Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Rôle de PIEZO1 au cours de la différentiation érythroïde normale et des stomatocytoses à cellules déshydratées

Hereditary xerocytosis (HX) is a dominant red cell membrane disorder mostly caused by gain-of-function mutations in PIEZO1. PIEZO1 encodes a mechanosensitive ion channel that translates a mechanic stimulus into calcium influx. We found that PIEZO1 is expressed early in human erythroid progenitors. We show for the first time that PIEZO1 activation enables a significant slowdown in erythroid maturation kinetics, both in cell lines and human primary cells. This effect is PIEZO1-dependant since reverted using a specific shRNA knockdown. In human primary cells, PIEZO1 maintains erythroid cells longer at an immature stage and modify the transcriptional balance in favor of genes associated with early erythropoiesis, as shown by a high GATA2/GATA1 ratio and a decreased α/β-globin expression. We also observe a reduced proliferation rate, with accumulation of cells in G0/G1 of cell cycle. PIEZO1-mediated effects require calcium-dependent activation of NFAT and ERK1/2 pathway. In primary erythroid cells, PIEZO1 activation synergizes with EPO to activate STAT5 and ERK, suggesting a modulation of signaling pathways downstream EPO-R activation. The delayed in vitro erythroid differentiation is confirmed in progenitors from 14 PIEZO1-mutated patients, from 11 families, carrying 10 different mutations, with heterogenous intensity. We are currently exploring calcium dependent pathways involved downstream PIEZO1, and PKCalpha represents a good candidate to make the link between calcium and EPO signaling. Our data demonstrate a new role for PIEZO1 during erythropoiesis, that may reveal new insights in the pathophysiology of HXLa stomatocytose héréditaire à cellules déshydratées, ou xérocytose héréditaire (XH) est une pathologie autosomique dominante de la membrane des globules rouges, essentiellement causée par des mutations "gain de fonction" de PIEZO1. PIEZO1 est un canal ionique mécanosensible capable de transformer un stimulus mécanique en un influx de calcium. Nous avons découvert que PIEZO1 est précocement exprimé au cours de l'érythropoïèse. Pour la première fois, nous montrons que l'activation de PIEZO1 ralentit significativement la différenciation érythroïde, à la fois des lignées cellulaires et des cellules primaires humaines. Cet effet est spécifique de PIEZO1 puisqu'il est bloqué par un knockdown spécifique via shRNA. Dans les cellules primaires humaines, PIEZO1 maintient plus longtemps les cellules érythroïdes à un stade immature, et favorise la transcription de gènes associés à l'érythropoïèse précoce, avec majoration du rapport GATA2/GATA1 et diminution de l'expression des α/β-globines. L'activation de PIEZO1 réduit la prolifération cellulaire, avec une accumulation de cellules en phase G0/G1 du cycle cellulaire. L'effet médié par PIEZO1 requiert un influx calcique, et l'activation des voies NFAT et ERK1/2 en aval. Dans les cellules érythroïdes primaires, l'activation de PIEZO1 potentialise la réponse à l'EPO pour activer STAT5 et ERK, ce qui suggère une modulation des voies de signalisation en aval de l'EPO-R. Le retard de différenciation érythroïde in vitro a été confirmé de façon hétérogène chez 14 patients présentant une mutation PIEZO1, issus de 11 familles, porteurs de 10 mutations différentes. Nous explorons actuellement les voies dépendantes du calcium potentiellement impliquées en aval de PIEZO1, et la PKCalpha représente un bon candidat pour établir le lien entre le calcium et la signalisation EPO-dépendante. Nos données soulignent un nouveau rôle pour PIEZO1 au cours de l'érythropoïèse, qui révèle ainsi de nouvelles connaissances sur la physiopathologie des X

Rôle de PIEZO1 au cours de la différentiation érythroïde normale et des stomatocytoses à cellules déshydratées

