Identification of genes involved in Ca(2+ )ionophore A23187-mediated apoptosis and demonstration of a high susceptibility for transcriptional repression of cell cycle genes in B lymphoblasts from a patient with Scott syndrome

BACKGROUND: In contrast to other agents able to induce apoptosis of cultured cells, Ca(2+ )ionophore A23187 was shown to elicit direct activation of intracellular signal(s). The phenotype of the cells derived from patients having the hemorrhagic disease Scott syndrome, is associated with an abnormally high proportion of apoptotic cells, both in basal culture medium and upon addition of low ionophore concentrations in long-term cultures. These features are presumably related to the mutation also responsible for the defective procoagulant plasma membrane remodeling. We analyzed the specific transcriptional re-programming induced by A23187 to get insights into the effect of this agent on gene expression and a defective gene regulation in Scott cells. RESULTS: The changes in gene expression upon 48 hours treatment with 200 nM A23187 were measured in Scott B lymphoblasts compared to B lymphoblasts derived from the patient's daughter or unrelated individuals using Affymetrix microarrays. In a similar manner in all of the B cell lines, results showed up-regulation of 55 genes, out of 12,000 represented sequences, involved in various pathways of the cell metabolism. In contrast, a group of 54 down-regulated genes, coding for histones and proteins involved in the cell cycle progression, was more significantly repressed in Scott B lymphoblasts than in the other cell lines. These data correlated with the alterations of the cell cycle phases in treated cells and suggested that the potent effect of A23187 in Scott B lymphoblasts may be the consequence of the underlying molecular defect. CONCLUSION: The data illustrate that the ionophore A23187 exerts its pro-apoptotic effect by promoting a complex pattern of genetic changes. These results also suggest that a subset of genes participating in various steps of the cell cycle progress can be transcriptionally regulated in a coordinated fashion. Furthermore, this research brings a new insight into the defect in cultured Scott B lymphoblasts, leading to hypothesize that a mutated gene plays a role not only in membrane remodeling but also in signal transduction pathway(s) leading to altered transcriptional regulation of cell cycle genes

Increased levels of circulating microparticles in primary Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis and relation with disease activity

INTRODUCTION: Cell stimulation leads to the shedding of phosphatidylserine (PS)-rich microparticles (MPs). Because autoimmune diseases (AIDs) are characterized by cell activation, we investigated level of circulating MPs as a possible biomarker in primary Sjögren's syndrome (pSS), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). METHODS: We measured plasma levels of total, platelet and leukocyte MPs by prothrombinase capture assay and flow cytometry in 43 patients with pSS, 20 with SLE and 24 with RA and in 44 healthy controls (HCs). Secretory phospholipase A2 (sPLA2) activity was assessed by fluorometry. Soluble CD40 ligand (sCD40L) and soluble P-selectin (sCD62P), reflecting platelet activation, were measured by ELISA. RESULTS: Patients with pSS showed increased plasma level of total MPs (mean +/- SEM 8.49 +/- 1.14 nM PS equivalent (Eq), P < 0.0001), as did patients with RA (7.23 +/- 1.05 n PS Eq, P = 0.004) and SLE (7.3 +/- 1.25 nM PS Eq, P = 0.0004), as compared with HCs (4.13 +/- 0.2 nM PS Eq). Patients with AIDs all showed increased level of platelet MPs (P < 0.0001), but only those with pSS showed increased level of leukocyte MPs (P < 0.0001). Results by capture assay and flow cytometry were correlated. In patients with high disease activity according to extra-glandular complications (pSS), DAS28 (RA) or SLEDAI (SLE) compared with low-activity patients, the MP level was only slightly increased in comparison with those having a low disease activity. Platelet MP level was inversely correlated with anti-DNA antibody level in SLE (r = -0.65; P = 0.003) and serum beta2 microglobulin level in pSS (r = -0.37; P < 0.03). The levels of total and platelet MPs were inversely correlated with sPLA2 activity (r = -0.37, P = 0.0007; r = -0.36, P = 0.002, respectively). sCD40L and sCD62P concentrations were significantly higher in pSS than in HC (P </= 0.006). CONCLUSIONS: Plasma MP level is elevated in pSS, as well as in SLE and RA, and could be used as a biomarker reflecting systemic cell activation. Level of leukocyte-derived MPs is increased in pSS only. The MP level is low in case of more severe AID, probably because of high secretory phospholipase A2 (sPLA2) activity, which leads to consumption of MPs. Increase of platelet-derived MPs, sCD40L and sCD62P, highlights platelet activation in pSS

Evaluation de l'analyseur Multiplate® dans le diagnostic et la caractérisation de la maladie de Willebrand et de certaines thrombopathies

