Physiopathologie Animale et bioThérapie du muscle et du système nerveux

Abstract Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory tract infection in infants, older adults and the immunocompromised. Effective directly acting antivirals are not yet available for clinical use. To address this, we screen the ReFRAME drug-repurposing library consisting of 12,000 small molecules against RSV. We identify 21 primary candidates including RSV F and N protein inhibitors, five HSP90 and four IMPDH inhibitors. We select lonafarnib, a licensed farnesyltransferase inhibitor, and phase III candidate for hepatitis delta virus (HDV) therapy, for further follow-up. Dose-response analyses and plaque assays confirm the antiviral activity (IC 50 : 10-118 nM). Passaging of RSV with lonafarnib selects for phenotypic resistance and fixation of mutations in the RSV fusion protein (T335I and T400A). Lentiviral pseudotypes programmed with variant RSV fusion proteins confirm that lonafarnib inhibits RSV cell entry and that these mutations confer lonafarnib resistance. Surface plasmon resonance reveals RSV fusion protein binding of lonafarnib and co-crystallography identifies the lonafarnib binding site within RSV F. Oral administration of lonafarnib dose-dependently reduces RSV virus load in a murine infection model using female mice. Collectively, this work provides an overview of RSV drug repurposing candidates and establishes lonafarnib as a bona fide fusion protein inhibitor.

Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus associated with a robust systemic infection and an acute inflammatory rheumatic disease. A high fiber diet has been widely promoted for its ability to ameliorate inflammatory diseases. Fiber is fermented in the gut into short chain fatty acids such as acetate, propionate, and butyrate, which enter the circulation providing systemic anti-inflammatory activities. Herein we show that mice fed a high fiber diet show a clear exacerbation of CHIKV arthropathy, with increased edema and neutrophil infiltrates. RNA-Seq analyses illustrated that a high fiber diet, in this setting, promoted a range of pro-neutrophil responses including Th17/IL-17. Gene Set Enrichment Analyses demonstrated significant similarities with mouse models of inflammatory psoriasis and significant depression of macrophage resolution phase signatures in the CHIKV arthritic lesions from mice fed a high fiber diet. Supplementation of the drinking water with butyrate also increased edema after CHIKV infection. However, the mechanisms involved were different, with modulation of AP-1 and NF-κB responses identified, potentially implicating deoptimization of endothelial barrier repair. Thus, neither fiber nor short chain fatty acids provided benefits in this acute infectious disease setting, which is characterized by widespread viral cytopathic effects and a need for tissue repair.

BACKGROUND: We recently developed and characterized a standardized and clinical grade human Platelet Lysate (hPL) that constitutes an advantageous substitute for fetal bovine serum (FBS) for human mesenchymal stem cell (hMSC) expansion required in cell therapy procedures, avoiding xenogenic risks (virological and immunological) and ethical issues. Because of the progressive use of pathogen-reduced (PR) labile blood components, and the requirement of ensuring the viral safety of raw materials for cell therapy products, we evaluated the impact of the novel procedure known as THERAFLEX UV-Platelets for pathogen reduction on hPL quality (growth factors content) and efficacy (as a medium supplement for hMSC expansion). This technology is based on short-wave ultraviolet light (UV-C) that induces non-reversible damages in DNA and RNA of pathogens while preserving protein structures and functions, and has the main advantage of not needing the addition of any photosensitizing additives (that might secondarily interfere with hMSCs). METHODOLOGY / PRINCIPAL FINDINGS: We applied the THERAFLEX UV-Platelets procedure on fresh platelet concentrates (PCs) suspended in platelet additive solution and prepared hPL from these treated PCs. We compared the quality and efficacy of PR-hPL with the corresponding non-PR ones. We found no impact on the content of five cytokines tested (EGF, bFGF, PDGF-AB, VEGF and IGF-1) but a significant decrease in TGF-ß1 (-21%, n = 11, p<0.01). We performed large-scale culture of hMSCs from bone marrow (BM) during three passages and showed that hPL or PR-hPL at 8% triggered comparable BM-hMSC proliferation as FBS at 10% plus bFGF. Moreover, after proliferation of hMSCs in an hPL- or PR-hPL-containing medium, their profile of membrane marker expression, their clonogenic potential and immunosuppressive properties were maintained, in comparison with BM-hMSCs cultured under FBS conditions. The potential to differentiate towards the adipogenic and osteogenic lineages of hMSCs cultured in parallel in the three conditions also remained identical. CONCLUSION / SIGNIFICANCE: We demonstrated the feasibility of using UV-C-treated platelets to subsequently obtain pathogen-reduced hPL, while preserving its optimal quality and efficacy for hMSC expansion in cell therapy applications.

