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Abstract The human population is ageing worldwide. The World Health Organization estimated that the world’s population of people aged 60 years and older will increase to at least 30%, coinciding with a growing frequency of cognitive and cardiovascular disease. Recently, in preclinical studies platelet Factor 4 (PF4) was presented as a pro-cognitive factor. This molecule is released by platelets in the circulation and could be present in blood products destined for transfusion. We wondered if PF4 levels are correlated to the age of the blood donor or to the storage time of platelet concentrates (PCs) intended for transfusion? We observed higher levels of PF4 in PCs from elderly donors compared to younger donors, while PC storage time did not determine PF4 levels expression.

Obesity is a major public health burden worldwide and is characterized by chronic low-grade inflammation driven by the cooperation of the innate immune system and dysregulated metabolism in adipose tissue and other metabolic organs. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a central regulator of inflammatory cell function that coordinates inflammation, apoptosis and necroptosis in response to inflammatory stimuli. Here we show that genetic polymorphisms near the human RIPK1 locus associate with increased RIPK1 gene expression and obesity. We show that one of these single nucleotide polymorphisms is within a binding site for E4BP4 and increases RIPK1 promoter activity and RIPK1 gene expression in adipose tissue. Therapeutic silencing of RIPK1 in vivo in a mouse model of diet-induced obesity dramatically reduces fat mass, total body weight and improves insulin sensitivity, while simultaneously reducing macrophage and promoting invariant natural killer T cell accumulation in adipose tissue. These findings demonstrate that RIPK1 is genetically associated with obesity, and reducing RIPK1 expression is a potential therapeutic approach to target obesity and related diseases.

Summary: Changes in lymphocyte production patterns occurring across human ontogeny remain poorly defined. In this study, we demonstrate that human lymphopoiesis is supported by three waves of embryonic, fetal, and postnatal multi-lymphoid progenitors (MLPs) differing in CD7 and CD10 expression and their output of CD127−/+ early lymphoid progenitors (ELPs). In addition, our results reveal that, like the fetal-to-adult switch in erythropoiesis, transition to postnatal life coincides with a shift from multilineage to B lineage-biased lymphopoiesis and an increase in production of CD127+ ELPs, which persists until puberty. A further developmental transition is observed in elderly individuals whereby B cell differentiation bypasses the CD127+ compartment and branches directly from CD10+ MLPs. Functional analyses indicate that these changes are determined at the level of hematopoietic stem cells. These findings provide insights for understanding identity and function of human MLPs and the establishment and maintenance of adaptative immunity.

Coronavirus disease (COVID)-19 is characterised in particular by vascular inflammation with platelet activation and endothelial dysfunction. During the pandemic, therapeutic plasma exchange (TPE) was used to reduce the cytokine storm in the circulation and delay or prevent ICU admissions. This procedure consists in replacing the inflammatory plasma by fresh frozen plasma from healthy donors and is often used to remove pathogenic molecules from plasma (autoantibodies, immune complexes, toxins, etc.). This study uses an in vitro model of platelet-endothelial cell interactions to assess changes in these interactions by plasma from COVID-19 patients and to determine the extent to which TPE reduces such changes. We noted that exposure of an endothelial monolayer to plasmas from COVID-19 patients post-TPE induced less endothelial permeability compared to COVID-19 control plasmas. Yet, when endothelial cells were co-cultured with healthy platelets and exposed to the plasma, the beneficial effect of TPE on endothelial permeability was somewhat reduced. This was linked to platelet and endothelial phenotypical activation but not with inflammatory molecule secretion. Our work shows that, in parallel to the beneficial removal of inflammatory factors from the circulation, TPE triggers cellular activation which may partly explain the reduction in efficacy in terms of endothelial dysfunction. These findings provide new insights for improving the efficacy of TPE using supporting treatments targeting platelet activation, for instance.

Platelet concentrate (PC) transfusion seeks to provide haemostasis in patients presenting severe central thrombocytopenia or severe bleeding. PCs may induce adverse reactions (AR) that can occasionally be severe (SAR). PCs contain active biomolecules such as cytokines and lipid mediators. The processing and storage of PCs creates so-called structural and biochemical storage lesions that accumulate when blood products reach their shelf life. We sought to investigate lipid mediators as bioactive molecules of interest during storage and review associations with adverse reactions post-transfusion. To facilitate understanding, we focused on single donor apheresis (SDA) PCs with approximately 31.8% of PCs being delivered in our setting. Indeed, pooled PCs are the most widely transfused products, but the study of a single donor lipid mediator is easier to interpret. We are investigating key lipid mediators involved in AR. Adverse reactions were closely monitored in accordance with current national and regional haemovigilance protocols. Residual PCs were analysed post-transfusion in a series of observations, both with and without severe reactions in recipients. A decrease in the lysophosphatidylcholine species to produce the lysophosphatidic acid species has been observed during storage and in the case of AR. Lysophosphatidic acid increased with primarily platelet-inhibitor lipids. Anti-inflammatory platelet-induced inhibition lipids were weakly expressed in cases of severe adverse reactions. We therefore propose that a decrease in lysophosphatidylcholine and an increase in lysophosphatidic acid can prospectively predict serious adverse transfusion reactions.

We conducted research to understand online trade in jaguar parts and develop tools of utility for jaguars and other species. Our research took place to identify potential trade across 31 online platforms in Spanish, Portuguese, English, Dutch, French, Chinese, and Vietnamese. We identified 230 posts from between 2009 and 2019. We screened the images of animal parts shown in search results to verify if from jaguar; 71 posts on 12 different platforms in four languages were accompanied by images identified as definitely jaguar, including a total of 125 jaguar parts (50.7% posts in Spanish, 25.4% Portuguese, 22.5% Chinese and 1.4% French). Search effort varied among languages due to staff availability. Standardizing for effort across languages by dividing number of posts advertising jaguars by search time and number of individual searches completed via term/platform combinations changed the proportions the rankings of posts adjusted for effort were led by Portuguese, Chinese, and Spanish. Teeth were the most common part; 156 posts offered at least 367 teeth and from these, 95 were assessed as definitely jaguar; 71 of which could be linked to a location, with the majority offered for sale from Mexico, China, Bolivia, and Brazil (26.8, 25.4, 16.9, and 12.7% respectively). The second most traded item, skins and derivative items were only identified from Latin America: Brazil (7), followed by Peru (6), Bolivia (3), Mexico (2 and 1 skin piece), and Nicaragua and Venezuela (1 each). Whether by number of posts or pieces, the most commonly parts were: teeth, skins/pieces of skins, heads, and bodies. Our research took place within a longer-term project to assist law enforcement in host countries to better identify potential illegal trade and presents a snapshot of online jaguar trade and methods that also may have utility for many species traded online.

