medium partially restored the enucleation rate. However, lipidomics failed to show any significant difference in the total membrane lipid content (and particularly in the cholesterol content) after RSL3 exposure in comparison to DMSO. Since cholesterol is particularly abundant in the lipid rafts, we investigated whether the lipid distribution was qualitatively altered within the cell membrane. We observed a disruption of membrane lipid rafts when cells were exposed to RSL3, as shown by a 60%±12 decrease in the mean cholera toxin fluorescence intensity. GPX4 presence in lipid rafts was confirmed using immunofluorescence showing their co-localization at cell surface in human primary erythroblasts. Since lipid rafts play a role in the contractile ring that separates pyrenocyte from reticulocyte, we evaluated the myosin-light chain phosphorylation using flow cytometry and Western Blot and found it drastically decreased in GPX4-knockdown conditions. In summary, we identified GPX4 as a new actor of human terminal erythroid differentiation, independently to its function in ferroptosis control. We described its interaction at cell surface with lipid rafts that are required for the assembly of the contractile ring and cytokinesis leading to the nucleus extrusion. Disclosures No relevant conflicts of interest to declare.

Awarding the 2021 Nobel to Ardem Patapoutian for the discovery of the PIEZO mechanotransducers has emphasized the importance of touch-sensing mechanisms in cell physiology. It is well known that PIEZO1 is expressed at the surface of red blood cells where it adjusts their hydration status under mechanical constraints. Besides this, recent findings suggest that PIEZO1 plays a broader role in erythroid lineage. This review aims to actualize the knowledge on PIEZO1 functions all along erythropoiesis.PIEZO1 is expressed in erythroid progenitors, and controls proliferation and differentiation of nucleated cells, as well as maturation of reticulocytes. As PIEZO1 detects displacements in the range of cell-cell interactions, it might mediate the interaction between the differentiating cells and their microenvironment through an inside-out activation of integrins on human erythroblasts as suggested by in-vitro data. Moreover, PIEZO1 is also expressed at the surface of macrophages where it regulates red blood cells clearance through erythrophagocytosis.These new findings on PIEZO1 suggest a continuous effect of mechanotransduction all over erythropoiesis from progenitors to clearance of red blood cells. Therefore, they open a new era in the understanding of hereditary xerocytosis pathophysiology, helping identify new potential therapeutic targets for the future.

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After allogeneic hematopoietic stem-cell transplantation (alloHSCT), the chimerism assay is used to monitor cell engraftment and quantify the respective proportions of donor/recipient cells in blood or bone-marrow samples. Here, we aimed to better assess the utility of determining CD3

Monoclonal gammopathy of unknown significance (MGUS), smouldering multiple myeloma (SMM), and multiple myeloma (MM) are very common neoplasms. However, it is often difficult to distinguish between these entities. In the present study, we aimed to classify the most powerful markers that could improve diagnosis by multiparametric flow cytometry (MFC). The present study included 348 patients based on two independent cohorts. We first assessed how representative the data were in the discovery cohort (123 MM, 97 MGUS) and then analysed their respective plasma cell (PC) phenotype in order to obtain a set of correlations with a hypersphere visualisation. Cluster of differentiation (CD)27 and CD38 were differentially expressed in MGUS and MM (P < 0·001). We found by a gradient boosting machine method that the percentage of abnormal PCs and the ratio PC/CD117 positive precursors were the most influential parameters at diagnosis to distinguish MGUS and MM. Finally, we designed a decisional algorithm allowing a predictive classification ≥95% when PC dyscrasias were suspected, without any misclassification between MGUS and SMM. We validated this algorithm in an independent cohort of PC dyscrasias (n = 87 MM, n = 41 MGUS). This artificial intelligence model is freely available online as a diagnostic tool application website for all MFC centers worldwide (https://aihematology.shinyapps.io/PCdyscrasiasToolDg/).

