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Limitations in the data used to define thromboprophylaxis for patients with antiphospholipid antibodies (aPLAbs) and thrombosis include uncertainties after an initial provoked venous thromboembolic event (VTE). We aimed to study such cases associated with combined oral contraceptive (COC) intake.We retrospectively analysed thrombotic outcomes after a first COC-associated VTE and positive aPLAbs, with a low risk HERDOO2 score, on low-dose aspirin (LDA) secondary thromboprophylaxis, seen from 2010 to 2021 in 3 tertiary referral centres, one in France and 2 in Russia. Data from 264 patients (distal deep vein thrombosis DVT: 62.9 %), cumulating in 1327.7 patient-years of observation, were collected.There were 22 cases of thrombosis: 16 distal DVTs, 3 proximal, 1 pulmonary embolism (PE) and 2 transient ischemic attacks. Recurrence rate was 1.66 per 100 patient-years (p-y; 95 % CI: 0.96-2.33). No major bleeding occurred. Risk factors affecting recurrence-free survival were the time between first COC intake and VTE (p 0.0001; the shortest, the lower), proximal DVT (p = 0.021), active smoking (p = 0.039), an associated systemic disease (p = 0.043) and circulating monocyte counts (p = 0.001).We observed a low risk of recurrence which was modulated by classical risk factors for VTE. These observational data may provide clues for future randomized controlled trials.

Background Few data are available on thrombotic outcomes during pregnancy and puerperium occurring after an initial provoked venous thromboembolic (VTE) event. Objectives To describe thrombotic outcomes during pregnancy after a first combined oral contraceptive (COC)-associated VTE and the factors associated with recurrence. Methods This was an international multicentric retrospective study on patients referred for thrombophilia screening from January 1, 2010 to January 1, 2021 following a first COC-associated VTE, including women with neither inherited thrombophilia nor antiphospholipid antibodies and focusing on those who had a subsequent pregnancy under the same thromboprophylaxis treatment. Thrombotic recurrences during pregnancy and puerperium as well as risk factors for recurrence were analyzed. Results We included 2,145 pregnant women. A total of 88 thrombotic events, 58 antenatal and 29 postnatal, occurred, mostly during the first trimester of pregnancy and the first 2 weeks of puerperium. Incidence rates were 49.6 (37–62) per 1,000 patient-years during pregnancy and 118.7 (78–159) per 1,000 patient-years during puerperium. Focusing on pulmonary embolism, incidence rates were 1.68 (1–4) per 1,000 patient-years during pregnancy and 65.5 (35–97) per 1,000 patient-years during puerperium.Risk factors for antenatal recurrences were maternal hypercholesterolemia and birth of a very small-for-gestational-age neonate. A risk factor for postnatal recurrence was the incidence of preeclampsia. Conclusion Our multicentric retrospective data show significant rates of VTE recurrence during pregnancy and puerperium in women with a previous VTE event associated with COC, despite a unique low-molecular-weight heparin-based thromboprophylaxis. These results may provide benchmarks and valuable information for designing future randomized controlled trials.

L’impact en santé publique des pathologies respiratoires chroniques ne cesse de croître. Dans ce contexte, il paraît indispensable d’améliorer les modèles d’études du poumon afin de reproduire au plus proche l’architecture pulmonaire complexe, garante des fonctions d’oxygénation et d’épuration du gaz carbonique. Les connaissances actuelles en physiopathologie respiratoire résultent en partie des études de modèles de reconstitution d’épithélium bronchique in vitro à partir de cellules primaires, en deux dimensions sur des inserts, ou en trois dimensions, en organoïdes mimant jusqu’à l’arborescence pulmonaire. Le développement de ces modèles in vitro a connu un nouvel essor grâce aux organoïdes pulmonaires issus de cellules souches pluripotentes et la démocratisation des outils d’édition du génome. Ces apports technologiques récents offrent de nouvelles perspectives en matière de thérapeutiques ou de compréhension physiopathologique et pourraient, dans le futur, ouvrir les portes de la médecine régénératrice pulmonaire.