Summary: Background: COVID-19 convalescent plasma (CCP) contains neutralising anti-SARS-CoV-2 antibodies that may be useful as COVID-19 passive immunotherapy in patients at risk of developing severe disease. Such plasma from convalescent patients may also have additional immune-modulatory properties when transfused to COVID-19 patients. Methods: CCP (n = 766) was compared to non-convalescent control plasma (n = 166) for soluble inflammatory markers, ex-vivo inflammatory bioactivity on endothelial cells, neutralising auto-Abs to type I IFNs and reported adverse events in the recipients. Findings: CCP exhibited a statistically significant increase in IL-6 and TNF-alpha levels (0.531 ± 0.04 vs 0.271 ± 0.04; (95% confidence interval [CI], 0.07371–0.4446; p = 0.0061) and 0.900 ± 0.07 vs 0.283 ± 0.07 pg/mL; (95% [CI], 0.3097–0.9202; p = 0.0000829) and lower IL-10 (0.731 ± 0.07 vs 1.22 ± 0.19 pg/mL; (95% [CI], −0.8180 to −0.1633; p = 0.0034) levels than control plasma. Neutralising auto-Abs against type I IFNs were detected in 14/766 (1.8%) CCPs and were not associated with reported adverse events when transfused. Inflammatory markers and bioactivity in CCP with or without auto-Abs, or in CCP whether or not linked to adverse events in transfused patients, did not differ to a statistically significant extent. Interpretation: Overall, CCP exhibited moderately increased inflammatory markers compared to the control plasma with no discernible differences in ex-vivo bioactivity. Auto-Abs to type I IFNs detected in a small fraction of CCP were not associated with reported adverse events or differences in inflammatory markers. Additional studies, including careful clinical evaluation of patients treated with CCP, are required in order to further define the clinical relevance of these findings. Funding: French National Blood Service—EFS, the Association “Les Amis de Rémi” Savigneux, France, the “Fondation pour la Recherche Médicale (Medical Research Foundation)–REACTing 2020”.

The northwest basins of the Atlantic and Pacific oceans are regions of intense western boundary currents (WBCs): the Gulf Stream and the Kuroshio. The variability of these poleward currents and their extensions in the open ocean is of major importance to the climate system. It is largely dominated by in-phase meridional shifts downstream of the points at which they separate from the coast. Tide gauges on the adjacent coastlines have measured the inshore sea level for many decades and provide a unique window on the past of the oceanic circulation. The relationship between coastal sea level and the variability of the western boundary currents has been previously studied in each basin separately, but comparison between the two basins is missing. Here we show for each basin that the inshore sea level upstream of the separation points is in sustained agreement with the meridional shifts of the western boundary current extension over the period studied, i.e. the past 7 (5) decades in the Atlantic (Pacific). Decomposition of the coastal sea level into principal components allows us to discriminate this variability in the upstream sea level from other sources of variability such as the influence of large meanders in the Pacific. Our result extends previous findings limited to the altimetry era and suggests that prediction of inshore sea-level changes could be improved by the inclusion of meridional shifts of the western boundary current extensions as predictors. Long-duration tide gauges, such as Key West, Fernandina Beach or Hosojima, could be used as proxies for the past meridional shifts of the western boundary current extensions.

We investigate the impact of the strength of the Atlantic Meridional Overturning Circulation (AMOC) at 26∘ N on the prediction of North Atlantic sea surface temperature anomalies (SSTAs) a season ahead. We test the dependence of sea surface temperate (SST) predictive skill in initialised hindcasts on the phase of the AMOC at 26∘N, invoking a seesaw mechanism driven by AMOC fluctuations, with positive SSTAs north of 26∘ N and negative SSTAs south of 26∘ N after a strong AMOC and vice versa. We use initialised simulations with the MPI-ESM-MR (where MR is mixed resolution) seasonal prediction system. First, we use an assimilation experiment between 1979–2014 to confirm that the AMOC leads a SSTA dipole pattern in the tropical and subtropical North Atlantic, with the strongest AMOC fingerprints after 2–4 months. Going beyond previous studies, we find that the AMOC fingerprint has a seasonal dependence and is sensitive to the length of the observational window used, i.e. stronger over the last decade than for the entire time series back to 1979. We then use a set of ensemble hindcast simulations with 30 members, starting each February, May, August and November between 1982 and 2014. We compare the changes in skill between composites based on the AMOC phase a month prior to each start date to simulations without considering the AMOC phase and find subtle influence of the AMOC mechanism on seasonal SST prediction skill. We find higher subtropical SST hindcast skill at a 2–4-month lead time for June–July–August (JJA) SSTA composites based on the AMOC phase at May start dates than for the full time period. In other regions and seasons, we find a negligible impact of the AMOC seesaw mechanism on seasonal SST predictions due to atmospheric influence, calling for caution when considering such a mechanism. Our method shows that, for May start dates following strong AMOC phases, summer SST hindcast skill over the subtropics increases significantly compared to that of weak AMOC phases. This suggests that in the assessment of SST skill for a season ahead an eye should be kept on the initial AMOC state.

