p63 regulates human keratinocyte proliferation via MYC-regulated gene network and differentiation commitment through cell adhesion-related gene network.

International audienceAlthough p63 and MYC are important in the control of epidermal homeostasis, the underlying molecular mechanisms governing keratinocyte proliferation or differentiation downstream of these two genes are not completely understood. By analyzing the transcriptional changes and phenotypic consequences of the loss of either p63 or MYC in human developmentally mature keratinocytes, we have characterized the networks acting downstream of these two genes to control epidermal homeostasis. We show that p63 is required to maintain growth and to commit to differentiation by two distinct mechanisms. Knockdown of p63 led to down-regulation of MYC via the Wnt/β-catenin and Notch signaling pathways and in turn reduced keratinocyte proliferation. We demonstrate that a p63-controlled keratinocyte cell fate network is essential to induce the onset of keratinocyte differentiation. This network contains several secreted proteins involved in cell migration/adhesion, including fibronectin 1 (FN1), interleukin-1β (IL1B), cysteine-rich protein 61 (CYR61), and jagged-1 (JAG1), that act downstream of p63 as key effectors to trigger differentiation. Our results characterized for the first time a connection between p63 and MYC and a cell adhesion-related network that controls differentiation. Furthermore, we show that the balance between the MYC-controlled cell cycle progression network and the p63-controlled cell adhesion-related network could dictate skin cell fate

Pathophysiology and Diagnosis of Drug-Induced Immune Thrombocytopenia

International audienceDrug-induced immune thrombocytopenia (DITP) is a life-threatening clinical syndrome that is under-recognized and difficult to diagnose. Many drugs can cause immune-mediated thrombocytopenia, but the most commonly implicated are abciximab, carbamazepine, ceftriaxone, eptifibatide, heparin, ibuprofen, mirtazapine, oxaliplatin, penicillin, quinine, quinidine, rifampicin, suramin, tirofiban, trimethoprim-sulfamethoxazole, and vancomycin. Several different mechanisms have been identified in typical DITP, which is most commonly characterized by severe thrombocytopenia due to clearance and/or destruction of platelets sensitized by a drug-dependent antibody. Patients with typical DITP usually bleed when symptomatic, and biological confirmation of the diagnosis is often difficult because detection of drug-dependent antibodies (DDabs) in the patient's serum or plasma is frequently not possible. This is in contrast to heparin-induced thrombocytopenia (HIT), which is a particular DITP caused in most cases by heparin-dependent antibodies specific for platelet factor 4, which can strongly activate platelets in vitro and in vivo, explaining why affected patients usually have thrombotic complications but do not bleed. In addition, laboratory tests are readily available to diagnose HIT, unlike the methods used to detect DDabs associated with other DITP that are mostly reserved for laboratories specialized in platelet immunology

Transcriptional Silencing of the TFPI-2 Gene by Promoter Hypermethylation in Choriocarcinoma Cells

International audienceTissue factor pathway inhibitor-2 (TFPI-2), a Kunitztype serine proteinase inhibitor associated with the extracellular matrix, has been shown to reduce tumor invasion. In the present study we identified the presence of a complete CpG island region spanning exon 1 and the three transcription initiation sites. We demonstrate that DNA demethylation by 5'-aza-2'-deoxycytidine restores TFPI-2 transcription in JAR choriocarcinoma cells. The effect of in vitro DNA methylation on TFPI-2 promoter function was also confirmed with TFPI-2/luciferase promoter constructs. Finally, we determined the precise methylation status of CpG sites of the TFPI-2 promoter in normal and tumor trophoblast cells using the bisulfite genomic sequencing method. We conclude that hypermethylation of the TFPI-2 gene is correlated with transcriptional silencing and that the TFPI-2 gene may be a candidate tumor suppressor gene

Characterization of 5B9, a chimeric monoclonal anti-PF4/H antibody with a human Fc fragment and which mimics the effects of HIT human antibodies