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease leading to irreversible destruction of the terminal bronchioles. Although the precise patho-physiological mechanisms remain to be elucidated, the bronchial epithelium seems to play a pivotal role in the disease. Recent studies have highlighted a great heterogeneity among COPD patients, with various disease courses including, in about half the cases, an origin in childhood. Modelling of COPD is a major goal but currently available models are imperfect. Our work aims to create a new in vitro cellular model to study the pathology of the disease. The differentiation of human induced pluripotential stem cells (hiPSCs) in bronchial epithelium is a step towards a better understanding of the developmental origin and the identification of new therapeutic targets., La bronchopneumopathie chronique obstructive (BPCO) est une atteinte chronique des voies aériennes distales caractérisée par une destruction irréversible des bronchioles terminales. Bien que les mécanismes physiopathologiques demeurent incompris à ce jour, l’épithélium bronchique semble être le chef d’orchestre de la maladie. De récentes études ont montré l’existence de différentes trajectoires de la maladie incluant dans la moitié des cas une origine pédiatrique. La modélisation de la BPCO constitue donc un enjeu majeur mais les modèles actuels sont imparfaits. La différenciation des cellules souches pluripotentes induites humaines (hiPSC) en épithélium bronchique représente un nouvel outil pour étudier les racines pédiatriques de la maladie et identifier de nouvelles cibles thérapeutiques.

Evidence highlights the concept of multiple trajectories leading to COPD. Early-life events (i.e., in utero lung development) may influence the maximally attained lung function and increase the risk to develop COPD. Human pluripotent stem cells (hiPSC) represent a unique opportunity to model lung development. We generated hiPSC lines from four highly characterized COPD patients with early onset and severe phenotype. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using integration-free Sendai Virus. The cell lines had normal karyotype, expressed pluripotency hallmarks, and differentiated into the three primary germ layers. These lines offer a tool to study early-life origins of COPD.

No reference values are currently available for coagulation assays performed for thrombophilia screening prescribed according to guidelines, after a first venous thromboembolic (VTE) event, and we have no idea of the intra-patient associations between results.We performed a retrospective study of consecutive prescriptions fulfilling guidelines in a French university hospital from 2010 to 2019 (n = 3842) from the Glims® laboratory information system. We collected results of 12 parameters: aPTT, PT, fibrinogen (Fg), one-stage clotting methods for factors VIII, IX, XI and II (FVIII, FIX, FXI, FII), antithrombin (using an amidolytic assay: AT), protein C and S (using clotting assays: PC and PS) and mixing tests of a lupus-anticoagulant sensitive aPTT and of DRVVT.We show the results of the 12 parameters from 3603 individual files with less than 6 missing values, then describe these distributions and correlations between results from 2930 files with no missing value. We give the frequency of results described as indicating a risk of first VTE or of VTE recurrence. We propose 2 quantitative scores linking the 12 parameters at the individual level and reflecting their degree of dispersion with respect to their mean, describe the values of these scores and their associations with thrombophilic results.These normal values should help laboratory workers to validate process results and to assess their degree of originality. Our 2 scores should help to determine the intra-patient plausibility of associations of results. The usefulness of these laboratory scores for predicting clinically-relevant outcomes deserves to be investigated.