Blood products in therapeutic transfusion are now commonly acknowledged to contain biologically active constituents during the processes of preparation. In the midst of a worldwide COVID-19 pandemic, preliminary evidence suggests that convalescent plasma may lessen the severity of COVID-19 if administered early in the disease, particularly in patients with profound B-cell lymphopenia and prolonged COVID-19 symptoms. This study examined the influence of photochemical Pathogen Reduction Treatment (PRT) using amotosalen‐HCl and UVA light in comparison with untreated control convalescent plasma (n= 72 – paired samples) - cFFP, regarding soluble inflammatory factors: sCD40L, IFN-alpha, IFN-beta, IFN-gamma, IL-1 beta, IL-6, IL-8, IL-10, IL-18, TNF-alpha and ex-vivo inflammatory bioactivity on endothelial cells. We didn’t observe significant modulation of the majority of inflammatory soluble factors (8 of 10 molecules tested) pre- or post-PRT. We noted that IL-8 concentrations were significantly decreased in cFFP with PRT, whereas the IL-18 concentration was increased by PRT. In contrast, endothelial cell release of IL-6 was similar whether cFFP was pre-treated with or without PRT. Expression of CD54 and CD31 in the presence of cFFP were similar to control levels, and both were significant decreased in when cFFP had been pre-treated by PRT. It will be interesting to continue investigations of IL-18 and IL-8, and the physiopathological effect of PRT- treated convalescent plasma and in clinical trials. But overall, it appears that cFFP post-PRT were not excessively pro-inflammatory. Further research, including a careful clinical evaluation of CCP-treated patients, will be required to thoroughly define the clinical relevance of these findings.

IntroductionSickle cell disease (SCD) is an inherited hemoglobinopathy disorder. The main consequence is synthesis of hemoglobin S leading to chronic hemolysis associated with morbidity. The aim of this study was to investigate Thrombin Generation Assay (TGA) to assess hypercoagulability in SCD and TGA parameters as biomarkers of vaso-occlusive crisis (VOC) risk and hospitalization within 1 year.Materials and methodsWe performed TGA in platelet poor plasma (PPP) with 1 pM of tissue factor and 4 μM of phospholipid-standardized concentration, in duplicate for patients and controls. We measured thrombomodulin (TM), soluble endothelial Protein C Receptor and Tissue Factor Pathway Inhibitor (TFPI).ResultsA total of 113 adult patients with SCD, 83 at steady state and 30 during VOC, and 25 healthy controls matched on age and gender were included. Among the 83 patients at steady state, (36 S/S-1 S/β0, 20 S/Sα3.7, and 19 S/C-7 S/β+) 28 developed a VOC within 1 year (median: 4 months [2.25–6]). We observed an increase of peak and velocity associated with a shortening of lagtime and time to peak (TTP) and no difference of endogenous thrombin potential (ETP) in patients compared to controls. TFPI (p < 0.001) and TM (p = 0.006) were significantly decreased. TGA confirmed hypercoagulability in all SCD genotypes and clinical status. The association of ETP > 1,207 nM.min and peak >228.5 nM presented a sensitivity of 73.5% and a specificity of 93.9% to predict VOC development within 1 year.ConclusionWe have demonstrated a hypercoagulable state in SCD associated with chronic hemolysis. These preliminary findings suggest that TGA parameters, as ETP and peak, could be used to predict VOC development within 1 year.

Background Extracellular vesicles (EVs) released by blood cells have proinflammation and procoagulant action. Patients with systemic lupus erythematosus (SLE) present high vascular inflammation and are prone to develop cardiovascular diseases. Therefore, we postulated that the EV populations found in blood, including platelet EVs (PEVs) and red blood cell EVs (REVs), are associated with SLE disease activity and SLE-associated cardiovascular accidents.Method We assessed autotaxin (ATX) plasma levels by ELISA, the platelet activation markers PAC1 and CD62P, ATX bound to platelets and the amounts of plasma PEVs and REVs by flow cytometry in a cohort of 102 patients with SLE, including 29 incident cases of SLE and 30 controls. Correlation analyses explored the associations with the clinical parameters.Result Platelet activation markers were increased in patients with SLE compared with healthy control, with the marker CD62P associated with the SLE disease activity index (SLEDAI). The incident cases show additional associations between platelet markers (CD62P/ATX and PAC1/CD62P) and the SLEDAI. Compared with controls, patients with SLE presented higher levels of PEVs, phosphatidylserine positive (PS+) PEVs, REVs and PS+ REVs, but there is no association with disease activity. When stratified according to the plasma level of PS+ REVs, the group of patients with SLE with a high level of PS+ REVs presented a higher number of past thrombosis events and higher ATX levels.Conclusion Incident and prevalent forms of SLE cases present similar levels of platelet activation markers, with CD62P correlating with disease activity. Though EVs are not associated with disease activity, the incidence of past thrombotic events is higher in patients with a high level of PS+ REVs.

Abstract Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil‐derived extracellular vesicles (NDEVs) involved in immune and thrombo‐inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID‐19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta.

Sickle cell disease is a complex genetic disease involving cell adhesion between red blood cells, white blood cells, platelets and endothelial cells, inducing painful vaso-occlusive crisis (VOC). We assessed reticulocyte and erythrocyte parameters in a cohort of confirmed SCD patients, and investigated whether a combination of these routine laboratory biomarkers of haemolysis could be used to predict VOC development. Reticulocyte and erythrocyte parameters were evaluated using the Sysmex XN-9000 analyser. A total of 98 patients with SCD were included, 72 in steady state and 26 in VOC. Among the 72 patients in steady state, 22 developed a VOC in the following year (median: 3 months [2–6]). The following parameters were increased in SCD patients with VOC development compared to SCD patients without VOC development in the following year: reticulocyte count (94.6 109/L [67.8–128] vs. 48.4 109/L [24.9–87.5]), immature reticulocyte count (259 109/L [181–334] vs. 152 109/L [129–208]) reticulocyte/immature reticulocyte fraction (IRF) ratio (6.63 109/(L*%) [4.67–9.56] vs. 4.94 109/(L*%) [3.96–6.61]), and medium fluorescence reticulocytes (MFR) (19.9% [17.4–20.7] vs. 17.1% [15.95–19.75]). The association of a reticulocyte count of >189.4 109/L and an MFR of >19.75% showed a sensitivity of 81.8% and a specificity of 88% to predict VOC development in the following year. Based on our findings, a combination of routine laboratory biomarkers, as reticulocyte count, immature reticulocyte count and fluorescent reticulocyte fraction at steady state, could be used to predict VOC development in SCD.