Hereditary xerocytosis (HX) is a dominant red cell membrane disorder mostly caused by gain-of-function mutations in PIEZO1. PIEZO1 encodes a mechanosensitive ion channel that translates a mechanic stimulus into calcium influx. We found that PIEZO1 is expressed early in human erythroid progenitors. We show for the first time that PIEZO1 activation enables a significant slowdown in erythroid maturation kinetics, both in cell lines and human primary cells. This effect is PIEZO1-dependant since reverted using a specific shRNA knockdown. In human primary cells, PIEZO1 maintains erythroid cells longer at an immature stage and modify the transcriptional balance in favor of genes associated with early erythropoiesis, as shown by a high GATA2/GATA1 ratio and a decreased α/β-globin expression. We also observe a reduced proliferation rate, with accumulation of cells in G0/G1 of cell cycle. PIEZO1-mediated effects require calcium-dependent activation of NFAT and ERK1/2 pathway. In primary erythroid cells, PIEZO1 activation synergizes with EPO to activate STAT5 and ERK, suggesting a modulation of signaling pathways downstream EPO-R activation. The delayed in vitro erythroid differentiation is confirmed in progenitors from 14 PIEZO1-mutated patients, from 11 families, carrying 10 different mutations, with heterogenous intensity. We are currently exploring calcium dependent pathways involved downstream PIEZO1, and PKCalpha represents a good candidate to make the link between calcium and EPO signaling. Our data demonstrate a new role for PIEZO1 during erythropoiesis, that may reveal new insights in the pathophysiology of HXLa stomatocytose héréditaire à cellules déshydratées, ou xérocytose héréditaire (XH) est une pathologie autosomique dominante de la membrane des globules rouges, essentiellement causée par des mutations "gain de fonction" de PIEZO1. PIEZO1 est un canal ionique mécanosensible capable de transformer un stimulus mécanique en un influx de calcium. Nous avons découvert que PIEZO1 est précocement exprimé au cours de l'érythropoïèse. Pour la première fois, nous montrons que l'activation de PIEZO1 ralentit significativement la différenciation érythroïde, à la fois des lignées cellulaires et des cellules primaires humaines. Cet effet est spécifique de PIEZO1 puisqu'il est bloqué par un knockdown spécifique via shRNA. Dans les cellules primaires humaines, PIEZO1 maintient plus longtemps les cellules érythroïdes à un stade immature, et favorise la transcription de gènes associés à l'érythropoïèse précoce, avec majoration du rapport GATA2/GATA1 et diminution de l'expression des α/β-globines. L'activation de PIEZO1 réduit la prolifération cellulaire, avec une accumulation de cellules en phase G0/G1 du cycle cellulaire. L'effet médié par PIEZO1 requiert un influx calcique, et l'activation des voies NFAT et ERK1/2 en aval. Dans les cellules érythroïdes primaires, l'activation de PIEZO1 potentialise la réponse à l'EPO pour activer STAT5 et ERK, ce qui suggère une modulation des voies de signalisation en aval de l'EPO-R. Le retard de différenciation érythroïde in vitro a été confirmé de façon hétérogène chez 14 patients présentant une mutation PIEZO1, issus de 11 familles, porteurs de 10 mutations différentes. Nous explorons actuellement les voies dépendantes du calcium potentiellement impliquées en aval de PIEZO1, et la PKCalpha représente un bon candidat pour établir le lien entre le calcium et la signalisation EPO-dépendante. Nos données soulignent un nouveau rôle pour PIEZO1 au cours de l'érythropoïèse, qui révèle ainsi de nouvelles connaissances sur la physiopathologie des X

A role of PIEZO1 activation in erythroid differentiation of normal and hereditary xerocytosis-derived human progenitors