Chronic obstructive pulmonary disease (COPD) is a common and preventable airway disease causing significant worldwide mortality and morbidity. Lifetime exposure to tobacco smoking and environmental particles are the two major risk factors. Over recent decades, COPD has become a growing public health problem with an increase in incidence. COPD is defined by airflow limitation due to airway inflammation and small airway remodelling coupled to parenchymal lung destruction. Most patients exhibit neutrophil-predominant airway inflammation combined with an increase in macrophages and CD8+ T-cells. Asthma is a heterogeneous chronic inflammatory airway disease. The most studied subtype is type 2 (T2) high eosinophilic asthma, for which there are an increasing number of biologic agents developed. However, both asthma and COPD are complex and share common pathophysiological mechanisms. They are known as overlapping syndromes as approximately 40% of patients with COPD present an eosinophilic airway inflammation. Several studies suggest a putative role of eosinophilia in lung function decline and COPD exacerbation. Recently, pharmacological agents targeting eosinophilic traits in uncontrolled eosinophilic asthma, especially monoclonal antibodies directed against interleukins (IL-5, IL-4, IL-13) or their receptors, have shown promising results. This review examines data on the rationale for such biological agents and assesses efficacy in T2-endotype COPD patients., Patients with severe COPD and eosinophilic inflammation experience uncontrolled symptoms despite optimal pharmaceutical treatment. The development of new biomarkers is needed for better phenotyping of patients to propose innovative targeted therapy. https://bit.ly/2KzWuNO

RationaleHighly reproducible in vitro generation of human bronchial epithelium from pluripotent stem cells is an unmet key goal for drug screening to treat lung diseases. The possibility of using induced pluripotent stem cells (hiPSC) to model normal and diseased tissue in vitro from a simple blood sample will reshape drug discovery for chronic lung, monogenic and infectious diseases.MethodsWe devised a simple and reliable method that drives a blood sample reprogrammed into hiPSC subsequently differentiated within 45 days into air-liquid interface bronchial epithelium (iALI), through key developmental stages, definitive-endoderm (DE) and Ventralized-Anterior-Foregut-Endoderm (vAFE) cells.ResultsReprogramming blood cells from one healthy and 3 COPD patients, and from skin-derived fibroblasts obtained in one PCD patient, succeeded in 100% of samples using Sendai viruses. Mean cell purity at DE and vAFE stages was greater than 80%, assessed by expression of CXCR4 and NKX2.1, avoiding the need of cell sorting. When transferred to ALI conditions, vAFE cells reliably differentiated within 4 weeks into bronchial epithelium with large zones covered by beating ciliated, basal, goblets, club cells and neuroendocrine cells as found in vivo. Benchmarking all culture conditions including hiPSCs adaptation to single-cell passaging, cell density and differentiation induction timing allowed for consistently producing iALI bronchial epithelium from the five hiPSC lines.ConclusionsReliable reprogramming and differentiation of blood-derived hiPSCs into mature and functional iALI bronchial epithelium is ready for wider use and this will allow better understanding lung disease pathogenesis and accelerating the development of novel gene therapies and drug discovery.

International audience; Recent advances in genome engineering based on the CRISPR/Cas9 technology have revolutionized our ability to manipulate genomic DNA. Its use in human pluripotent stem cells (hPSCs) has allowed a wide range of mutant cell lines to be obtained at an unprecedented rate. The combination of these two groundbreaking technologies has tremendous potential, from disease modeling to stem cell-based therapies. However, the generation, screening and molecular characterization of these cell lines remain a cumbersome and multi-step endeavor. Here, we propose a pipeline of strategies to efficiently generate, sub-clone, and characterize CRISPR/Cas9-edited hPSC lines in the function of the introduced mutation (indels, point mutations, insertion of large constructs, deletions).

Thrombosis is the most common, most feared complication of paroxysmal nocturnal haemoglobinuria (PNH), a striking example of the effect of uncontrolled complement activation and haemolysis on vascular biology and haemostasis. Uncontrolled complement activation and haemolysis may occur at any site and there is a higher incidence of thrombosis at atypical sites. The mechanisms are highly multifactorial and still not fully understood. Eculizumab (a complement C5 inhibitor) has been observed to reduce the number of thrombotic events and, consequently, it is thought that the signalling pathways that depend on the activation of complement C5 may be involved. Complement inhibition, initially using eculizumab together with anticoagulation, is thus the key to treating and preventing thrombosis. New proximal complement inhibitors targeting complement C3 have also shown promising results with further improvements in the clinical prognosis and quality of life of patients. Screening for PNH in patients with unexplained thrombosis must be systematically considered in well characterized cases.