Platelets are anucleate cytoplasmic fragments derived from the fragmentation of medullary megakaryocytes. Activated platelets adhere to the damaged endothelium by means of glycoproteins on their surface, forming the platelet plug. Activated platelets can also secrete the contents of their granules, notably the growth factors contained in the α-granules, which are involved in platelet aggregation and maintain endothelial activation, but also contribute to vascular repair and angiogenesis. Platelets also have a major inflammatory and immune function in antibacterial defence, essentially through their Toll-like Receptors (TLRs) and Sialic acid-binding immunoglobulin-type lectin (SIGLEC). Platelet activation also contributes to the extensive release of anti- or pro-inflammatory mediators such as IL-1β, RANTES (Regulated on Activation, Normal T Expressed and Secreted) or CD154, also known as the CD40-ligand. Platelets are involved in the direct activation of immune cells, polynuclear neutrophils (PNNs) and dendritic cells via the CD40L/CD40 complex. As a general rule, all of the studies presented in this review show that platelets are capable of covering most of the stages of inflammation, primarily through the CD40L/CD40 interaction, thus confirming their own role in this pathophysiological condition.

Stent thrombosis remains an infrequent but significant complication following percutaneous coronary intervention. Preclinical models to rapidly screen and validate therapeutic compounds for efficacy are lacking. Herein, we describe a reproducible, high throughput and cost-effective method to evaluate candidate therapeutics and devices for either treatment or propensity to develop stent thrombosis in an in vitro bench-top model. Increasing degree of stent malapposition (0.00 mm, 0.10 mm, 0.25 mm and 0.50 mm) was associated with increasing thrombosis and luminal area occlusion (4.1 ± 0.5%, 6.3 ± 0.5%, 19.7 ± 4.5%, and 92.6 ± 7.4%, p < 0.0001, respectively). Differences in stent design in the form of bare-metal, drug-eluting, and bioresorbable vascular scaffolds demonstrated differences in stent thrombus burden (14.7 ± 3.8% vs. 20.5 ± 3.1% vs. 86.8 ± 5.3%, p < 0.01, respectively). Finally, thrombus burden was significantly reduced when healthy blood samples were incubated with Heparin, ASA/Ticagrelor (DAPT), and Heparin+DAPT compared to control (DMSO) at 4.1 ± 0.6%, 6.9 ± 1.7%, 4.5 ± 1.2%, and 12.1 ± 1.8%, respectively (p < 0.01). The reported model produces high throughput reproducible thrombosis results across a spectrum of antithrombotic agents, stent design, and degrees of apposition. Importantly, performance recapitulates clinical observations of antiplatelet/antithrombotic regimens as well as device and deployment characteristics. Accordingly, this model may serve as a screening tool for candidate therapies in preclinical evaluation.

Secreted phospholipase A2-IIA (sPLA2-IIA) hydrolyzes phospholipids to liberate lysophospholipids and fatty acids. Given its poor activity toward eukaryotic cell membranes, its role in the generation of proinflammatory lipid mediators is unclear. Conversely, sPLA2-IIA efficiently hydrolyzes bacterial membranes. Here, we show that sPLA2-IIA affects the immune system by acting on the intestinal microbial flora. Using mice overexpressing transgene-driven human sPLA2-IIA, we found that the intestinal microbiota was critical for both induction of an immune phenotype and promotion of inflammatory arthritis. The expression of sPLA2-IIA led to alterations of the intestinal microbiota composition, but housing in a more stringent pathogen-free facility revealed that its expression could affect the immune system in the absence of changes to the composition of this flora. In contrast, untargeted lipidomic analysis focusing on bacteria-derived lipid mediators revealed that sPLA2-IIA could profoundly alter the fecal lipidome. The data suggest that a singular protein, sPLA2-IIA, produces systemic effects on the immune system through its activity on the microbiota and its lipidome.

Introduction: Cerebral venous thrombosis (CVT) is a rare disease with highly variable clinical presentation and outcome. Etiological assessment may be negative. The clinical and radiological presentation and evolution can be highly variable. The mechanisms involved in this variability remain unknown.Objective: The aim of this multicenter French study registered on ClinicalTrials.gov (NCT02013635) was therefore to prospectively recruit a cohort of patients with cerebral venous thrombosis (FPCCVT) in order to study thrombin generation and clot degradation, and to evaluate their influence on clinical radiological characteristics. The first part of the study was to compare our cohort with a reference cohort.Methods: This prospective, multicenter, French study was conducted from July 2011 to September 2016. Consecutive patients (aged >15 years) referred to the stroke units of 21 French centers and who had a diagnosis of symptomatic CVT were included. All patients gave their written informed consent. The diagnosis of CVT had to be confirmed by imaging. Clinical, radiological, biological, and etiological characteristics were recorded at baseline, at acute phase, at 3 months and at last follow-up visit. Thrombophilia screening and the choice of treatment were performed by the attending physician. All data were compared with data from the International Study on CVT published by Ferro et al.Results: Two hundred thirty-one patients were included: 117 (50.6%) had isolated intracranial hypertension, 96 (41.5%) had focal syndrome. During hospitalization, 229 (99.1%) patients received anticoagulant treatment. Median length of hospital stay was 10 days. Five patients died during hospitalization (2.2%). At 3 months, 216 patients (97.0%) had follow-up with neurological data based on an outpatient visit. The mean duration of antithrombotic treatment was 9 months, and the mean time to last follow-up was 10.5 months. At the end of follow-up, eight patients had died, and 26 patients were lost to follow-up. At least one risk factor was identified in 200 patients.Conclusions: We demonstrated that the FPCCVT cohort had radiological, biological, and etiological characteristics similar to the historical ISCVT cohort. Nevertheless, the initial clinical presentation was less severe in our study probably due to an improvement in diagnostic methods between the two studies.