Heparin-induced thrombocytopenia (HIT) is a frequent drug-adverse event caused in the majority of patients by platelet-activating antibodies (Abs) directed against complexes of heparin (H) bound to platelet factor 4 (PF4). In most cases, HIT Abs are IgG which are potentially pathogenic as they are able to activate platelets directly in the presence of heparin via FcgRIIA receptors. The diagnosis of HIT is based on both clinical and biological criteria. However, despite recent improvements in HIT laboratory assays, a standard is always lacking for both immunological and functional assays. First, platelets from healthy donors exhibit wide variability in their response to HIT antibodies. Second, many patients who synthesize significant levels of antibodies to PF4/H while being treated with heparin do not develop HIT and the mechanisms that regulate the pathogenicity of HIT antibodies have not been fully defined. However, several studies suggested that epitope specificity of IgG antibodies is critical for the pathophysiology of HIT, especially by influencing the stability of PF4 tetramers.We therefore aimed to develop a monoclonal anti-PF4/H antibody with a human Fc fragment using transgenic mice homozygous for Cg gene of a G-class human immunoglobulin and that directly produce chimeric IgG antibodies.After immunization, several clones were found to synthesize anti-PF4 IgG antibodies but only one (5B9) produced an IgG1 that specifically bind PF4/H complexes without reactivity against PF4 alone. 5B9 was able to induce dose-dependent platelet activation and aggregation in the presence of low concentrations of UFH (0.1 and 0.5 IU/mL), and this effect was not observed without UFH or with high concentration of UFH (10 IU/ml), and was fully inhibited by IV.3 antibody. In addition, 5B9 with UFH (0.5 IU/mL) induced strong TF mRNA synthesis in isolated monocytes. Injection of 5B9 with UFH to transgenic mice expressing human PF4 and FcgRIIA receptors was followed by significant thrombocytopenia, similar to that observed with KKO, a murine IgG2b anti-PF4/H antibody. Plasma levels of thrombin/anti-thrombin (TAT) also significantly increased after injection of heparin in all mice having received 5B9 or KKO, but this effect was more pronounced on day 1 in 5B9-treated mice (mean value= 47 vs. 5 ng/mL at day 0) than in mice injected with KKO (33 vs. 11 ng/mL, respectively).Competitive immunoassays were also developed and 15 of 25 plasma samples (60%) collected in HIT patients were shown to inhibit (by at least 20%) the binding of 5B9 to PF4/H complexes compared to 3/25 (12%) samples containing non pathogenic anti-PF4/H antibodies (obtained in patients without HIT). Similar experiments were performed with KKO and its binding to PF4/H was also inhibited by 32% of HIT plasma samples (8/25), and none of the non-HIT plasmas. However, the levels of 5B9 and KKO binding inhibition to PF4/H were highly correlated (R2=0.85), thereby suggesting that the epitopes recognized by the antibodies are similar. Noticeably, KKO was also shown to inhibit in a concentration-dependent manner the binding of 5B9 to PF4/H complexes, further supporting this hypothesis.5B9 was then sequenced and a docking model was elaborated based on VH and VL sequences of 5B9 obtained and a recently described crystal structure of KKO/PF4 tetramer complex. According to this model, 5B9 Fab likely interacts with the B and D monomers of PF4, thus possibly contributing to the stability of tetramers. In addition, the binding of 5B9 to PF4 involves 28 aa, 16 in the B monomer including Asp-7, Gln-9, Pro-34 and Pro-37 and 12 in the D monomer including Gln-9. Importantly, 13 of these 28 aa have also been identified as critical in the formation of PF4/KKO complex (Cai et al, Nat Commun. 2015;6:8277). Three regions (Asp-7 to Thr-15 and Ala-32 to Lys-41 in the B monomer and Gln-9 to Asp-18 in the D monomer) therefore appear particularly important in the binding of both 5B9 and KKO on PF4 tetramers.In conclusion, 5B9 is the first anti-PF4/H monoclonal antibody that has a human Fc fragment, and fully mimics the effect of HIT human antibodies. 5B9 could therefore be used as a standard in HIT laboratory assays. Moreover, our results support that three regions both recognized by 5B9 and KKO within PF4 tetramers are critical for the binding and pathogenicity of HIT antibodies

Point-of-care diagnostics for ricin exposure

International audienc

Detection of T wave beta-to-beat variations prior to ventricular arrythmias onset in ICD-stored intracardiac electrograms: the endocardial T-wave alternans study (ETWAS)

International audienceBackground: The aim of the Endocardial T-Wave Alternans Study was to prospectively assess thepresence of T-wave alternans (TWA) or beat-to-beat repolarization changes on implantable cardioverter-defibrillator (ICD)-stored electrograms (EGMs) immediately preceding the onset of spontaneous ventriculartachycardia (VT) or fibrillation (VF). Methods: Thirty-seven VT/VF episodes were compared to 116 baseline reference EGMs from the same57 patients. A Bayesian model was used to estimate the T-wave waveform in each cardiac beat and a setof 10 parameters was selected to segment each detected T wave. Beat-by-beat differences in each T-waveparameter were computed using the absolute value of the difference between each beat and the followingone. Fisher criterion was used for determining the most discriminant T-wave parameters, then top-Mranked parameters yielding a normalized cumulative Fisher score>95% were selected, and analysis wasapplied on these selected parameters. Simulated TWA EGMs were used to validate the algorithm. Results: In the simulation study, TWA was detectable even in the case of the smallest simulatedalternans of 25μV. In 13 of the 37 episodes (35%) occurring in nine of 16 patients, significant largerbeat-to-beat variations before arrhythmia onset were detected compared to their respective references(median one positive episode per patient). Parameters including the T-wave apex amplitude seem themore discriminant parameters.Conclusions:Detection of beat-by-beat repolarization variations in ICD-stored EGMs is feasible ina significant subset of cases and may be used for predicting the onset of ventricular arrhythmias