Resume Les folates et cobalamines sont des cofacteurs necessaires a la synthese de l’ADN via des reactions de transfert d’unites monocarbonees. Les consequences d’une carence en l’une ou l’autre de ces vitamines portent d’abord sur les processus tissulaires a haut niveau de renouvellement, au premier rang desquels l’hematopoiese et les tissus muqueux. L’atteinte hematologique est variable, de la classique « anemie megaloblastique » a des formes moins expressives telles que des thrombopenies isolees. Mais le diagnostic doit egalement etre evoque devant des manifestations extrahematologiques qui dependent de fonctions metaboliques propres a chacune de ces vitamines. Ainsi, la vitamine B12 est impliquee dans la synthese de la myeline, et les carences peuvent induire des manifestations neuropsychiatriques parfois irreversibles. Les carences en folates chez la femme enceinte ont, elles, ete associees a des defauts de fermeture du tube neural, et une supplementation systematique des la phase de conception est recommandee. Le diagnostic de routine est base sur les dosages immunologiques des folates et de la vitamine B12 seriques dont l’interpretation doit toujours tenir compte du tableau clinique car ils manquent de specificite et parfois de sensibilite. Dans des situations diagnostiques difficiles, des tests complementaires tels que le dosage de l’homocysteine dans ces deux carences, de l’holo-transcobalamine et de l’acide methyl-malonique dans les carences en B12, ou des folates erythrocytaires dans les carences en folates peuvent etre proposes.

Primary lymphedema, a rare disease, has a genetic cause in ~40% of patients. Recently, loss-of-function mutations in PIEZO1, which encodes the mechanotransducer protein PIEZO1, were described as causing primary lymphedema, when gain-of-function PIEZO1 mutations were attributed to dehydrated hereditary stomatocytosis type-1 (DHS), a dominant red cell hemolytic disorder, with ~20% of patients having perinatal edema. Lymphedema was diagnosed in a 36-year-old man from a three-generation DHS family, with a PIEZO1-allele harboring 3 missense mutations in cis. Four affected family members had severe fetal and neonatal edema, most severe in the proband, whose generalized edema with prevailing ascites resolved after 8 months. Our patient's intermittent lower limb-lymphedema episodes during hot periods appeared at puberty; they became persistent and bilateral at age 32. Clinical Stemmer's sign confirmed lymphedema. Lower leg lymphoscintigraphy showed substantial dermal backflow in both calves, predominantly on the right. Noncontrast magnetic resonance lymphography showed bilateral lower limb lymphedema, dilated dysplastic lymphatic iliac, and inguinal trunks. Exome-sequencing analysis identified no additional pathogenic variation in primary lymphedema-associated genes. This is the first description of well-documented lymphedema in an adult with PIEZO1-DHS. The pathophysiology of PIEZO1-associated primary lymphedema is poorly understood. Whether it infers overlapping phenotypes or different mechanisms of gain- and loss-of-function PIEZO1 mutations deserves further investigation.

International audience; No abstract available

Simple Summary Lung cancer is recognized for having a very poor prognosis with an overall survival rate of 5-years not exceeding 15%. Platinum-doublet therapy is the most current chemotherapeutic treatment used to treat lung tumors. However, resistance to such drugs evolves rapidly in patients with non-small cell lung cancer (NSCLC) and is one of the major reasons behind therapy failure. Tumor recurrence due to chemoresistance is mainly attributed to the presence of cancer stem cells (CSCs) subpopulations. Thus, the identification of resistance actors and markers is necessary. The Orai3 channel has been recently identified as a predictive marker of metastasis and survival in resectable NSCLC tumors. Our results show, for the first time, that the Orai3 channel is able to induce chemoresistance by enriching CSCs population. Our findings present Orai3 as a promising predictive biomarker which could help with selecting chemotherapeutic drugs. Abstract The development of the resistance to platinum salts is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). Among the reasons underlying this resistance is the enrichment of cancer stem cells (CSCs) populations. Several studies have reported the involvement of calcium channels in chemoresistance. The Orai3 channel is overexpressed and constitutes a predictive marker of metastasis in NSCLC tumors. Here, we investigated its role in CSCs populations induced by Cisplatin (CDDP) in two NSCLC cell lines. We found that CDDP treatment increased Orai3 expression, but not Orai1 or STIM1 expression, as well as an enhancement of CSCs markers. Moreover, Orai3 silencing or the reduction of extracellular calcium concentration sensitized the cells to CDDP and led to a reduction in the expression of Nanog and SOX-2. Orai3 contributed to SOCE (Store-operated Calcium entry) in both CDDP-treated and CD133+ subpopulation cells that overexpress Nanog and SOX-2. Interestingly, the ectopic overexpression of Orai3, in the two NSCLC cell lines, lead to an increase of SOCE and expression of CSCs markers. Furthermore, CD133+ cells were unable to overexpress neither Nanog nor SOX-2 when incubated with PI3K inhibitor. Finally, Orai3 silencing reduced Akt phosphorylation. Our work reveals a link between Orai3, CSCs and resistance to CDDP in NSCLC cells.