Abstract Background Duchenne muscular dystrophy (DMD) is an X-linked inherited disease caused by mutations in the gene encoding dystrophin that leads to a severe and ultimately life limiting muscle-wasting condition. Recombinant adeno-associated vector (rAAV)-based gene therapy is promising, but the size of the full-length dystrophin cDNA exceeds the packaging capacity of a rAAV. Alternative or complementary strategies that could treat DMD patients are thus needed. Intracellular calcium overload due to a sarcolemma permeability to calcium (SPCa) increase is an early and critical step of the DMD pathogenesis. We assessed herein whether TRPC1 and TRPC3 calcium channels may be involved in skeletal muscle SPCa alterations and could represent therapeutic targets to treat DMD. Methods All experiments were conducted in the DMD mdx rat, an animal model that closely reproduces the human DMD disease. We measured the cytosolic calcium concentration ([Ca 2+ ] c ) and SPCa in EDL ( Extensor Digitorum Longus ) muscle fibers from age-matched WT and DMD mdx rats of 1.5 to 7 months old. TRPC1 and TRPC3 expressions were measured in the EDL muscles at both the mRNA and protein levels, by RT-qPCR, western blot and immunocytofluorescence analysis. Results As expected from the malignant hyperthermia like episodes observed in several DMD mdx rats, calcium homeostasis alterations were confirmed by measurements of early increases in [Ca 2+ ] c and SPCa in muscle fibers. TRPC3 and TRPC1 protein levels were increased in DMD mdx rats. This was observed as soon as 1.5 months of age for TRPC3 but only at 7 months of age for TRPC1. A slight but reliable shift of the TRPC3 apparent molecular weight was observed in DMD mdx rat muscles. Intracellular localization of both channels was not altered. We thus focused our attention on TRPC3. Application of Pyr10, a specific inhibitor of TRPC3, abolished the differences between SPCa values measured in WT and DMD mdx . Finally, we showed that a rAAV-microdystrophin based treatment induced a high microdystrophin expression but only partial prevention of calcium homeostasis alterations, skeletal muscle force and TRPC3 protein increase. Conclusions All together our results show that correcting TRPC3 channel expression and/or activity appear to be a promising approach as a single or as a rAAV-based complementary therapy to treat DMD.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked muscle disease that leads to fibre necrosis and progressive paralysis. At present, DMD remains a lethal disease without any effective treatment, requiring a better understanding of the pathophysiological processes and comprehensive assessment of the newly identified therapeutic strategies. MicroRNAs including members of the muscle-specific myomiR family have been identified as being deregulated in muscle of DMD patients and in mdx mice used as a model for DMD. In recent years, the Golden Retriever muscular dystrophy (GRMD) dog has appeared as the crucial animal model for objectively assessing the potential of new innovative approaches. Here, we first aim at establishing the muscle expression pattern of five selected miRNAs in this clinically relevant model to determine if they are similarly affected compared with other DMD contexts. Second, we attempt to show whether these miRNAs could be impacted by the systemic delivery of a promising stem cell candidate (referred to as MuStem cells) to implement our knowledge on its mode of action and/or identify markers associated with cell therapy efficacy. METHODS: A comparative study of miRNAs expression levels and cellular localization was performed on 9-month-old healthy dogs, as well as on three sub-sets of GRMD dog (without immunosuppression or cell transplantation, with continuous immunosuppressive regimen and with MuStem cell transplantation under immunosuppression), using RT-qPCR and in situ hybridization. RESULTS: We find that miR-222 expression is markedly up-regulated in GRMD dog muscle compared to healthy dog, while miR-486 tends to be down-expressed. Intriguingly, the expression of miR-1, miR-133a and miR-206 does not change. In situ hybridization exploration reveals, for the first time, that miR-486 and miR-206 are mainly localized in newly regenerated fibres in GRMD dog muscle. In addition, we show that cyclosporine-based immunosuppression, classically used in allogeneic cell transplantation, exclusively impacts the miR-206 expression. Finally, we demonstrate that intra-arterial administration of MuStem cells results in up-regulation of miR-133a and miR-222 concomitantly with a down-expression of two sarcomeric proteins corresponding to miR-222 targets. CONCLUSION: We point out a differential muscle expression of miR-222 and miR-486 associated with the pathophysiology of the clinically relevant GRMD dog model with a tissue localization focused on regenerated fibres. We also establish a modified expression of miR-133a and miR-222 subsequent to MuStem cell infusion.

