Deregulation of the protocadherin gene FAT1 alters muscle shapes: implications for the pathogenesis of facioscapulohumeral dystrophy.

International audienceGeneration of skeletal muscles with forms adapted to their function is essential for normal movement. Muscle shape is patterned by the coordinated polarity of collectively migrating myoblasts. Constitutive inactivation of the protocadherin gene Fat1 uncoupled individual myoblast polarity within chains, altering the shape of selective groups of muscles in the shoulder and face. These shape abnormalities were followed by early onset regionalised muscle defects in adult Fat1-deficient mice. Tissue-specific ablation of Fat1 driven by Pax3-cre reproduced muscle shape defects in limb but not face muscles, indicating a cell-autonomous contribution of Fat1 in migrating muscle precursors. Strikingly, the topography of muscle abnormalities caused by Fat1 loss-of-function resembles that of human patients with facioscapulohumeral dystrophy (FSHD). FAT1 lies near the critical locus involved in causing FSHD, and Fat1 mutant mice also show retinal vasculopathy, mimicking another symptom of FSHD, and showed abnormal inner ear patterning, predictive of deafness, reminiscent of another burden of FSHD. Muscle-specific reduction of FAT1 expression and promoter silencing was observed in foetal FSHD1 cases. CGH array-based studies identified deletion polymorphisms within a putative regulatory enhancer of FAT1, predictive of tissue-specific depletion of FAT1 expression, which preferentially segregate with FSHD. Our study identifies FAT1 as a critical determinant of muscle form, misregulation of which associates with FSHD

Rôle de la signalisation ErbB/Neurégulines dans la propagation de PEA3 dans les motoneurones de la moelle épinière

Les signaux environnementaux ont une grande influence sur le devenir de certaines populations de motoneurones. J'étudie la population qui exprime le facteur de transcription PEA3, située au niveau brachial, caractérisée et spécifiée par ce facteur, et qui innerve les muscles dorsaux des membres (Livet et al., 2002 ; Vrieseling et al., 2006). Cette population représente un des exemples les mieux compris de l'acquisition d'une identité neuronale par des signaux provenant du muscle cible. Au cours du développement, l'expression de PEA3 se met en place de manière séquentielle. PEA3 est d'abord exprimé dans un premier sous-groupe de neurones localisé en position postérieure dans le domaine (neurones pionniers), puis dans un deuxième sous-groupe de neurones situé en position plus antérieure. Le développement de cette population implique des échanges de signaux entre les neurones pionniers, instruits par le muscle cible, et le deuxième groupe de neurones antérieurs, instruit par les neurones pionniers. Le GDNF, produit par les cellules du futur muscle cible, induit PEA3 dans les neurones pionniers (Haase et al., 2002). Puis le HGF, un autre facteur dérivé du membre, induit les neurones pionniers à sécréter un ‘signal de propagation’, qui agit à distance et induit l'expression de PEA3 dans le deuxième groupe de neurones (neurones recrutés) (Helmbacher et al., 2003). L'objectif initial de ma thèse a été basé sur l'identification de ce signal de propagation. J'ai d'abord utilisé une approche pharmacologique dans un système in vitro de cultures d'explants de moelles épinières d'embryons de souris. En y inhibant la voie EGF, j'ai démontré que le signal de propagation appartient à cette famille de molécules. Les récepteurs de la voie EGF (ErbB1 à ErbB4) sont exprimés chez l'embryon de poulet et de souris dans la moelle épinière brachiale, et spécifiquement dans les motoneurones, au moment où PEA3 est exprimé. Parmi les ligands de la voie EGF, je me suis intéressée aux neurégulines, une famille de glycoprotéines connue pour son implication dans la mise en place du système nerveux. J'ai montré que des isoformes du gène neuréguline1 (nrg1), possédant un domaine immunoglobuline (type I) sont capables d'induire l'expression de pea3 dans la moelle épinière brachiale, et spécifiquement dans les neurones recrutés. J'ai pu démontrer, en utilisant des souris mutantes pour le récepteur à l’HGF (metd/d), que le signal de propagation est vraisemblablement une isoforme NRG1, de type I.Signals derived from the environment have an important influence on development of some motorneurons populations. I study the population that expresses the transcription factor PEA3, localized at the brachial level, characterized and specified by this factor. This population innervates the limb dorsal muscles (Livet et al., 2002 ; Vrieseling et al., 2006). This population represents one of the best understood examples of an acquisition of a neuronal identity induced by signals derived from the target muscle. During development, PEA3 expression is made in two times in motorneurons. Initially, PEA3 is expressed in a first population, localized in the posterior part of the domain (pionneers neurons), then in a second population, localized in a more anterior position. Development of this population implies exchanges of signals between pionneers neurons, instructed by the target muscle, and anterior neurons, instructed by pionneers neurons. GDNF, produced by cells of the future target muscle, induces PEA3 in the pioneers neurons (Haase et al., 2002). Then, HGF, another limb-derived factor, induces pioneers neurons to secrete a propagation signal’, which induces PEA3 expression in the second population of neurons (recruited neurons) (Helmbacher et al., 2003). The initial purpose of my phD was to identify this ‘propagation signal’. First, I used a pharmacological approach in an in vitro assay of mouse embryos spinal cord explants culture. I did inhibition of the EGF pathway in this assay, and I showed that the propagation signal belongs to this family. EGF receptors (ErbB1 à ErbB4) are expressed in chick and mouse embryos, and especially in motorneurons, when PEA3 is expressed. Among the EGF ligands, I studied neuregulins, a family of glycoproteins involved in the nervous system development. I showed that isoforms of neuregulin1 gene (nrg1), which have an immunoglobulin domain (type I) induce pea3 expression in the brachial spinal cord, and especially in the recruited neurons. I observed, by using mice mutants for HGF receptor (metd/d), that the propagation signal is plausibly a typeI NRG1 isoform. Keywords : motorneurons, PEA3, recruitment, ErbB