Background and Aims Chronic kidney disease (CKD) is a global health condition characterized by a progressive deterioration of renal function due to high serum levels of uremic toxins. Anemia is a major trouble in CKD patients that contributes to a faster deterioration of renal failure, leading to cardiovascular disease and increasing morbimortality. Erythropoietin (EPO) is known to contribute to CKD-associated anemia. Thus, accumulation of uremic toxins in blood impairs EPO synthesis, leading to a subsequent impairment of erythropoiesis in the bone marrow. Very few molecular clues explain why erythropoiesis is affected in CKD or explain why erythropoiesis-stimulating agents (ESA) are not efficient in some patients with CKD. The current study aims to characterize the impact of one of the most representative uremic toxins, Indoxyl Sulfate (IS), in CKD-related anemia. IS is a protein-bound uremic toxin derived from the tryptophan dietary metabolism which is difficult to remove by dialysis. Our study demonstrates the molecular effects of IS on the growth and the differentiation of red blood cells in an erythroid cell line and in primary cell cultures CD34+. Method Firstly, we examined in vitro the time-courses of IS under clinically relevant concentrations of IS (250 µM -1 mM) in a human leukemic cell line in which proliferation is induced by EPO, the UT7/EPO cell line. Cell apoptosis, proliferation, differentiation and cell cycle analysis were assessed by the MACSQuant flow cytometry. Erythroid gene expression analysis was assessed by RT-qPCR (Quantstudio 7 flex). The ratio A260/280 assessed the quality of nucleic acids. Western blotting experiments were performed to study protein expression. Human primary CD34+ cells were obtained from mobilized peripheral blood mononuclear cells (MNC) of healthy subjects and were isolated by magnetic microbeads separation on MACS columns. Results IS at 250 µM and 1 mM increased apoptosis of UT7/EPO cell line at 48h compared to control condition. On the other hand, we found no significant effect of IS on the phenotype of UT7/EPO, when using CD235a (Glycophorin A), as a marker for the detection of the erythroid cell lineage. Ki67 cellular levels, a cell proliferation marker, was not altered between control and IS experiments. This indicated that IS did not affect proliferation in UT7/EPO. At 48h, at the clinically relevant concentration of IS (250 µM), we observed an increase of the cell phase cycle Sub-G1. The analysis of erythropoiesis related genes shows that HIF2α was deregulated with IS (250 µM). Finally, in the Epo-EpoR signalling pathway, we studied the activation of the Jak2/Stat5 proteins. Results in human primary CD34+ cells confirmed the apoptotic effect of IS observed in UT7/EPO. Conclusion Our findings suggest that IS, a representative protein-bound uremic toxin, could affect cell viability, apoptosis and the cell cycle. This study suggests clues to develop new therapies for CKD-associated anemia.

Hereditary xerocytosis is a rare red blood cell disease related to gain-of-function mutations in the FAM38A gene, encoding PIEZO1, in 90% of cases. PIEZO1 is a broadly expressed mechano-transducer that plays a major role in many cell systems and tissues that respond to mechanical stress. In erythrocytes, PIEZO1 adapts the intracellular ionic content and cell hydration status to the mechanical constraints induced by the environment. Until recently, the pathophysiology of hereditary xerocytosis was mainly believed to be based on the "PIEZO1-Gardos channel axis" in erythrocytes, according to which PIEZO1-activating mutations induce a calcium influx that secondarily activates the Gardos channel, leading to potassium and water efflux and subsequently to red blood cell dehydration. However, recent studies have demonstrated additional roles for PIEZO1 during early erythropoiesis and reticulocyte maturation as well as roles in other tissues and cells such as lymphatic vessels, hepatocytes, macrophages and platelets that may affect the pathophysiology of the disease. These findings, presented and discussed in this review, broaden our understanding of hereditary xerocytosis beyond that of primarily being a red blood cell disease and identify potential therapeutic targets. This article is protected by copyright. All rights reserved.