Venous thromboembolism (VTE) is the third leading cardiovascular cause of death and is conventionally treated with anticoagulants that directly antagonize coagulation. However, recent data have demonstrated that also platelets play a crucial role in VTE pathophysiology. In the current review, we outline how platelets are involved during all stages of experimental venous thrombosis. Platelets mediate initiation of the disease by attaching to the vessel wall upon which they mediate leukocyte recruitment. This process is referred to as immunothrombosis, and within this novel concept inflammatory cells such as leukocytes and platelets directly drive the progression of VTE. In addition to their involvement in immunothrombosis, activated platelets can directly drive venous thrombosis by supporting coagulation and secreting procoagulant factors. Furthermore, fibrinolysis and vessel resolution are (partly) mediated by platelets. Finally, we summarize how conventional antiplatelet therapy can prevent experimental venous thrombosis and impacts (recurrent) VTE in humans.

Current guidelines recommend monitoring the anticoagulant effect of unfractionated heparin (UFH) by measuring anti-Xa activity rather than activated partial thromboplastin time (aPTT) in intensive care unit (ICU) patients. The primary objective of this study was to evaluate the correlation of aPTT, anti-Xa activity, and thrombin generation in UFH-treated ICU patients. A prospective observational pilot study was conducted in adult surgical ICU patients treated with UFH. aPTT and anti-Xa activity were monitored daily. The therapeutic target was aPTT between 50 s and 84 s, and/or anti-Xa between 0.3 and 0.7 U/mL. Correlation among aPTT, anti-Xa activity, and thrombin generation was determined by measuring endogenous thrombin potential (ETP), with the inflammatory response evaluated. C-reactive protein (CRP) was used as a marker of inflammatory response. The plasma of 107 samples from 30 ICU patients was analyzed. The correlation between aPTT and anti-Xa activity was 0.66, CI95% [0.54;0.76] (p < 0.0001). Although thrombin generation, aPTT, and anti-Xa were correlated with inflammatory responses, the correlation was higher with thrombin generation and anti-Xa activity compared to aPTT. When aPTT was in a therapeutic range, a low thrombin generation was observed but was 50% inhibited when anti-Xa was in a therapeutic range. Coagulation testing with aPTT, anti-Xa correlated with thrombin generation. A 50% decrease in thrombin generation was observed when anti-Xa was within a therapeutic range. Further work is needed to evaluate coagulation biomarker responses and clinical outcomes in specific ICU populations.

Since December 2019, a pandemic caused by a new coronavirus has spread to more than 170 countries around the world. Worsening infected patients requiring intensive care unit (ICU) admission associated with 30% of mortality. A part of worsening is induced by hemostasis deregulation. The aim of this study was to investigate the association of coagulation activation in COVID-19 progression. Thirty-five of the 99 patients got clinically worse. The final model of the logistic regression analysis revealed that O2 requirement (RR = 7.27 [1.50–19.31]), monocytes below 0.2G/L (RR = 2.88 [1.67–3.19]), fibrinogen levels (RR = 1.45 [1.17–1.82] per g/L increase), prothrombin fragments 1+2 higher than 290 pM (RR = 2.39 [1.20–3.30]), and thrombin peak (RR = 1.28 [1.03–1.59] per 50 nM increase) were associated with an increased risk of clinical worsening. A fibrinogen level threshold of 5.5 g/L, a thrombin peak measurement threshold of 99 pM, and O2 requirement associated with clinical outcome in more than 80% of our cohort. In conclusion, we identified fibrinogen and thrombin peak at admission as coagulation biomarkers associated with an increased risk of ICU admission or death. This finding allows initiating steroids and triage for worsening patients. Our results should therefore be considered as exploratory and deserve confirmation.

The production of extracellular vesicles (EV) is a ubiquitous feature of eukaryotic cells but pathological events can affect their formation and constituents. We sought to characterize the nature, profile and protein signature of EV in the plasma of Parkinson's disease (PD) patients and how they correlate to clinical measures of the disease. EV were initially collected from cohorts of PD (n = 60; Controls, n = 37) and Huntington's disease (HD) patients (Pre-manifest, n = 11; manifest, n = 52; Controls, n = 55) – for comparative purposes in individuals with another chronic neurodegenerative condition – and exhaustively analyzed using flow cytometry, electron microscopy and proteomics. We then collected 42 samples from an additional independent cohort of PD patients to confirm our initial results. Through a series of iterative steps, we optimized an approach for defining the EV signature in PD. We found that the number of EV derived specifically from erythrocytes segregated with UPDRS scores corresponding to different disease stages. Proteomic analysis further revealed that there is a specific signature of proteins that could reliably differentiate control subjects from mild and moderate PD patients. Taken together, we have developed/identified an EV blood-based assay that has the potential to be used as a biomarker for PD.

Background: Idarucizumab has been included in guidelines for the management of bleeding or surgical procedure in dabigatran-treated patients without need for biological monitoring. The aim of the study was to assess the prognostic value of dabigatran plasma level before reversal to test the hemostatic efficacy of idarucizumab. The secondary objectives were (i) to analyze plasma dabigatran level according to the risk of rebound and (ii) to evaluate the incidence of post-reversal non-favorable clinical outcomes (including thromboembolism, bleeding, antithrombotic, and death) and antithrombotic resumption.Methods and Results: This was an observational multicentric cohort study, which included all French patients who required idarucizumab for dabigatran reversal. Between May 2016 and April 2019, 87 patients from 21 French centers were enrolled. Patients received idarucizumab for overt bleeding (n = 61), urgent procedures (n = 24), or overdose without bleeding (n = 2). Among patients with major bleeding (n = 57), treatment with idarucizumab was considered effective in 44 (77.2%) of them. Patients who did not achieve effective hemostasis after reversal had a significantly higher mean level of plasma dabigatran at baseline (524.5 ± 386 vs. 252.8 ng/mL ± 235, p = 0.033). Furthermore, patients who did not achieve effective hemostasis after reversal had less favorable outcomes during follow-up (46.2 vs. 81.8%, p = 0.027). ROC curve identified a cutoff of 264 ng/mL for dabigatran level at admission to be predictive of ineffective hemostasis. No plasma dabigatran rebound was observed after reversal in patients with dabigatran plasma level < 264 ng/mL at baseline.Conclusion: This retrospective study shows that dabigatran level before reversal could predict hemostatic effectiveness and dabigatran plasma rebound after idarucizumab injection.