Toward a consideration of homework in elementary schools.

Publié en anglais sous le titre: Toward a consideration of homework in elementary schools. Abridged version : brief to the Minister of education, recreation and sportBibliogr.: p. 35-3

Pour soutenir une réflexion sur les devoirs à l'école primaire : avis à la ministre de l'éducation, du loisir et du sport /

Bibliogr.: p. 101-10

Assessment of neutrophil subsets and immune checkpoint inhibitor expressions on T lymphocytes in liver transplantation: A preliminary study beyond the neutrophil-lymphocyte ratio

International audienceBackground: Advanced stages of cirrhosis are characterized by the occurrence of progressive immune alterations known as CAID (Cirrhosis Associated Immune Dysfunction). In advanced cirrhosis, liver transplantation (LT) remains the only curative treatment. Sepsis, shares many similarities with decompensated cirrhosis in terms of immuno-inflammatory response. In both conditions, the neutrophil-lymphocyte ratio (NLR) is associated with poor outcomes. Based on alterations in sepsis, we hypothesized that we could observe in cirrhotic and LT patients more detailed neutrophil and lymphocyte phenotypes. To this end, along with leukocyte count, we assessed immature neutrophils, LOX-1 + MDSC and PD-1 and TIM-3 lymphocyte expressions in cirrhotic patients before transplantation in association with liver disease severity and during the first month after transplantation.Methods: We conducted a prospective monocentric study including cirrhotic patients registered on LT waiting-list. Blood samples were collected at enrolment before LT and for 1 month post-LT. In addition to NLR, we assessed by whole blood flow cytometry the absolute count of immature neutrophils and LOX-1 + MDSC as well as the expressions of immune checkpoint receptors PD-1 and TIM-3 on T lymphocytes. Results: We included 15 healthy volunteers (HV) and 28 patients. LT was performed for 13 patients. Pre-LT patients presented with a higher NLR compared to HV and NLR was associated with cirrhosis severity. Increased immature neutrophils and LOX-1 + MDSC counts were observed in the most severe patients. These alterations were mainly associated with acute decompensation of cirrhosis. PD-1 and TIM-3 expressions on T lymphocytes were not different between patients and HV. Post-LT immune alterations were dominated by a transitory but tremendous increase of NLR and immature neutrophils during the first days post-LT. Then, immune checkpoint receptors and LOX-1 + MDSC tended to be overexpressed by the second week after surgery.Conclusion: The present study showed that NLR, immature neutrophils and LOX-1 + MDSC counts along with T lymphocyte count and checkpoint inhibitor expression were altered in cirrhotic patients before and after LT. These data illustrate the potential interest of immune monitoring of cirrhotic patients in the context of LT in order to better define risk of sepsis. For this purpose, larger cohorts of patients are now necessary in order to move forward a more personalised care of LT patients

Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

Hepatitis C virus (HCV) is an oncogenic virus associated with the onset of hepatocellular carcinoma (HCC). The present study investigated the possible link between HCV infection and Netrin-1, a ligand for dependence receptors that sustains tumorigenesis, in particular in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV+ liver biopsies compared to hepatitis B virus (HBV+) and uninfected samples. Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues. Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression. In vitro, HCV increased the level and translation of Netrin-1 in a NS5A-La-related protein 1 (LARP1)-dependent fashion. Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells. Netrin-1 enhanced infectivity of HCV particles and promoted viral entry by increasing the activation and decreasing the recycling of the epidermal growth factor receptor (EGFR), a protein that is dysregulated in HCC. Netrin-1 and HCV are, therefore, reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions. This functional association involving a cancer-related virus and Netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species

Circulating microRNAs improve bacterial infection diagnosis and overall survival prediction in acute decompensation of liver cirrhosis

Summary: Bacterial infections are the most frequent precipitating event in patients with acute decompensation of cirrhosis (AD) and are associated with high mortality. Early diagnosis is challenging due to cirrhosis-related systemic inflammation. Here we investigated the potential of circulating microRNAs to diagnose bacterial infections and predict survival in cirrhotic patients with AD. High throughput profiling of circulating microRNAs was performed using the Nanostring technology in 57 AD patients and 24 patients with compensated cirrhosis (CC). Circulating miRs profiling showed that: (a) miRs differentially detected in AD vs. CC were mostly down-regulated; (b) a composite score including absolute neutrophil count, C reactive protein and miR-362-3p could diagnose bacterial infection with an excellent performance (AUC of 0.825 [95% CI = 0.671–0.980; p < 0.001]); (c) a composite score including miR-382-5p, miR-592 and MELD-Na improved 6-month survival prediction. Circulating miRs are strongly dysregulated in patients with AD and may help to improve bacterial infection diagnosis and survival prediction

Direct-acting antiviral therapy decreases hepatocellular carcinoma recurrence rate in cirrhotic patients with chronic hepatitis C

International audienceBACKGROUND AND AIMS: Arrival of direct-acting antiviral (DAA) agents against hepatitis C virus (HCV) with high-sustained virological response (SVR) rates and very few side effects has drastically changed the management of HCV infection. The impact of DAA exposure on hepatocellular carcinoma (HCC) recurrence after a first remission in patients with advanced fibrosis remains to be clarified. METHODS: 68 consecutive HCV patients with a first HCC diagnosis and under remission, subsequently treated or not with a DAA combination, were included. Clinical, biological, and virological data were collected at first HCC diagnosis, at remission and during the surveillance period. RESULTS: All patients were cirrhotic. Median age was 62 years and 76% of patients were male. Twenty-three patients (34%) were treated with DAAs and 96% of them achieved SVR. Median time between HCC remission and DAA initiation was 7.2 months (IQR: 3.6 - 13.5; range: 0.3 - 71.4) and median time between DAA start and HCC recurrence was 13.0 months (IQR: 9.2 - 19.6; range: 3.0 - 24.7). Recurrence rate was 1.7/100 person-months among treated patients vs 4.2/100 person-months among untreated patients (p=0.008). In multivariate survival analysis, the hazard ratio for HCC recurrence after DAA exposure was 0.24 (95% confidence interval: 0.10-0.55; p\textless0.001). CONCLUSIONS: HCC recurrence rate was significantly lower among patients treated with DAA compared with untreated patients. Given the potential impact of our observation, large-scale prospective cohort studies are needed to confirm these results. This article is protected by copyright. All rights reserve

<i>Fat1</i> controls the shape of subsets of scapular muscle by modulating myoblast polarity during planar migration.