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Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), causing the COVID-19, has been declared as pandemic by the World Health Organization. Epidemiological and clinical characteristics of patients with COVID-19 have been largely reported but biological risk factors have not yet been well described. In this retrospective and monocentric study, we explored 35 hematological and biochemical parameters, routinely measured at the Amiens University Hospital laboratory, between February 21, 2020 and March 30, 2020 for patients diagnosed with COVID-19. 154 patients were included in this study. We compared biological parameters collected at hospital admission between patients who survived or not after hospitalization. Non survivor patients displayed lower hemoglobin (p=0.02) and bicarbonate concentrations (p=0.03) and higher potassium concentration (p=0.03) compared to the survivors. We then compared these biological parameters between patients hospitalized in conventional care units and patients hospitalized in intensive care units (ICU). Numerous biological examinations had significant variations, including lymphocyte and neutrophil counts, bicarbonate, calcium and C Reactive Protein concentrations. In multivariate Cox analysis, risk factors for aggravation (defined as ICU admission or death) included low bicarbonate levels and hyponatremia. A significant worse overall survival was associated with hyponatremia, hyperkaliemia and prothrombin time > 16.8 seconds. We then proposed a prognostic score, to be validated in a future prospective study. Thus, these biological parameters, easily available, could help clinicians to identify high risk patients at an early stage of infection.

Acute lymphoblastic leukemias (ALL) are characterized by a large number of genetic abnormalities of prognostic and theranostic interest. Their detection requires the use of several conventional and molecular cytogenetic techniques in order to establish the most exhaustive genetic profile: conventional karyotype, FISH, SNP-Array and RT-MLPA for detection of fusion transcripts. Optical genome mapping technique is based on analysis of ultra-high molecular weight DNA (UHMW: 150 kbp-2.5 Mbp). It allows an automatic, high resolution, genome-wide detection of structural anomalies, including balanced translocations and copy number anomalies, making this technology a promising tool in ALL. However, very few data are available in ALL so far. Our objective was to compare optical mapping (Bionano Genomics) to standard techniques performed routinely (combination of karyotype, FISH, SNP-array and RT-MLPA) in 10 selected B or T-ALL patients carrying at least one major genetic abnormality including t(10;14) TLX1-TRA, t(9;22) BCR-ABL1 (e1a2 and e14a2 transcripts), t(10;11) MLLT10-PICALM, t(11;19) KMT2A-MLLT1, typical hyperdiploid profile, iAMP21, SET-NUP214 fusion, various deletions involving CDKN2A/CDKN2B, IKZF1, PAX5, PTEN, ERG and FBXW7. Abnormalities on SNP-array were retained when allele fraction was > 10% and larger than 1Mbp or > 20 kbp and involving one of the 186 genes defined as potentially relevant in leukemogenesis. A total of 79 anomalies meeting these criteria in SNP-array, and/or found by other standard techniques were identified (45 deletions, 16 whole chromosome gains, 10 duplications or partial gains, and 8 translocations). Bone marrow DMSO pellets from the 10 patients, all treated at the CHU Amiens-Picardie, were used for this study. The UHMW DNA extraction technique provided molecules with an average N50 (≥ 150 kbp) of 286 kbp, and an average coverage of 339x. Direct imaging of the DNA molecules was performed on the Bionano Saphyr system and structural variants were automatically detected using the Rare Variant Pipeline. Abnormalities analysis was blindly performed by two independent operators on the Bionano Access software (v 1.5.2.), one cytogeneticist with no training in optical mapping (operator 1) and the other optical mapping technician without training in cytogenetics (operator 2). Among the 79 relevant anomalies, 72 (91.1%) were found by operator 1 and 71 (89.9%) by operator 2. All (8/8) translocations, 43/45 (95.6%) of deletions, 8/10 (80%) of gains were found by both operators whereas 13/16 (81.25%) and 12/16 (75%) of chromosomal gains were found by operator 1 and 2 respectively. Concerning the 8 discrepancies between optical mapping and standard techniques, 4 of them were filtering issues. Three were low allelic frequency large gains clearly visible in raw data but rejected by software's CNV filters, totally, or partially for a trisomy 3 that caused the discrepancy between the two observers. The fourth was a deletion of VPREB1 gene, erroneously eliminated by operators because included in recurrent deletion zones of IGL locus. The 4 others discrepancies were non detected anomalies. Two of them were copy number variations in Xp22.33 (pseudoautosomal region), a non-covered area with the optical mapping technology, one of which was a P2RY8-CRLF2 fusion. Another sub-clonal gain (trisomy 21) found by karyotype and FISH (quantified at 7% of all nuclei) but not by SNP-array, was missed, even in raw data. The last was a near-tetraploid subclone, found only on karyotype, that might be strictly tetraploid and by definition undetectable by quantitative techniques. Interestingly, new anomalies, not detected by standard techniques, were revealed, including a translocation t(11;14)(p13;q11.2) LMO2-TRA, a translocation with inversion t(7;8)(q34;q24.1) TRB-MYC and a translocation t(14;20)(q32.33;q13.13) IGH-CEBPB. At last, in 3 cases, unidentified complex rearrangements by cytogenetics could be resolved using optical mapping. Thus, optical mapping represents an innovative, time and cost effective alternative to the different cytogenetic techniques currently performed routinely for ALL genetic characterization. It enables identification of complex cytogenetic events, including those currently inaccessible to standard techniques such as chromotripsis or certain complex translocations. Disclosures Lee: Bionano Genomics, San Diego: Current Employment. Ferret:Bionano Genomics: Research Funding.