This study investigated the long-term effectiveness and safety of a variant of the transobturator vaginal tape inside-out technique for acquired urinary incontinence. Twelve spayed female dogs were operated over a 2 yr period. No intraoperative complications were encountered. Transient dysuria was the most common postoperative complication (7 out of 12 dogs). On the 12th day postoperatively, incontinence was completely cured in 11 out of 12 dogs (92%). At the time of the second evaluation (median follow-up time was 21 mo), patients classified as "cured," "greatly improved," or "improved" were 25, 50, and 25% of the total, respectively. At the time of either the fourth evaluation or at the time of death (median follow-up time was 52 mo), 50% of the bitches (6 out of 12) had the same results as previously but the other 50% had leakage that reappeared sporadically. A fistula appeared on the path of the tape in two bitches at 28 and 32 mo postsurgically. The technique presented is effective and more cost effective than the standard technique and could constitute an attractive alternative; however, it could be associated with an immediate postoperative dysuria, delayed fistula formation, and a partial recurrence of clinical signs.

Preclinical data acquired for human muscle stem (hMuStem) cells indicate their great repair capacity in the context of muscle injury. However, their clinical potential is limited by their moderate ability to survive after transplantation. To overcome these limitations, their encapsulation within protective environment would be beneficial. In this study, tunable calcium-alginate hydrogels obtained through molding method using external or internal gelation were investigated as a new strategy for hMuStem cell encapsulation. The mechanical properties of these hydrogels were characterized in their fully hydrated state by compression experiments using Atomic Force Microscopy. Measured elastic moduli strongly depended on the gelation mode and calcium/alginate concentrations. Values ranged from 1 to 12.5 kPa and 3.9 to 25 kPa were obtained for hydrogels prepared following internal and external gelation, respectively. Also, differences in mechanical properties of hydrogels resulted from their internal organization, with an isotropic structure for internal gelation, while external mode led to anisotropic one. It was further shown that viability, morphological and myogenic differentiation characteristics of hMuStem cells incorporated within alginate hydrogels were preserved after their release. These results highlight that hMuStem cells encapsulated in calcium-alginate hydrogels maintain their functionality, thus allowing to develop muscle regeneration protocols to improve their therapeutic efficacy. • Comparative study of the impact of gelation mode (external vs. internal) on physical properties of Ca-alginate hydrogels • Evaluation of the mechanical, structural and diffusion properties of Ca-alginate hydrogels for Muscle Stem cell encapsulation • Ca-alginate hydrogels possess distinct internal structures (anisotropic vs isotropic) depending on the gelation mode • Ca-alginate hydrogels with elastic modulus close to the skeletal muscle preserve properties of encapsulated hMuStem cells

The development of innovative immune cell therapies relies on efficient cell tracking strategies. For this, multiscale fluorescence-based analyses of transferred cells into the host with complementary techniques, including flow cytometry for high-throughput cell analysis and two-photon microscopy for deep tissue imaging would be highly beneficial. Ideally, cells should be labelled with a single fluorescent probe combining all the properties required for these different techniques. Due to the intrinsic autofluorescence of most tissues and especially the liver, far-red emission is also an important asset. However, the development of far-red emitting probes suitable for two-photon microscopy and compatible with clearing methods to track labelled immune cells in thick samples, remains challenging. A newly-designed water-soluble far-red emitting polymer probe, 19K-6H, with a large Stokes shift, was thus evaluated for the tracking of primary immune CD8 T cells. These cells, prepared from mouse spleen, were efficiently labelled with the 19K-6H probe, which was internalized via endocytosis and was highly biocompatible at concentrations up to 20 μM. Labelled primary CD8 T cells were detectable in culture by both confocal and two-photon microscopy as well as flow cytometry, even after 3 days of active proliferation. Finally, 19K-6H-labelled primary CD8 T cells were injected to mice in a classical model of immune mediated hepatitis. The efficient tracking of the transferred cells in the liver by flow cytometry (on purified non-parenchymal cells) and by two-photon microscopy on 800 μm thick cleared sections, demonstrated the versatility of the 19K-6H probe.

Nanoparticles (NPs), offering high specific surface, are considered as the best potential anti-microbial agents for a wide range of medical nanotechnology. Among them, silver NPs (AgNPs) allow high biocidal activity. A design of ecological capsules is proposed where AgNPs of 22 nm in diameter are grafted on the surface of biobased nanocrystals obtained from cellulose (CNC) and chitin (ChiNC). These silver nanohybrids are dispersed in calcium-alginate microgels of 45-50 µm in diameter using microfluidic tools. Such double level of immobilization of AgNPs leads to highly stable carriers, prolongs shelf life and raises bioactivity, with a precise control of well dispersed AgNPs as determined by scanning transmission electron microscopy, UV-Vis spectroscopy and atomic absorption spectroscopy. Preliminary tests for antimicrobial activities of these new microgels have shown a significant inhibitory effect on Candida albicans, the most common fungal pathogen, responsible for thrush and vaginal yeast infections, for very low levels of silver.