Summary Genomic integrity of human pluripotent stem cells (hPSCs) is essential for research and clinical applications. However, genetic abnormalities can accumulate during hPSC generation and routine culture and following gene editing. Their occurrence should be regularly monitored, but the current assays to assess hPSC genomic integrity are not fully suitable for such regular screening. To address this issue, we first carried out a large meta-analysis of all hPSC genetic abnormalities reported in more than 100 publications and identified 738 recurrent genetic abnormalities (i.e., overlapping abnormalities found in at least five distinct scientific publications). We then developed a test based on the droplet digital PCR technology that can potentially detect more than 90% of these hPSC recurrent genetic abnormalities in DNA extracted from culture supernatant samples. This test can be used to routinely screen genomic integrity in hPSCs., Graphical Abstract, Highlights • A meta-analysis was carried out to list genetic abnormalities detected in hPSCs • Recurrent genetic abnormalities in hPSCs were precisely defined • ddPCR is a robust and sensitive approach to screen these recurrent abnormalities • In culture supernatant digital test can be used to rapidly screen iPSC clones, In this article, De Vos and colleagues performed a comprehensive meta-analysis of genetic abnormalities detected in hPSC lines. They then used this information to devise a ddPCR test targeting the majority of recurrent abnormalities and show that this test can be applied to the cell culture supernatant. Finally, they illustrate the interest of this test for routine hPSC screening.

Introduction La production in vitro, de maniere reproductible et en larges quantite, d’un epithelium bronchique a partir de cellules souches pluripotentes est un objectif cle pour le criblage de medicaments a haut debit pour identifier de nouvelles molecules et la production de cellules pour une therapie cellulaire en pneumologie (Pharmacol Ther 2018 :183, 58-77). Methodes Nous avons concu une methode simple et reproductible qui conduit un simple echantillon de sang en hiPSC par reprogrammation, puis differencie en 45 jours en epithelium bronchique en interface air-liquide (iALI), en suivant successivement les etapes cles du developpement que sont la formation de l’endoderme definitif (DE) et de l’endoderme anterieur ventralise (vAFE). Resultats La reprogrammation des cellules sanguines d’un patient sain et de 3 patients atteints de BPCO, ainsi que de fibroblastes derives de la peau obtenus chez un patient PCD, a ete obtenue en utilisant des virus Sendai. La purete moyenne des cellules aux stades DE et vAFE evaluee par l’expression de CXCR4 et NKX2.1, respectivement, etait superieure a 85 %, evitant ainsi la necessite d’un tri cellulaire. Lorsqu’elles sont transferees dans des conditions de culture d’interface air-liquide (ALI), les cellules vAFE se differencient en 4 semaines en epithelium bronchique avec de grandes zones couvertes par les principales sous-populations cellulaires de l’epithelium bronchique : cellules basales (KRT5 + ), ciliees (TubIV + ), a mucus (Muc5AC + ), Club (CCSP + ), ainsi que des cellules neuroendocrines (CHGA + )(epithelium bronchique iALI). La microscopie electronique confirme la presence de cils motiles et leur frequence de battement est similaire a celui observe dans des HBEC. Certains iALI ont ete maintenus pendant plus de 400 jours. Conclusions La reprogrammation et la differenciation fiables des hiPSC derives du sang, quel que soit leur contexte clinique, en epithelium bronchique iALI mature et fonctionnel est maintenant accessible a une utilisation plus large, ce qui permettra de mieux comprendre la pathogenese des maladies pulmonaires et d’accelerer le developpement de nouvelles therapies geniques et la decouverte de medicaments.