Multiple molecular disorders can affect mechanisms regulating proliferation and differentiation of growth plate chondrocytes. Mutations in the TRIM37 gene cause the Mulibrey nanism, a heritable growth disorder. Since chondrocytes are instrumental in long bone growth that is deficient in nanism, we hypothesized that TRIM37 defect could contribute to dysregulation of the chondrocyte cell cycle. Western blotting, confocal microscopy and imaging flow cytometry determined TRIM37 expression in CHON-002 cell lineage. We showed that TRIM37 is expressed during mitosis of chondrocytes and directly impacted their proliferation. During the chondrocyte cell cycle, TRIM37 was present in both nucleus and cytoplasm. During M phase we observed an increase of the TRIM37-Tubulin co-localization in comparison with G1, S and G2 phases. TRIM37 knock down inhibited proliferation, together with cell cycle anomalies and increased autophagy, while overexpression accordingly enhanced cell proliferation. We demonstrated that microRNA-223 directly targets TRIM37, and suggest that miR-223 regulates TRIM37 gene expression during the cell cycle. In summary, our results give clues to explain why TRIM37 deficiency in chondrocytes impacts bone growth. Modulating TRIM37 using miR-223 could be an approach to increase chondrogenesis.

Introduction: Southeast Asian Ovalocytosis is a dominantly inherited pathology due to a red blood cell membrane abnormal band 3 protein which is prevalent in Southeast Asia. It is associated with no clinical manifestations or induced hemolytic anemia. Case Report: We report herein a case of a 30 years old patient with both Southeast Asian Ovalocytosis and sickle cell trait who had three splenic infarctions without hypoxic situation. Since the last episode, there was no anemia and reticulocyte count and bilirubin reached normal levels. Conclusion: This abnormal band 3 protein lead to a red blood cell deshydratation which could explain polymerization of HbS and clinical manifestations. Only one case of a retinal stroke in a patient from the Comoros Islands with both Southeast Asian Ovalocytosis and a sickle cell trait has been previously reported in 2009. Splenic infarctions could be therefore another clinical manifestation of this association.

International audience; We describe the clinical, hematologic and genetic characteristics of a retrospective series of 126 subjects from 64 families with hereditary xerocytosis. Twelve patients from six families carried a KCNN4 mutation, five had the recurrent p.Arg352His mutation and one had a new deletion at the exon 7-intron 7 junction. Forty-nine families carried a PIEZO1 mutation, which was a known recurrent mutation in only one-third of the cases and private sequence variation in others; 12 new probably pathogenic missense mutations were identified. The two dominant features leading to diagnosis were hemolysis that persisted after splenectomy and hyperferritinemia, with an inconstant correlation with liver iron content assessed by magnetic resonance imaging. PIEZO1-hereditary xerocytosis was characterized by compensated hemolysis in most cases, perinatal edema of heterogeneous severity in more than 20% of families and a major risk of post-splenectomy thrombotic events, including a high frequency of portal thrombosis. In KCNN4-related disease, the main symptoms were more severe anemia, hemolysis and iron overload, with no clear sign of red cell dehydration; therefore, this disorder would be better described as a ‘Gardos channelopathy’. These data on the largest series to date indicate that PIEZO1-hereditary xerocytosis and Gardos channelopathy are not the same disease although they share hemolysis, a high rate of iron overload and inefficient splenectomy. They demonstrate the high variability in clinical expression as well as genetic bases of PIEZO1-hereditary xerocytosis. These results will help to improve the diagnosis of hereditary xerocytosis and to provide recommendations on the clinical management in terms of splenectomy, iron overload and pregnancy follow-up.