La stomatocytose héréditaire à cellules déshydratées, ou xérocytose héréditaire (XH) est une pathologie autosomique dominante de la membrane des globules rouges, essentiellement causée par des mutations "gain de fonction" de PIEZO1. PIEZO1 est un canal ionique mécanosensible capable de transformer un stimulus mécanique en un influx de calcium. Nous avons découvert que PIEZO1 est précocement exprimé au cours de l'érythropoïèse. Pour la première fois, nous montrons que l'activation de PIEZO1 ralentit significativement la différenciation érythroïde, à la fois des lignées cellulaires et des cellules primaires humaines. Cet effet est spécifique de PIEZO1 puisqu'il est bloqué par un knockdown spécifique via shRNA. Dans les cellules primaires humaines, PIEZO1 maintient plus longtemps les cellules érythroïdes à un stade immature, et favorise la transcription de gènes associés à l'érythropoïèse précoce, avec majoration du rapport GATA2/GATA1 et diminution de l'expression des α/β-globines. L'activation de PIEZO1 réduit la prolifération cellulaire, avec une accumulation de cellules en phase G0/G1 du cycle cellulaire. L'effet médié par PIEZO1 requiert un influx calcique, et l'activation des voies NFAT et ERK1/2 en aval. Dans les cellules érythroïdes primaires, l'activation de PIEZO1 potentialise la réponse à l'EPO pour activer STAT5 et ERK, ce qui suggère une modulation des voies de signalisation en aval de l'EPO-R. Le retard de différenciation érythroïde in vitro a été confirmé de façon hétérogène chez 14 patients présentant une mutation PIEZO1, issus de 11 familles, porteurs de 10 mutations différentes. Nous explorons actuellement les voies dépendantes du calcium potentiellement impliquées en aval de PIEZO1, et la PKCalpha représente un bon candidat pour établir le lien entre le calcium et la signalisation EPO-dépendante. Nos données soulignent un nouveau rôle pour PIEZO1 au cours de l'érythropoïèse, qui révèle ainsi de nouvelles connaissances sur la physiopathologie des XHHereditary xerocytosis (HX) is a dominant red cell membrane disorder mostly caused by gain-of-function mutations in PIEZO1. PIEZO1 encodes a mechanosensitive ion channel that translates a mechanic stimulus into calcium influx. We found that PIEZO1 is expressed early in human erythroid progenitors. We show for the first time that PIEZO1 activation enables a significant slowdown in erythroid maturation kinetics, both in cell lines and human primary cells. This effect is PIEZO1-dependant since reverted using a specific shRNA knockdown. In human primary cells, PIEZO1 maintains erythroid cells longer at an immature stage and modify the transcriptional balance in favor of genes associated with early erythropoiesis, as shown by a high GATA2/GATA1 ratio and a decreased α/β-globin expression. We also observe a reduced proliferation rate, with accumulation of cells in G0/G1 of cell cycle. PIEZO1-mediated effects require calcium-dependent activation of NFAT and ERK1/2 pathway. In primary erythroid cells, PIEZO1 activation synergizes with EPO to activate STAT5 and ERK, suggesting a modulation of signaling pathways downstream EPO-R activation. The delayed in vitro erythroid differentiation is confirmed in progenitors from 14 PIEZO1-mutated patients, from 11 families, carrying 10 different mutations, with heterogenous intensity. We are currently exploring calcium dependent pathways involved downstream PIEZO1, and PKCalpha represents a good candidate to make the link between calcium and EPO signaling. Our data demonstrate a new role for PIEZO1 during erythropoiesis, that may reveal new insights in the pathophysiology of H

Hemoglobinopathies and hemolytic anemias

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Having myeloma before the age of 40: a French retrospective study