L objectif de notre travail a été d étudier les performances du MultiplateÒ, agrégomètre par impédance de dernière génération, dans la détection des anomalies constitutionnelles de l hémostase primaire chez 30 patients atteints d une maladie de Willebrand définie par une activité cofacteur de la ristocétine < 40%. Nous avons comparé les résultats obtenus par le MultiplateÒ avec ceux obtenus par agrégométrie optique classique. L'étude a également inclus 30 volontaires bien portants. D autre part, nous avons également étudié 4 patients atteints d une thrombopathie constitutionnelle (2 thrombasthénies de Glanzmann et 2 syndromes de Bernard et Soulier). En conclusion, nos résultats suggèrent que le MultiplateÒ a une sensibilité équivalente à celle de l agrégométrie optique pour le diagnostic de la maladie de Willebrand, met en évidence les maladies de Willebrand de type 2B ainsi que les thrombopathies constitutionnelles. Ces résultats préliminaires encourageants doivent être confirmés sur une plus large population de patients atteints de maladie de Willebrand et de thrombopathies.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

Use of platelet-rich plasma in sports medicine in 2019. Guidelines from the French Society of Sports Traumatology

peer reviewedL'apparition des plasmas riches en plaquettes dans l'arsenal thérapeutique au début des années 2000 a été suivie par un développement exponentiel de son usage, notamment en pathologie sportive. La Société française de traumatologie du sport a souhaité faire une mise au point des connaissances sur ce sujet et des recommandations pour un bon usage. La composition des PRP utilisés en traumatologie sportive est très hétérogène et trop souvent opaque. La conséquence est l'entretien d'une confusion et un manque de clarté dans le débat scientifique quant à l'intérêt de cette technique chez les sportifs. La définition des PRP est rappelée : couche profonde du plasma riche en plaquettes (entre 6 et 9 105 PQ/mL) après centrifugation qui ne doit contenir aucune autre cellule dans toute la mesure du possible (facteurs de la coagulation non activés, leuco- cytes < 1 %, érythrocytes < 1 %). À ce jour, l'injection de PRP dans les douleurs des lésions cartilagineuses apporte une amélioration égale ou supérieure à l'acide hyaluronique sur un grand nombre d'études de haut niveau de preuve. Le PRP est une option thérapeutique intéressante pour les tendinopathies patellaires et, dans une moindre mesure, pour les tendinopathies des épicondyliens latéraux, de l'aponévrose plantaire, les enthésopathies proximales des ischiojam- biers, les tendinopathies du moyen glutéal ou de la coiffe des rotateurs. Concernant toutes les autres localisations, il n'existe pas ou peu de données. L'utilisation de PRP en pathologie du sport doit suivre une démarche diagnostique exhaustive et rigoureuse, la plupart du temps multidisci- plinaire et appliquer des règles d'asepsie strictes. Dans la grande majorité des cas, l'injection doit bénéficier d'un guidage par échographie. De futures études de haut niveau de preuve sont encore nécessaires afin de mieux définir les bonnes indications et les meilleures modalités d'injection.Since its advent as part of the therapeutic armamentarium early in the 2000s, platelet-rich plasma (PRP) products have been widely used in sports pathology. The French Society of Sports Traumatology has published guidelines for good clinical practices when using PRP. PRP products used in sports medicine have various compositions that are not always explicitly described. There is thus a certain confusion in the scientific debate on their usefulness for athletes. PRP can be defined as a deep layer of plasma with a high platelet count (6 to 9 105 PQ/ml) after centrifugation and as far as possible no cells (non-activated coagulation factors, white cells < 1%, red cells < 1%). According to large-scale studies with a high level of proof, PRP injections provide relief for cartilage injury-related pain that is as good as or better than that obtained with hyaluronic acid. PRP is an attractive therapeutic option for patellar tendinopathy and to a lesser degree for certain other localizations: lateral epicondyles, plantar aponeurosis,hamstring proximal enthesis, gluteus medius, rotator cuff. Data are not available for other localizations. A rigorous exhaustive diagnostic approach is required for using PRP in sports medicine. A multidisciplinary concertation is usually needed, together with strict asepsis. Ultrasound guidance is generally recommended. Further studies with a high level of proof are still necessary to better define appropriate indications and injection modalities

A Novel Single-Domain Antibody Against von Willebrand Factor A1 Domain Resolves Leukocyte Recruitment and Vascular Leakage During Inflammation—Brief Report