International audience

Le muscle squelettique est organisé en faisceau de fibres musculaires de différentes tailles et parcouru par des réseaux vasculaires et nerveux. Les cellules satellites sont des cellules souches logées le long des fibres musculaires et sont à la base des progéniteurs myogéniques (myoblastes). Les cellules satellites peuvent être aisément extraites du muscle et cultivées. Les modèles typiques de culture en deux dimensions (2D) de cellules dérivées du muscle squelettique ne peuvent pas recréer complètement l'organisation et la fonction des tissus musculaires vivants, ce qui limite leurs utilités dans les études physiologiques approfondies. Le développement de modèles de culture 3D fonctionnels offre une opportunité unique pour mimer les tissus vivants et modéliser les maladies musculaires. A cet égard, ce nouveau type de modèles in vitro augmente significativement notre compréhension de l'implication des différentes populations cellulaires dans la formation du muscle squelettique et de leurs interactions, ainsi que les modalités de réponse d'un muscle pathologique à de nouvelles thérapies. Ce deuxième point pourrait conduire à l'identification de traitements efficaces. Dans cette synthèse, nous traitons des progrès significatifs qui ont été réalisés ces dernières années pour concevoir des structures ressemblant à des tissus musculaires, fournissant des outils utiles pour étudier le comportement des cellules souches résidentes. Nous nous intéressons plus particulièrement au développement de systèmes basés sur des « myosphères » et des faisceaux de fibres ou « myobundles » ainsi que sur les systèmes de bio-impression. Les protocoles de stimulation électrique/mécanique et les systèmes de co-culture développés pour améliorer le processus et les fonctionnalités de maturation des tissus seront également présentés. La formation de tissus musculaires biomimétiques représente une nouvelle technologie pour étudier la fonction et l'organisation spatiale des muscles squelettiques dans un grand nombre de contextes physiologiques, pathologiques et agronomiques.

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Session : Pathologie et Prévention

L’excitation multiphotonique permet d’obtenir des signaux de génération de seconde et troisième harmonique ((SHG et THG). Il s’agit d’un processus optique non linéaire pour lequel deux ou trois photons d’une fréquence donnée sont convertis en un seul photon avec une fréquence doublée et/ou triplée [1]. Les signaux de SHG peuvent être produits uniquement par des arrangements denses de molécules non-centro-symétriques. Le collagène fibrillaire et les myofilaments sont les sources majeures de SHG générées dans le tissu musculaire. D’un point de vue histologique, l’imagerie de la SHG permet d’explorer la fibrose au sein des muscles sans réalisation préalable d’immunomarquage. Les signaux SHG sont également exploités pour analyser les modifications d’organisation et d’orientation des fibres de collagène en lien avec une maladie [17]. Les signaux THG sont générés lorsqu’il y a des interfaces [(eau/huile) ou (air/eau)] dans un échantillon tissulaire. C’est pourquoi, ils permettent d’imager les vésicules lipidiques ou les bulles d’air. L’intérêt biologique de la détection des signaux THG est que ces derniers permettent d’observer le tissu adipeux dans le muscle dystrophique et la myéline riche en corps gras pour l’exploration du système nerveux. Il existe des nanoparticules harmoniques capables de générer à la fois des signaux SHG et THG. Ces outils sont très prometteurs pour suivre des agents thérapeutiques cellulaires tels que les cellules souches adultes dérivées du muscle développées par l’équipe de Karl Rouger [24, 26].

International audience

International audience

Plasma Symmetric DiMethyl Arginine (SDMA) concentration in cats with Acute Kidney Injury due to Urethral Obstruction (UO). IVECCS

absent

International audience

Pompe disease (glycogen storage disease type II) is a lysosomal storage disorder caused by the mutation of acid α-glucosidase (Gaa), the unique enzyme degrading glycogen in glucose into lysosomes. A massive glycogen overload is described in Pompe patients, mainly in skeletal and cardiac muscles. Furthermore, severe impairment of autophagic flux has been described, highlighted by autophagic buildup. Contrary to cardiac muscle, no treatment currently allows to cure efficiently and durably the skeletal muscle. We have identified the transcription factor Forkhead box O3 (FoxO3a) as a potential target to alleviate skeletal muscle impairments through its key role on regulation of both glycogen homeostasis and autophagy.\nThe objectives of the study were:\n1/ to explore the preventive effect of FoxO3a overexpression on :[br/]\n- glycogen overload\n- autophagic flux impairments\n2/ to investigate the impact of FoxO3a overexpression on skeletal muscle remodeling