Background : Patients with acute myeloid leukemia in relapse or refractory to induction therapy have dismal prognosis. Response rates to common salvage regimens are low and allogenic hematopoietic stem cell transplantation is the only curative option. Several studies have demonstrated that salvage chemotherapy with sequential conditioning could reduce leukemia relapse risk with an acceptable toxicity profile for unfavorable acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). [1] Therefore, we decided to assess this procedure in our center at Amiens University Hospital. Methods We conducted a monocentric retrospective study, including 53 patients aged over 18 years undergoing a hematopoietic stem cell transplant (HSCT) with sequential conditioning between January 2012 and December 2018, for relapse/refractory AML or high risk MDS. 44 (83%) patients received sequential conditioning containing clofarabine (SET-RIC) or Amsacrine (FLAMSA) and 9 (17%) thiotepa based (TEC-RIC) with post-transplant cyclophosphamide for mismatched donors. Patients who were GVHD free after immunosuppressors withdrawal received immunomodulation as relapse prevention with azacytidine 37.5mg/m²/day 5 days every 4 weeks for 12 cycles with 3 donor lymphocyte infusions (DLI) alterned between azacytidine cycles. Results The median age was 52 years (range 18-70). Before conditioning, 48 patients had unfavorable AML with ELN intermediate score refractory to at least one course of induction therapy or in relapse, or unfavorable ELN score; 5 patients had high risk MDS with complex karyotype. 32 patients (60,5%) had active disease and 21 (39,5%) were in complete remission (CR) including 12 with positive MRD. 13 (24,5%) patients had HLA-identical sibling donors, 27 (51%) match unrelated donors (MUD), 4 (7,5%) mismatch unrelated donors (MMD) and 9 (17%) haploidentical donors. Majority of patients (90,5%) received peripheral blood stem cell (PBSC) PBSC with median CD34+ count of 7,94.106/kg (1,84-8,44). Acute GvHD prophylaxis with Ciclosporin A, in combination with Mycophenolate mofetil for MUD/MMR/Haplo, was withdrawal with a median time of 90 days. With a median follow-up of 40 months, overall survival (OS) at 1 and 2 years was 68% and 52%. Median OS was 18,7 months (0-72,4 months) and median disease free survival (DFS) was 14,9 months (0-72,4 months). 17 patients (32%) experienced relapse after HSCT with a median time from HSCT to relapse of 6 months (1-35 months). 22 (41,5%) of patients presented with grade I-II acute graft versus host disease (GVHD) and 6 (11,3%) with grade III IV aGVHD . GVHD free relapse free survival (GRFS) at 1 and 2 years was 53% and 34,2%. One-year cumulative incidence of disease related death and non-relapse mortality was 12,6% and 17% respectively. 19 patients received immunomodulation with 5 Azacitidine and DLI if no GVHD occurred within day 120. OS was 79 % in the 19 patients receiving DLI. In univariate analysis immunomodulation post HSCT (Figure 1) was significantly associated with overall survival and leukemia free survival (p=0,0164 and p=0,0359 respectively) but not the disease status before HSCT (p=0,7). Immunomodulation administration with azacytidine and DLI was not significantly associated with cGVHD occurrence (p=0.31). Benefit of immunomodulation OS persisted in multivariate analysis (p=0.0284). Conclusion: Sequential conditioning regimen on refractory AML with secondary immunomodulation with azacytidine and DLI shows very good results with an acceptable toxicity profile in unfavorable AML. We achieve very good OS and DFS whatever disease status before HSCT. GRFS is also encouraging comparing to previously report datas [1]. Reference: [1] Similar outcome of allogeneic stem cell transplantation after myeloablative and sequential conditioning regimen in patients with refractory or relapsed acute myeloid leukemia: A study from the Société Francophone de Greffe de Moelle et de Thérapie Cellulaire. Decroocq et al. Am J Hematol 2018 ;93 :416-423. Figure 1 Disclosures No relevant conflicts of interest to declare.