Treatment of voluminous and complicated superficial slow-flow vascular malformations with sirolimus (PERFORMUS): protocol for a multicenter phase 2 trial with a randomized observational-phase design

International audienceBackground: Slow-flow superficial vascular malformations (VMs) are rare congenital anomalies that can be responsible for pain and functional impairment. Currently, we have no guidelines for their management, which can involve physical bandages, sclerotherapy, surgery, anti-inflammatory or anti-coagulation drugs or no treatment The natural history is progressive and worsening. Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that acts as a master switch in cell proliferation, apoptosis, metabolism and angio/lymphangiogenesis. Sirolimus directly inhibits the mTOR pathway, thereby inhibiting cell proliferation and angio/lymphangiogenesis. Case reports and series have reported successful use of sirolimus in children with different types of vascular anomalies, with heterogeneous outcomes.Objective: The objective of this trial is to evaluate the efficacy and safety of sirolimus in children with complicated superficial slow-flow VMs.Methods/design: This French multicenter randomized observational-phase, phase 2 trial aims to include 50 pediatric patients 6 to 18 years old who have slow-flow (lymphatic, venous or lymphatico-venous) voluminous complicated superficial VM. Patients will be followed up for 12 months. All patients will start with an observational period (no treatment). Then at a time randomly selected between month 4 and month 8, they will switch to the experimental period (switch time), when they will receive sirolimus until month 12. Each child will undergo MRI 3 times: at baseline, at the switch time, and at month 12. For both periods (observational and treatment), we will calculate the relative change in volume of the VM divided by the study period duration. This relative change weighted by the study period duration will constitute the primary endpoint. VM will be measured by MRI images, which will be centralized and interpreted by the same radiologist who will be blinded to the study period. Hence, each patient will be his/her own control. Secondary outcomes will include assessment of safety and efficacy by viewing standardized digital photographs and according to the physician, the patient or proxy; impact on quality of life; and evolution of biological makers (coagulation factors, vascular endothelial growth factor, tissue factor).Discussion: The main benefit of the study will be to resolve uncertainty concerning the efficacy of sirolimus in reducing the volume of VMs and limiting related complications and the safety of the drug in children with slowflow VMs. This trial design is interesting in these rare conditions because all included patients will have the opportunity to receive the drug and the physician can maintain it after the end of the protocol if is found efficient (which would not be the case in a classical cross-over study)

Additional file 3: of Treatment of voluminous and complicated superficial slow-flow vascular malformations with sirolimus (PERFORMUS): protocol for a multicenter phase 2 trial with a randomized observational-phase design

SPIRIT figure. (DOC 50 kb

Relation of outcomes to ABC (Atrial Fibrillation Better Care) pathway adherent care in European patients with atrial fibrillation: an analysis from the ESC-EHRA EORP Atrial Fibrillation General Long-Term (AFGen LT) Registry

International audienceAbstract Aims There has been an increasing focus on integrated, multidisciplinary, and holistic care in the treatment of atrial fibrillation (AF). The ‘Atrial Fibrillation Better Care’ (ABC) pathway has been proposed to streamline integrated care in AF. We evaluated the impact on outcomes of an ABC adherent management in a contemporary real-life European-wide AF cohort. Methods and results Patients enrolled in the ESC-EHRA EURObservational Research Programme in AF General Long-Term Registry with baseline data to evaluate ABC criteria and available follow-up data were considered for this analysis. Among the original 11 096 AF patients enrolled, 6646 (59.9%) were included in this analysis, of which 1996 (30.0%) managed as ABC adherent. Patients adherent to ABC care had lower CHA2DS2-VASc and HAS-BLED scores (mean ± SD, 2.68 ± 1.57 vs. 3.07 ± 1.90 and 1.26 ± 0.93 vs. 1.58 ± 1.12, respectively; P < 0.001). At 1-year follow-up, patients managed adherent to ABC pathway compared to non-adherent ones had a lower rate of any thromboembolic event (TE)/acute coronary syndrome (ACS)/cardiovascular (CV) death (3.8% vs. 7.6%), CV death (1.9% vs. 4.8%), and all-cause death (3.0% vs. 6.4%) (all P < 0.0001). On Cox multivariable regression analysis, ABC adherent care showed an association with a lower risk of any TE/ACS/CV death [hazard ratio (HR): 0.59, 95% confidence interval (CI): 0.44–0.79], CV death (HR: 0.52, 95% CI: 0.35–0.78), and all-cause death (HR: 0.57, 95% CI: 0.43–0.78). Conclusion In a large contemporary cohort of European AF patients, a clinical management adherent to ABC pathway for integrated care is associated with a significant lower risk for cardiovascular events, CV death, and all-cause death