Poloxomer 188 Has a Deleterious Effect on Dystrophic Skeletal Muscle Function

Duchenne muscular dystrophy (DMD) is an X-linked, fatal muscle wasting disease for which there is currently no cure and limited palliative treatments. Poloxomer 188 (P188) is a tri-block copolymer that has been proposed as a potential treatment for cardiomyopathy in DMD patients. Despite the reported beneficial effects of P188 on dystrophic cardiac muscle function, the effects of P188 on dystrophic skeletal muscle function are relatively unknown. Mdx mice were injected intraperitoneally with 460 mg/kg or 30 mg/kg P188 dissolved in saline, or saline alone (control). The effect of single-dose and 2-week daily treatment was assessed using a muscle function test on the Tibialis Anterior (TA) muscle in situ in anaesthetised mice. The test comprises a warm up, measurement of the force-frequency relationship and a series of eccentric contractions with a 10% stretch that have previously been shown to cause a drop in maximum force in mdx mice. After 2 weeks of P188 treatment at either 30 or 460 mg/kg/day the drop in maximum force produced following eccentric contractions was significantly greater than that seen in saline treated control mice (P = 0.0001). Two week P188 treatment at either dose did not significantly change the force-frequency relationship or maximum isometric specific force produced by the TA muscle. In conclusion P188 treatment increases susceptibility to contraction-induced injury following eccentric contractions in dystrophic skeletal muscle and hence its suitability as a potential therapeutic for DMD should be reconsidered

Lack of compensatory mitophagy in skeletal muscles during sepsis

Abstract Skeletal muscle dysfunction is a major problem in critically ill patients suffering from sepsis. This condition is associated with mitochondrial dysfunction and increased autophagy in skeletal muscles. Autophagy is a proteolytic mechanism involved in eliminating dysfunctional cellular components, including mitochondria. The latter process, referred to as mitophagy, is essential for maintaining mitochondrial quality and skeletal muscle health. Recently, a fluorescent reporter system called mito-QC (i.e. mitochondrial quality control) was developed to specifically quantify mitophagy levels. In the present study, we used mito-QC transgenic mice and confocal microscopy to morphologically monitor mitophagy levels during sepsis. To induce sepsis, Mito-QC mice received Escherichia coli lipopolysaccharide (10 mg kg–1 i.p.) or phosphate-buffered saline and skeletal muscles (hindlimb and diaphragm) were excised 48 h later. In control groups, there was a negative correlation between the basal mitophagy level and overall muscle mitochondrial content. Sepsis increased general autophagy in both limb muscles and diaphragm but had no effect on mitophagy levels. Sepsis was associated with a downregulation of certain mitophagy receptors (Fundc1, Bcl2L13, Fkbp8 and Phbb2). The present study suggests that general autophagy and mitophagy can be dissociated from one another, and that the characteristic accumulation of damaged mitochondria in skeletal muscles under the condition of sepsis may reflect a failure of adequate compensatory mitophagy. Key points - There was a negative correlation between the basal level of skeletal muscle mitophagy and the mitochondrial content of individual muscles. - Mitophagy levels in limb muscles and the diaphragm were unaffected by lipopolysaccharide (LPS)-induced sepsis. - With the exception of BNIP3 in sepsis, LPS administration induced either no change or a downregulation of mitophagy receptors in skeletal muscles

Clostridium difficile infection.

Infection of the colon with the Gram-positive bacterium Clostridium difficile is potentially life threatening, especially in elderly people and in patients who have dysbiosis of the gut microbiota following antimicrobial drug exposure. C. difficile is the leading cause of health-care-associated infective diarrhoea. The life cycle of C. difficile is influenced by antimicrobial agents, the host immune system, and the host microbiota and its associated metabolites. The primary mediators of inflammation in C. difficile infection (CDI) are large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), and, in some bacterial strains, the binary toxin CDT. The toxins trigger a complex cascade of host cellular responses to cause diarrhoea, inflammation and tissue necrosis - the major symptoms of CDI. The factors responsible for the epidemic of some C. difficile strains are poorly understood. Recurrent infections are common and can be debilitating. Toxin detection for diagnosis is important for accurate epidemiological study, and for optimal management and prevention strategies. Infections are commonly treated with specific antimicrobial agents, but faecal microbiota transplants have shown promise for recurrent infections. Future biotherapies for C. difficile infections are likely to involve defined combinations of key gut microbiota

Dystrophin expression in muscle following gene transfer with a fully deleted ("Gutted") adenovirus is markedly improved by Trans-acting adenoviral gene products