International audienceObjective—von Willebrand factor (VWF) is crucial to hemostasis, but also plays a role in inflammatory processes. Unfortunately, no proper monoclonal antibodies to study VWF function in mice are currently available. We therefore aimed to generate single-domain antibodies (sdAbs) recognizing murine VWF and blocking its function in vivo.Approach and Results—Llama-derived sdAbs recognizing both human and murine VWF were isolated via phage display technology. One of them (designated KB-VWF-006) recognized the VWF A1 domain with picomolar affinity. This sdAb avidity was strongly enhanced via dimerization using a triple Ala linker (KB-VWF-006bi). When administered in vivo to wild-type mice, KB-VWF-006bi dose dependently induced bleeding in a tail clip model. In 2 distinct models of inflammation, KB-VWF-006bi efficiently interfered with leukocyte recruitment and vascular leakage.Conclusions—KB-VWF-006bi is an sdAb recognizing the A1 domain of human VWF and murine VWF that interferes with VWF–platelet interactions in vivo. By using this sdAb, we now also show that the A1 domain is pertinent to the participation of VWF in the inflammatory response

The IgG-degrading enzyme, Imlifidase, restores the therapeutic activity of FVIII in inhibitor-positive hemophilia A mice

Neutralizing anti-factor VIII (FVIII) antibodies, known as FVIII inhibitors, represent a major drawback of replacement therapy in persons with congenital hemophilia A (PwHA), rendering further infusions of FVIII ineffective. FVIII inhibitors can also appear in non-hemophilic individuals causing acquired hemophilia A (AHA). The use of non-FVIII bypassing agents in cases of bleeds or surgery in inhibitor-positive patients is complicated by the lack of reliable biological monitoring and increased thrombotic risk. Imlifidase (IdeS) is an endopeptidase that degrades human immunoglobulin G (IgG); it was recently approved for hyperimmune patients undergoing renal transplants. Here we investigated the ability of IdeS to eliminate FVIII inhibitors in vitro and in a model of inhibitor-positive HA mice. IdeS cleaved anti-FVIII plasma IgG from PwHA and AHA patients, and hydrolyzed recombinant human anti-FVIII IgG independently from their subclass or specificity for the A2, A3, C1 or C2 domains of FVIII. In HA mice passively immunized with recombinant human anti-FVIII IgG, IdeS restored the hemostatic efficacy of FVIII, as evidenced by the correction of the bleeding tendency. Our results provide the proof of concept for the transient removal of FVIII inhibitors by IdeS, thereby opening a therapeutic window for efficient FVIII replacement therapy in inhibitor-positive patients

FVIII dosages in persons with haemophilia A treated with extended half‐life products: From local biology to optimized patient management

International audienceManagement of persons with haemophilia A (PwHA) has advanced considerably with the introduction of modified recombinant factor VIII (FVIII) with extended half-life (EHL). Monitoring such products may be challenging due to discrepancies between FVIII levels measured by chronometric one-stage assays (OSAs) and chromogenicstage assays (CSAs). CSA is considered to be the most consistent method because it is used for potency labelling of FVIII concentrates according to the European Pharmacopeia recommendations

Macrophage scavenger receptor SR-AI contributes to the clearance of von Willebrand factor

International audiencePreviously, we found that LDL-receptor related protein-1 on macrophages mediated shear stress-dependent clearance of von Willebrand factor. In control experiments, however, we observed that von Willebrand factor also binds to macrophages independently of this receptor under static conditions, suggesting the existence of additional clearance-receptors. In search for such receptors, we focused on the macrophage-specific scavenger-receptor SR-AI. von Willebrand factor displays efficient binding to SR-AI (half-maximum binding 14±5 nM). Binding is calcium-dependent and is inhibited by 72±4% in the combined presence of antibodies against the A1- and D4-domains. Association with SR-AI was confirmed in cell-binding experiments. In addition, binding to bone marrow-derived murine SR-AI-deficient macrophages was strongly reduced compared to binding to wild-type murine macrophages. Following expression via hydrodynamic gene transfer, we determined ratios for von Willebrand factor-propeptide over von Willebrand factor-antigen, a marker of von Willebrand factor clearance. Propeptide/antigen ratios were significantly reduced in SR-AI-deficient mice compared to wild-type mice (0.6±0.2 versus 1.3±0.3; P<0.0001), compatible with a slower clearance of von Willebrand factor in SR-AI-deficient mice. Interestingly, mutants associated with increased clearance (von Willebrand factor/p.R1205H and von Willebrand factor/p.S2179F) had significantly increased binding to purified SR-AI and SR-AI expressed on macrophages. Accordingly, propeptide/antigen ratios for these mutants were reduced in SR-AI-deficient mice. In conclusion, we have identified SR-AI as a novel macrophage-specific receptor for von Willebrand factor. Enhanced binding of von Willebrand factor mutants to SR-AI may contribute to the increased clearance of these mutants