Background & Aim Genetic integrity of human pluripotent stem cells (hPSCs) is essential for the accuracy of the disease models and for the security of cell therapies that are use those cells. Because genetic abnormalities can accumulate during the generation of hPSC, their long term maintenance or during genome editing, it is mandatory to regularly check the genome of hPSCs. However, the current methods to assess genomic integrity of hPSC are not fully suitable for such regular screening. These approaches can be limited in term of resolution, flexibility, cost and time to process. Our aim is improving genetic screening of hPSC. Methods, Results & Conclusion Based on a large meta-analysis of all hPSC genetic abnormalities reported in more than 100 publications, we were able to exhaustively identify recurrent genetic abnormalities accumulating in hPSCs. We then developed a test (iCS-digital test) based on the droplet digital PCR technology that can detect more than 90% of these hPSC recurrent genetic abnormalities in DNA extracted from culture supernatant samples. This test was successfully used to detect copy number variations that arose during cell culture or gene editing. Hence, the iCS-digital test can be used to routinely screen genomic integrity in hPSCs, contribute to secure genome editing workflow, and reduce time and financial losses induced by the use of an abnormal hPSC lines.

International audience; Primary ciliary dyskinesia (PCD) is a rare and heterogeneous genetic disorder that affects the structure and function of motile cilia. In the airway epithelium, impaired ciliary motion results in reduced or absent mucociliary clearance that leads to the appearance of chronic airway infection, sinusitis and bronchiectasis. Currently, there is no effective treatment for PCD, and research is limited by the lack of convenient models to study this disease and investigate innovative therapies. Furthermore, the high heterogeneity of PCD genotypes is likely to hinder the development of a single therapy for all patients. The generation of patient-derived induced pluripotent stem cells (iPSC) and their differentiation into airway epithelium as well as genome editing technologies could represent major tools for in vitro PCD modelling and for developing personalized therapies. Here, we review PCD pathogenesis, and then discuss how human iPSC could be used to model this disease for the development of innovative patient-specific biotherapies.

Wallis Island is part of a French Territory in the South Pacific. In 1980 the prevalence of hypertension and type 2 diabetes mellitus (T2DM) was low, consistent with a subsistence economy. Considerable social and economic changes have occurred over the last 30 years.Survey data from 1980 and 2009 were analysed by sex in 10-year age groups, and 25-64 years age-standardised to the 2008 Census. Means and prevalences were calculated for blood pressure, fasting plasma glucose, body mass index (BMI), blood cholesterol and triglycerides as risk factors contributing to cardiovascular disease.During 1980-2009 there were significant increases (p 0.05) in age-standardised means and prevalences of blood pressure and hypertension, fasting plasma glucose and T2DM, BMI and obesity, blood cholesterol (men) and triglycerides; and non-significant increases in mean diastolic blood pressure and fasting plasma glucose in women. Mean cholesterol and the prevalence of elevated cholesterol declined in women. Hypertension prevalence increased from 12% to 43% in men and from 15% to 30% in women, with 42% of the increase in men and 33% of the increase in women statistically explained by increases in BMI. T2DM increased from 2.3% to 12.2% in men and from 4.0% to 15.8% in women, with 35% of the increase in men and 26% of the increase in women statistically explained by increases in BMI.Risk factors for cardiovascular disease have increased considerably in Wallis Island over the past 30 years, consistent with modernisation in way of life.

A new method was developed by Roche for the measurement of plasma calcium using the chromophore 5-nitro-5'-methyl-(1,2-bis(o-aminophenoxy)ethan-N,N,N',N'-tetraacetic acid (NM-BAPTA) which could have several advantages over the CPC method. The aim of our study was to evaluate the analytical performances of the NM-BAPTA assay from Roche on c701/Cobas 8000® and to perform a comparison study of calcium values with CPC and Arsenazo III methods.The analytical performance including imprecision study, linearity, and stability of the NM-BAPTA assay was tested on the c701/Cobas 8000®analyzer. The most frequent interferences such as magnesium and gadolinium-based contrast agents (Gd-CAs) were examined with spiked human plasma on the selected method. The calcium Arsenazo III method from Horiba (Montpellier, France) installed on ABX Pentra 400® was used as a reference method. Linear regression analysis was performed to compare data from the different methods.The CV of the NM-BAPTA assay showed good analytical performances with CV1.5%, in agreement with the proposed and interim European biologic goals. We found no interference neither with gadobenate dimeglumine nor with gadoteric acid considering significant findings as interference greater than 5%. In the analytical range from 0.85 to 3.80 mmol/L, the NM-BAPTA method was closely correlated to the Arsenazo III method.Our data demonstrate that this new calcium NM-BAPTA method developed by Roche analyzers perform as well as the conventional method, especially for the outermost values. Thus, this new colorimetric assay could substitute the CPC method on Roche analyzers.