Background: Thrombosis is frequent during COVID-19 disease, and thus, identifying predictive factors of hemostasis associated with a poor prognosis is of interest. The objective was to explore coagulation disorders as early predictors of worsening critical conditions in the intensive care unit (ICU) using routine and more advanced explorations. Materials: Blood samples within 24 h of ICU admission for viscoelastic point-of-care testing, (VET), advanced laboratory tests: absolute immature platelet count (A-IPC), von Willebrand-GPIb activity (vWF-GpIb), prothrombin fragments 1 + 2 (F1 + 2), and the thrombin generation assay (TGA) were used. An association with worse outcomes was explored using univariable and multivariable analyses. Worsening was defined as death or the need for organ support. Results: An amount of 85 patients with 33 in critical condition were included. A-IPC were lower in worsening patients (9.6 [6.4–12.5] vs. 12.3 [8.3–20.7], p = 0.02) while fibrinogen (6.9 [6.1–7.7] vs. 6.2 [5.4–6.9], p = 0.03), vWF-GpIb (286 [265–389] vs. 268 [216–326], p = 0.03) and F1 + 2 (226 [151–578] vs. 155 [129–248], p = 0.01) were higher. There was no difference observed for D-dimer, TGA or VET. SAPS-II and A-IPC were independently associated with worsening (OR = 1.11 [1.06–1.17] and OR = 0.47 [0.25–0.76] respectively). The association of a SAPS-II ≥ 33 and an A-IPC ≤ 12.6 G/L predicted the worsening of patients (sensitivity 58%, specificity 89%). Conclusions: Immature platelets are early predictors of worsening in severe COVID-19 patients, suggesting a key role of thrombopoiesis in the adaption of an organism to SARS-CoV-2 infection.

Steady state peripheral blood (SSPB) contains hematopoietic stem and progenitor cells (HSPCs) presenting characteristics of real hematopoietic stem cells, and thus represents an interesting alternative cell supply for hematopoietic cell transplantation. Development of ex vivo expansion strategies could overcome the low HSPC numbers usually rescued from SSPB. We investigated the effect of alpha lipoic acid (ALA) on ex vivo culture of SSPB CD34 positive (CD34pos) cells on primitive cell expansion, cell cycle, and oxidative metabolism as estimated by determining the ROS and GSH content. ALA increased the ex vivo expansion of total CD34pos cells and of phenotypically defined CD34pos HSPCs subpopulations that retained in vivo repopulating capacity, concomitantly to a decreased expansion of differentiating cells. ALA did not modify cell cycle progression nor the proliferation of ex vivo expanded CD34pos cells, and coherently did not affect the ROS level. On the contrary, ALA decreased the proliferation and disturbed cell cycle progression of cells reaching a differentiated status, a phenomenon that seems to be associated with a drop in ROS level. Nonetheless, ALA affected the redox status of hematopoietic primitive cells, as it reproducibly increased GSH content. In conclusion, ALA represents an interesting molecule for the improvement of ex vivo expansion strategies and further clinical application in hematopoietic cell transplantation (HCT).

It is known that cancer stem cells (CSCs) with the largest proliferative capacity survive the anoxic and/or ischemic conditions present inside tumorous tissue. In this study we test whether normal stem cells can survive under the same conditions due to cancer cell-like metabolic adaptations. We cultivated a CD34+ population with a majority of hematopoietic progenitors, and a CD34+CD38lowCD133+CD90+CD45RA− population, highly enriched in hematopoietic stem cells (HSCs), under anoxic, anoxic/aglycemic (“ischemia-like”), or physiological conditions (3% O2). Results showed, despite a reduction in total cell fold expansion proportionate to the decrease in O2 concentration; CD34+ cells, aldehyde dehydrogenase-expressing primitive cells, and committed progenitors expanded, even in anoxia. Interestingly, under ischemia-like conditions, stem and CD34+ cell populations are maintained at day-0 level. Cell-cycle analysis further revealed an accumulation of cells in the G0/G1 phase in anoxia or anoxia/aglycemia, with a fraction of cells (~40%) actively cycling (SG2M phases). Also stem cell analysis showed that in these conditions a long-term Scid Repopulating activity was equal to that found with 3% O2. In addition stem cells with the highest proliferative capacity were maintained in anoxia/aglycemia and in anoxia. The estimated ATP profile, active mitochondrial content, and succinate accumulation are indicative of anaerobic mitochondrial respiration in both HSCs and CD34+ progenitors under ischemia-like conditions. We demonstrate here that primitive hematopoietic cells show similar metabolic flexibility to CSCs, allowing them to survive a lack of O2 and O2/glucose. Our study reveals that this feature is not the consequence of malignant transformation, but an attribute of stemness.

Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as biological markers and cell-free biotherapies in cardiovascular and oncologic diseases. However, their therapeutic perspectives remain limited due to the lack of reliable data regarding their systemic biodistribution after intravenous administration. Methods: Applied to a mouse model of peripheral ischemia, radiolabeled endothelial LEVs were tracked and their in vivo whole-body distribution was quantified by microSPECT/CT imaging. Hindlimb perfusion was followed by LASER Doppler and motility impairment function was evaluated up to day 28 post-ischemia. Results: Early and specific homing of LEVs to ischemic hind limbs was quantified on the day of ischemia and positively correlated with reperfusion intensity at a later stage on day 28 after ischemia, associated with an improved motility function. Conclusions: This concept is a major asset for investigating the biodistribution of LEVs issued from other cell types, including cancer, thus partly contributing to better knowledge and understanding of their fate after injection.