Purpose of the Study: To identify policy gaps in the delivery and availability of assistive health technology (AHT) and medical devices (MD) for aging populations, particularly in low- and middle-income countries (LMICs). Design and Methods: The findings presented in this paper are the results of several narrative overviews. They provide a contextual analysis of the conclusions and evidence from WHO commissioned research and expert consultations in 2013 and 2014, as well as a synthesis of literature reviews conducted on AHT and MD. Results: Practical, life-enhancing support for older people through AHT, MD, and related health and social services is a neglected issue. This is particularly so in LMICs where the biggest increases in aging populations are occurring, and yet where there is commonly little or no access to these vital components of healthy aging. Implications: Health technologies, especially medical and assistive health technology, are essential to ensure older people's dignity and autonomy, but their current and potential benefits have received little recognition in LMICs. Viewing these technologies as relevant only to disabled people is an inadequate approach. They should be accessible to both older adults with disabilities and older adults with functional limitation. Many countries need much greater official awareness of older adults' needs and preferences. Such attitudinal changes should then be reflected in laws and regulations to address the specificities of care for older people.

International audience

Background: Despite being a signatory since 2004, Japan has not yet fully implemented Article 8 of the World Health Organization’s Framework Convention on Tobacco Control regarding 100% protection against exposure to second-hand smoke (SHS). The Japanese government still recognizes designated smoking rooms (DSRs) in public space as a valid control measure. Furthermore, subnational initiatives for tobacco control in Japan are of limited effectiveness. Through an analysis of the Hyogo initiative in 2012, we identified key barriers to the achievement of a smoke-free environment. Methods: Using a descriptive case-study approach, we analyzed the smoke-free policy development process. The information was obtained from meeting minutes and other gray literature, such as public records, well as key informant interviews. Results: Hyogo Prefecture established a committee to propose measures against SHS, and most committee members agreed with establishing completely smoke-free environments. However, the hospitality sector representatives opposed regulation, and tobacco companies were allowed to make a presentation to the committee. Further, political power shifted against completely smoke-free environments in the context of upcoming local elections, which was an obvious barrier to effective regulation. Throughout the approving process, advocacy by civil society for stronger regulation was weak. Eventually, the ordinance approved by the Prefectural Assembly was even weaker than the committee proposal and included wide exemptions. Conclusions: The analysis of Hyogo’s SHS control initiative shed light on three factors that present challenges to implementing tobacco control regulations in Japan, from which other countries can also draw lessons: incomplete national legislation, the weakness of advocacy by the civil society, and the interference of the tobacco industry.

Introductory paragraphThe successful production of Hematopoietic Stem and Progenitor Cells (HSPCs) from human pluripotent sources is conditioned by transgene delivery1-5. We describe here a dedicated and tractable one step, GMP-grade, transgene-free and stroma-free protocol to produce HSPCs from human induced pluripotent stem cells (hiPSCs). This procedure, applied to several sources of hiPSCs with equal efficiency, is based on a directed differentiation with morphogens and cytokines to generate a cell population close to nascent HSPCs or their immediate forerunners i.e., hemogenic endothelial cells6-9. Following engraftment into immunocompromised recipients, this cell population was proved capable of a robust myeloid, lymphoid and definitive red blood cell production in sequential recipients for at least 40 weeks. Further identification of the repopulating cells show that they express the G protein–coupled receptor APELIN (APLNR) and the homing receptor CXCR4. This demonstrates that the generation of bona fide HSPCs from hiPSCs without transgenes is possible and passes through an early endo-hematopoietic intermediate. This work opens the way to the generation of clinical grade HSPCs for the treatment of hematological diseases and holds promise for the analysis of HSPC development in the human species.

To the editor: Red cells of patients with hereditary spherocytosis (HS) have a decreased surface-to-volume ratio,[1][1] leading to their trapping and destruction during their passage through the splenic cords.[2][2][⇓][3]-[4][4] Therefore, surgical total splenectomy (TS) by removing the main site