<p>(<b>A–C</b>) Reporter gene expression in the forelimb and flank of mouse embryos between E11.5 and E13.5. (<b>A</b>) <i>Gdnf-lacZ</i> staining labels myoblasts of the <i>latissimus dorsee</i> (LD) and <i>cutaneous maximus</i> (CM). CM myoblasts migrate away from the brachial plexus to form a subcutaneous muscle sheath, composed of radially-oriented chains of myoblasts. (<b>B</b>) At E13.5, <i>MLC3f-lacZ</i> staining reveals the characteristic fan-shaped form of the CM (dotted white purple line) as compared to other limb muscles. (<b>C</b>) <i>Fat1</i> expression detected using the <i>lacZ</i> gene trap allele KST249 (<i>Fat1^LacZ</i>) is selectively localized within the CM and in surrounding tissue (pink arrow). (<b>D</b>) CM myoblasts express <i>Fat1</i> and migrate towards an increasing gradient of <i>Fat1</i> expression. Alternate vibratome cross-sections of a wild type E12.5 embryo were hybridized with <i>Fat1</i> (left column) and <i>MyoD</i> (purple, right column) RNA probes. Photographs of adjacent sections were superimposed (photoshop) after conversion of <i>Fat1</i> staining color in pink (right column; <i>Fat1</i> in pink, <i>MyoD</i> in purple). <i>MyoD</i> expression is used as a marker of the muscle lineage. Superimposition was meant to compare the relative levels of <i>Fat1</i> expression within and around the <i>cutaneous maximus</i> (CM) muscle (indicated with purple arrows), at three consecutive antero-posterior positions, respectively within the CM (top row), at the posterior end (middle row), and posterior to the caudal extremity of the CM at that stage. CM myoblasts, migrating from anterior to posterior, express lower levels of <i>Fat1</i> RNA than the surrounding subcutaneous cell layer (pink arrows). Intensity of <i>Fat1</i> staining in this subcutaneous layer increases gradually in caudal sections. (<b>E–H</b>) Orientation of CM myoblast migration in whole-mounts of E12.5 <i>Fat1^LacZ/LacZ</i> and control embryos detected using <i>MyoD in situ</i> hybridization. In all panels anterior is to the left, dorsal is to the top. (<b>E</b>) The CM muscle (purple dotted line) in <i>Fat1^LacZ/LacZ</i> embryos displays reduced size and altered shape as compared to wild type. Higher magnification images (right hand panels) show that within the CM muscle, radial organization of myoblast chains was perturbed by <i>Fat1</i>-deficiency, resulting in a fuzzy migration front and irregular distribution of myoblasts (red arrows). In addition, ectopic clusters of myoblasts (orange arrows) are detected in the shoulder area (dotted orange line). (<b>F</b>) Quantification of the abnormal orientation of <i>Fat1</i> mutant myoblasts. The angle between the longest diameter of each myoblast nucleus and the axis of the closest myoblast chain was measured on flat-mounted CM muscles. The bar graph presents mean (± s.e.m.) percentages of <i>myoD</i>⁺ nuclei displaying a given angle (by angle ranges of 10°) for wild type (gray) and <i>Fat1^LacZ/LacZ</i> (black) embryos. (<b>G, H</b>) High magnification images of <i>MyoD</i>-expressing myoblasts in equivalent positions – within the chains (<b>G</b>) or at the leading edge (migration front, <b>H</b>) – in the CM of mutants and controls. Scale bars: (<b>A–C</b>), 0.8 mm; (<b>D</b>) 300 µm; (<b>E</b>), left: 0.5 mm; (<b>E</b>), right: 100 µm; (<b>G, H</b>) 10 µm.</p