Antiphospholipid syndrome (APS) is associated with thrombotic events (tAPS) and/or obstetrical morbidity (oAPS), with persisting antiphospholipid antibodies (aPL). Despite an update of aPL in 2006, several patients had typical clinical events without the classical biological criteria. The aim of our study was to evaluate the hypercoagulability state with both thrombin generation (TG) profiles and activated protein C resistance (aPCR) in different types of APS. Methods: We retrospectively included 41 patients with Sydney criteria classification (tAPS, oAPS) and no clinical manifestation of APS with persistent aPL (biological APS). A thrombin generation assay was performed with a Fluoroskan Ascent fluorometer in platelet-poor plasma (PPP). Activated protein C resistance was measured as a ratio: ETP+aPC/ETP-aPC × 100. Results: Thrombotic APS and oAPS had an increase of global thrombin generation (ETPcontrol = 808 nM.min (756–853) vs. 1265 nM.min (956–1741) and 1863 nM.min (1434–2080), respectively) (Peakcontrol = 78 nM (74–86) vs. 153 nM (109–215) and 254 nM.min (232–289), respectively). Biological APS had only a lag time increase (Tcontrol = 4.89 ± 1.65 min vs. 13.6 ± 3.9 min). An increased aPCR was observed in tAPS (52.7 ± 16.4%), oAPS (64.1 ± 14.6%) as compared to the control group (27.2 ± 13.8%). Conclusion: Our data suggest an increase of thrombin generation in thrombotic and obstetrical APS and no hypercoagulable states in patients with biological APS. The study of a prospective and a larger controlled cohort could determine the TGA useful for APS monitoring and could confirm an aPCR evaluation in PPP.

On activation, platelets release vesicles called microparticles (MPs). MPs are heterogeneous with regard to the presence or absence of mitochondria. We quantified MPs in platelet concentrates (PCs) taking their mitochondrial content into account. Platelet-rich plasma (PRP), buffy coat (BC) and apheresis (AP) PCs were tested through 7 days of storage. A combination of flow cytometry and spanning-tree progression analysis of density-normalized events (SPADE) was used to determine MP and mitochondrial release during storage. All the PC biochemical parameters complied with transfusion standards at all times. Platelet activation markers increased during storage and were higher for PRP than other types of PCs. Concentrations of MPs and extracellular mitochondria interpreted by SPADE algorithm were significantly higher in PRP than other in PCs and were stable throughout storage. The mode of preparation, rather than storage duration, impacts the release of MPs and mitochondria in PCs.

Antiphospholipid antibodies (aPL) promote endothelial dysfunction, inflammation and procoagulant state. We investigated the effect of hydroxychloroquine (HCQ) on prothrombotic state and endothelial function in mice and in human aortic endothelial cells (HAEC). Human aPL were injected to C57BL/6 mice treated or not with HCQ. Vascular endothelial function and eNOS were assessed in isolated mesenteric arteries. Thrombosis was assessed both in vitro by measuring thrombin generation time (TGT) and tissue factor (TF) expression and in vivo by the measurement of the time to occlusion in carotid and the total thrombosis area in mesenteric arteries. TGT, TF, and VCAM1 expression were evaluated in HAEC. aPL increased VCAM-1 expression and reduced endothelium dependent relaxation to acetylcholine. In parallel, aPL shortened the time to occlusion and extended thrombus area in mice. This was associated with an overexpression of TF and an increased TGT in mice and in HAEC. HCQ reduced clot formation as well as TGT, and improved endothelial-dependent relaxations. Finally, HCQ increased the p-eNOS/eNOS ratio. This study provides new evidence that HCQ improves procoagulant status and vascular function in APS by modulating eNOS, leading to an improvement in the production of NO.

Alpha tocopherol acetate (αTOA) is an analogue of alpha tocopherol (αTOC) that exists in the form of an injectable drug. In the context of the metabolic hypothesis of stem cells, we studied the impact of αTOA on the metabolic energetic profile and functional properties of hematopoietic stem and progenitor cells. In ex vivo experiments performed on cord blood CD34+ cells, we found that αTOA effectively attenuates oxidative phosphorylation without affecting the glycolysis rate. This effect concerns complex I and complex II of the mitochondrial respiratory chain and is related to the relatively late increase (3 days) in ROS (Reactive Oxygen Species). The most interesting effect was the inhibition of Hypoxia-Inducible Factor (HIF)-2α (Hexpression, which is a determinant of the most pronounced biological effect—the accumulation of CD34+ cells in the G0 phase of the cell cycle. In parallel, better maintenance of the primitive stem cell activity was revealed by the expansion seen in secondary cultures (higher production of colony forming cells (CFC) and Severe Combined Immunodeficiency-mice (scid)-repopulating cells (SRC)). While the presence of αTOA enhanced the maintenance of Hematopoietic Stem Cells (HSC) and contained their proliferation ex vivo, whether it could play the same role in vivo remained unknown. Creating αTOC deficiency via a vitamin E-free diet in mice, we found an accelerated proliferation of CFC and an expanded compartment of LSK (lineagenegative Sca-1+cKit+) and SLAM (cells expressing Signaling Lymphocytic Activation Molecule family receptors) bone marrow cell populations whose in vivo repopulating capacity was decreased. These in vivo data are in favor of our hypothesis that αTOC may have a physiological role in the maintenance of stem cells. Taking into account that αTOC also exhibits an effect on proliferative capacity, it may also be relevant for the ex vivo manipulation of hematopoietic stem cells. For this purpose, low non-toxic doses of αTOA should be used.

From ten years of observations of the Atlantic meridional overturning circulation (MOC) at 26° N (2004–2014), we revisit the question of flow compensation between components of the circulation. Contrasting with early results from the observations, transport variations of the Florida Current (FC) and upper mid-ocean (UMO) transports (top 1000 m east of the Bahamas) are now found to compensate on sub-annual timescales. The observed compensation between the FC and UMO transports is associated with horizontal circulation and means that this part of the correlated variability does not project onto the MOC. A deep baroclinic response to wind-forcing (Ekman transport) is also found in the lower North Atlantic Deep Water (LNADW; 3000–5000 m) transport. In contrast, co-variability between Ekman and the LNADW transports does contribute to overturning. On longer timescales, the southward UMO transport has continued to strengthen, resulting in a continued decline of the MOC. Most of this interannual variability of the MOC can be traced to changes in isopycnal displacements on the western boundary, within the top 1000 m and below 2000 m. Substantial trends are observed in isopycnal displacements in the deep ocean, underscoring the importance of deep boundary measurements to capture the variability of the Atlantic MOC.