Les modeles actuels utilises en laboratoire pour mimer les pathologies pulmonaires, et notamment la BPCO comportent des inconvenients majeurs : – le modele murin du fait des discordances fonctionnelles et anatomiques avec la pathologie humaine ; – les cellules epitheliales primaires bronchiques humaines (HBEC) issue de fibroscopie et cultivees en interface air–liquide (ALI) du fait des quantites limitees produites. Les cellules souches humaines pluripotentes induites (iPSC) sont des cellules caracterisees a la fois par une proliferation in vitro illimitee et une capacite a se differencier en tout type cellulaire. Leur utilisation permettrait d’envisager une production illimitee d’epithelium bronchique humain, etape critique pour envisager des programmes de recherche innovant, des programmes de R&D pharmacologiques a large echelle voire de la therapie cellulaire. Nous avons pu reprogrammer du sang peripherique d’un individu sain et de trois patients porteurs de BPCO tres severe en lignees iPSC. Les cellules iPSC ont ete differentiees successivement en sillon primitif anterieur (APS), puis en endoderme definitif (DE), puis en endoderme intestinal anterieur ventralise (vAFE). L’induction du DE est robuste et caracterise par un taux de CXCR4 > 95 % (n = 6). L’etape de vAFE permet d’obtenir des taux de purete > 80–90 % des progeniteurs bronchiques NKX2.1+ (n = 3). Apres polarisation et passage en ALI, les marquages en immunofluorescence et les PCR a 50 jours de differenciation attestent de la presence de cellules a mucus (Muc5AC), de cellules basales (KRT5), de cellules Club (CCSP) et de cellules ciliees (FoxJ1). L’ajustement de parametres de matrice extracellulaire nous a permis des cellules multiciliees mobiles a partir de la lignee normale HY03, documente par video microscopie, et attestant de leur fonctionnalite (n = 1). En conclusion, nous parvenons a obtenir a partir d’iPSC des progeniteurs bronchiques NKX2.1+ en une semaine avec une excellente purete. Ces progeniteurs peuvent ensuite etre differencies en epithelium bronchique comportant des cellules multiciliees fonctionnelles.

Serum free light chain (FLC) levels are correlated with chronic kidney disease (CKD) stages and are highest in patients on hemodialysis (HD). Aim of this study was to assess the FLC removal efficiency of Elisio™-210H dialyzer using either high-flux HD or on line high efficiency hemodiafiltration (HDF) modalities in CKD-5D patients. In this prospective and comparative study, 20 CKD-5D patients free from multiple myeloma were randomized in two groups: HD versus on line HDF. All patients were dialyzed with Elisio™-210H dialyzer. Serum samples were collected before and after the midweek dialysis session, before randomization and at the end of the study to measure κ and λ FLC concentrations. Reduction ratios were corrected for net ultrafiltration. For both HD and HDF mode, κ and λ FLC concentrations were significantly lower after dialysis than before but median reductions in κ and λ FLC levels were significantly higher in HDF versus HD groups (κ 73.5 vs. 65.5 %, p = 0.04 and λ 51.0 vs. 36.6 %, p = 0.07). After dialysis, all κ/λ ratio values were between 0.26 and 1.65 which is the reference range described in subjects with normal kidney function, for both HD and HDF groups (median κ/λ ratios were 0.80 [0.47–1.22] and 0.67 [0.50–0.79] respectively). This study shows the superiority of on line HDF compared with HD to remove both κ and λ FLC. Moreover, all post-dialysis κ/λ ratios reached normal reference range.