International audienceMyeloma is rarely diagnosed at a young age, with less than 2% of patients under the age of 40. The characteristics and outcome of this particular population are poorly understood. Here we present 214 patients who were 40 years of age or younger at the time of diagnosis of myeloma and related conditions in the 2000s. Of these, 189 had symptomatic myeloma, and their baseline characteristics were broadly similar to those of older patients, except for a higher proportion with a poor prognostic ISS score (ISS-1). 90% received intensified therapy followed by first-line autologous stem cell transplantation, and nearly 25% allograft mainly at first or second relapse. With a median follow-up of 76 months, the estimated median survival was 14.5 years. At five years, overall survival was 84%. In multivariate analysis, a high ISS score (ISS-3; HR = 2.14; p = 0.03), unfavourable cytogenetics (HR = 4.54; p < 0.0001), bone lesions (HR = 3.95; p = 0.01), or disease progression (HR = 12.78; p < 0.0001) conferred a shorter survival. Given the low risk of death in the general population of the same age, the relative survival of these patients was relatively close to overall survival (83%), with a 70-fold increased risk of mortality despite their prolonged survival

Recent advances in the pathophysiology of PIEZO1-related hereditary xerocytosis

International audienceHereditary 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

Artificial intelligence to empower diagnosis of myelodysplastic syndromes by multiparametric flow cytometry

International audienceThe diagnosis of myelodysplastic syndromes (MDS) might be challenging and relies on the convergence of cytological, cytogenetic, and molecular arguments. Multiparametric flow cytometry (MFC) helps diagnose MDS, especially when other features are non-contributory, but remains underestimated mostly due to a lack of standardization of cytometers. We present here an innovative model integrating artificial intelligence (AI) with MFC to improve the diagnosis and the classification of MDS. We develop a machine learning model by elasticnet algorithm trained on a cohort of 191 patients and only based on flow cytometry parameters selected by Boruta algorithm, to build a simple but reliable prediction score with 5 parameters. Our MDS prediction score assisted by AI greatly improves the sensitivity of Ogata score while keeping an excellent specificity validated on an external cohort of 89 patients with an AUC = 0.935. This model allows the diagnosis of both high and low risk MDS with 91.8% sensitivity and 92.5% specificity. Interestingly, it highlights a progressive evolution of the score from clonal hematopoiesis of indeterminate potential (CHIP) to highrisk MDS, suggesting a linear evolution between these different stages. By significantly decreasing the overall misclassification of 52% for patients with MDS and of 31.3% for those without MDS (p=0.02), our AI-assisted prediction score outperforms the Ogata score and positions itself as a reliable tool to help diagnose myelodysplastic syndromes

A one-step assay for sorted CD3(+) cell purity and chimerism after hematopoietic stem cell transplantation

International audienceA hematopoietic chimerism assay is the laboratory test for monitoring engraftment and quantifying the proportions of donor and recipient cells after hematopoietic stem cell transplantation recipients. Flow cytometry is the reference method for determining the purity of CD3(+) cells on the chimerism of selected CD3(+) cells. In the present study, we developed a single-step procedure that combines the CD3(+) purity assay (using the PCR-based Non-T Genomic Detection Kit from Accumol, Calgary, Canada) and the qPCR chimerism monitoring assay (the QTRACE qPCR assay from Jeta Molecular, Utrecht, the Netherlands). First, for the CD3(+) purity assay, we used a PCR-friendly protocol by changing the composition of the ready-to-use reaction tubes (buffer and taq polymerase) and obtained a satisfactory calibration plot (R-2 = 0.8924) with a DNA reference scale of 2 ng/mu l. Next, 29 samples (before and after CD3 positive selection) were analyzed, the mean cell purity was, respectively, 19.6% +/- 6.45 and 98.9% +/- 1.07 in the flow cytometry assay; 26.8% +/- 7.63 and 98.5% +/- 1.79 in the PCR-based non-T genomic detection assay. Our results showed that the CD3(+) purity assay using a qPCR kit is a robust alternative to the flow cytometry assay and is associated with time savings when combined with a qPCR chimerism assay

Fast CMV and EBV Reactivations after Ruxolitinib for Graft-Versus-Host Disease Treatment

61st Annual Meeting and Exposition of the American-Society-of-Hematology (ASH), Orlando, FL, DEC 07-10, 2019International audienc