The feasibility of ex vivo expansion allows us to consider the steady-state peripheral blood as an alternative source of hematopoietic stem progenitor cells for transplantation when growth factor-induced cell mobilization is contraindicated or inapplicable. Ex vivo expansion dramatically enhances the in vivo reconstituting cell population from steady-state blood. In order to investigate phenotype and the expression of homing molecules, the expression of CD34, CD133, CD90, CD45RA, CD26 and CD9 was determined on sorted CD34+ cells according to CXCR4 (“neg”, “low” “bright”) and CD133 expression before and after ex vivo expansion. Hematopoietic stem cell activity was determined in vivo on the basis of hematopoietic repopulation of primary and secondary recipients - NSG immuno-deficient mice. In vivo reconstituting cells in the steady-state blood CD34+ cell fraction before expansion belong to the CD133+ population and are CXCR4low or, to a lesser extent, CXCR4neg, while after ex vivo expansion they are contained only in the CD133+CXCR4low cells. The failure of the CXCR4bright population to engraft is probably due to the exclusive expression of CD26 by these cells. The limiting-dilution analysis showed that both repopulating cell number and individual proliferative capacity were enhanced by ex vivo expansion. Thus, steady-state peripheral blood cells exhibit a different phenotype compared to mobilized and cord blood cells, as well as to those issued from the bone marrow. These data represent the first phenotypic characterization of steady-state blood cells exhibiting short- and long-term hematopoietic reconstituting potential, which can be expanded ex vivo, a sine qua non for their subsequent use for transplantation.

Among immunoglobulins (Igs), IgE can powerfully contribute to antimicrobial immunity and severe allergy despite its low abundance. IgE protein and gene structure resemble other Ig classes, making it unclear what constrains its production to thousand-fold lower levels. Whether class-switched B cell receptors (BCRs) differentially control B cell fate is debated, and study of the membrane (m)IgE class is hampered by its elusive in vivo expression. Here, we demonstrate a self-controlled mIgE+ B cell stage. Primary or transfected mIgE+ cells relocate the BCRs into spontaneously internalized lipid rafts, lose mobility to chemokines, and change morphology. We suggest that combined proapoptotic mechanisms possibly involving Hax1 prevent mIgE+ memory lymphocyte accumulation. By uncoupling in vivo IgE switching from cytokine and antigen stimuli, we show that these features are independent from B cell stimulation and instead result from mIgE expression per se. Consequently, few cells survive IgE class switching, which might ensure minimal long-term IgE memory upon differentiation into plasma cells.

The bovine gastrointestinal tract (GIT) is the main reservoir for enterohaemorrhagic Escherichia coli (EHEC) responsible for food-borne infections. Therefore, it is crucial to develop strategies, such as EHEC suppression by antagonistic microorganisms, to reduce EHEC survival in the GIT of cattle and to limit shedding and food contamination. Most human-derived Lactobacillus reuteri strains produce hydroxypropionaldehyde (HPA), an antimicrobial compound, during anaerobic reduction of glycerol. The capacity of L. reuteri LB1-7, a strain isolated from raw bovine milk, to produce HPA and its antimicrobial activity against an O157:H7 EHEC strain (FCH6) were evaluated in bovine rumen fluid (RF) under strict anaerobiosis. EHEC was totally suppressed when incubated in RF inoculated with L. reuteri LB1-7 and supplemented with 80 mM glycerol (RF-Glyc80). The addition of LB1-7 or glycerol alone did not modify EHEC survival in RF. Glycerol was converted to HPA (up to 14 mM) by LB1-7 during incubation in RF-Glyc80, and HPA production appeared to be responsible for EHEC suppression. The bactericidal activity of L. reuteri LB1-7, the concentration of glycerol required and the level of HPA produced depended on physiological and ecological environments. In vitro experiments also showed that EHEC inoculated in rumen fluid and exposed to L. reuteri and glycerol had a very limited growth in rectal contents. However, L. reuteri exerted an antimicrobial activity against the rumen endogenous microbiota and perturbed feedstuff degradation in the presence of glycerol. The potential administration of L. reuteri and glycerol in view of application to finishing beef cattle at the time of slaughter is discussed. Further in vivo studies will be important to confirm the efficiency of L. reuteri and glycerol supplementation against EHEC shedding in ruminants.

The Atlantic meridional overturning circulation (AMOC) has been observed continuously at 26° N since April 2004. The AMOC and its component parts are monitored by combining a transatlantic array of moored instruments with submarine-cable-based measurements of the Gulf Stream and satellite derived Ekman transport. The time series has recently been extended to October 2012 and the results show a downward trend since 2004. From April 2008 to March 2012, the AMOC was an average of 2.7 Sv (1 Sv = 106 m3 s−1) weaker than in the first four years of observation (95% confidence that the reduction is 0.3 Sv or more). Ekman transport reduced by about 0.2 Sv and the Gulf Stream by 0.5 Sv but most of the change (2.0 Sv) is due to the mid-ocean geostrophic flow. The change of the mid-ocean geostrophic flow represents a strengthening of the southward flow above the thermocline. The increased southward flow of warm waters is balanced by a decrease in the southward flow of lower North Atlantic deep water below 3000 m. The transport of lower North Atlantic deep water slowed by 7% per year (95% confidence that the rate of slowing is greater than 2.5% per year).

The coastal circulation in the Gulf of Lions (GoL) is influenced by the Northern Current (NC), forced by a complex wind system and also affected by important river discharges from the Rhône River. Correct modelling of this current is therefore important for obtaining a good representation of the gulf circulation. An observing system simulation experiment using the SEEK filter data assimilation method was used in a regional 1/16° configuration of the GoL in the NEMO model. The synthetic observation database used for the experiment comprised altimetric data in addition to in-situ temperature and salinity profiles. Statistical diagnostics and other physical criteria based on the improvement of NC representation were set up in order to assess the quality of this experiment. Comparisons between the free 1/16° simulation and the experience with assimilation show that data assimilation significantly improved the description of the characteristics of the NC as well as its seasonal and mesoscale variability, which in turn improved the description of the water exchanges between the coastal region and the open sea.