Helper-dependent adenoviruses (HDAd) are Ad vectors lacking all or most viral genes. They hold great promise for gene therapy of diseases such as Duchenne muscular dystrophy (DMD), because they are less immunogenic than E1/E3-deleted Ad (first-generation Ad or FGAd) and can carry the full-length (Fl) dystrophin (dys) cDNA (12 kb). We have compared the transgene expression of a HDAd (HDAdCMVDysFl) and a FGAd (FGAdCMV-dys) in cell culture (HeLa, C2C12 myotubes) and in the muscle of mdx mice (the mouse model for DMD). Both vectors encoded dystrophin regulated by the same cytomegalovirus (CMV) promoter. We demonstrate that the amount of dystrophin expressed was significantly higher after gene transfer with FGAdCMV-dys compared to HDAdCMVDysFl both in vitro and in vivo. However, gene transfer with HDAdCMVDysFl in the presence of a FGAd resulted in a significant increase of dystrophin expression indicating that gene products synthesized by the FGAd increase, in trans, the amount of dystrophin produced. This enhancement occurred in cell culture and after gene transfer in the muscle of mdx mice and dystrophic golden retriever (GRMD) dogs, another animal model for DMD. The E4 region of Ad is required for the enhancement, because no increase of dystrophin expression from HDAdCMVDysFl was observed in the presence of an E1/E4-deleted Ad in vitro and in vivo. The characterization of these enhancing gene products followed by their inclusion into an HDAd may be required to produce sufficient dystrophin to mitigate the pathology of DMD by HDAd-mediated gene transfer

Mechanisms Involved in Alleviation of Intestinal Inflammation by Bifidobacterium Breve Soluble Factors

Objectives: Soluble factors released by Bifidobacterium breve C50 (Bb) alleviate the secretion of pro-inflammatory cytokines by immune cells, but their effect on intestinal epithelium remains elusive. To decipher the mechanisms accounting for the cross-talk between bacteria/soluble factors and intestinal epithelium, we measured the capacity of the bacteria, its conditioned medium (Bb-CM) and other Gram(+) commensal bacteria to dampen inflammatory chemokine secretion. Methods: TNFa-induced chemokine (CXCL8) secretion and alteration of NF-kB and AP-1 signalling pathways by Bb were studied by EMSA, confocal microscopy and western blotting. Anti-inflammatory capacity was also tested in vivo in a model of TNBS-induced colitis in mice. Results: Bb and Bb-CM, but not other commensal bacteria, induced a time and dose-dependent inhibition of CXCL8 secretion by epithelial cells driven by both AP-1 and NF-kB transcription pathways and implying decreased phosphorylation of p38-MAPK and IkB-a molecules. In TNBS-induced colitis in mice, Bb-CM decreased the colitis score and inflammatory cytokine expression, an effect reproduced by dendritic cell conditioning with Bb-CM. Conclusions: Bb and secreted soluble factors contribute positively to intestinal homeostasis by attenuating chemokine production. The results indicate that Bb down regulate inflammation at the epithelial level by inhibiting phosphorylation

Dystrophin deficiency in canine X-linked muscular dystrophy in Japan (CXMDJ) alters myosin heavy chain expression profiles in the diaphragm more markedly than in the tibialis cranialis muscle

<p>Abstract</p> <p>Background</p> <p>Skeletal muscles are composed of heterogeneous collections of muscle fiber types, the arrangement of which contributes to a variety of functional capabilities in many muscle types. Furthermore, skeletal muscles can adapt individual myofibers under various circumstances, such as disease and exercise, by changing fiber types. This study was performed to examine the influence of dystrophin deficiency on fiber type composition of skeletal muscles in canine X-linked muscular dystrophy in Japan (CXMD_J), a large animal model for Duchenne muscular dystrophy.</p> <p>Methods</p> <p>We used tibialis cranialis (TC) muscles and diaphragms of normal dogs and those with CXMD_Jat various ages from 1 month to 3 years old. For classification of fiber types, muscle sections were immunostained with antibodies against fast, slow, or developmental myosin heavy chain (MHC), and the number and size of these fibers were analyzed. In addition, MHC isoforms were detected by gel electrophoresis.</p> <p>Results</p> <p>In comparison with TC muscles of CXMD_J, the number of fibers expressing slow MHC increased markedly and the number of fibers expressing fast MHC decreased with growth in the affected diaphragm. In populations of muscle fibers expressing fast and/or slow MHC(s) but not developmental MHC of CXMD_Jmuscles, slow MHC fibers were predominant in number and showed selective enlargement. Especially, in CXMD_Jdiaphragms, the proportions of slow MHC fibers were significantly larger in populations of myofibers with non-expression of developmental MHC. Analyses of MHC isoforms also indicated a marked increase of type I and decrease of type IIA isoforms in the affected diaphragm at ages over 6 months. In addition, expression of developmental (embryonic and/or neonatal) MHC decreased in the CXMD_Jdiaphragm in adults, in contrast to continuous high-level expression in affected TC muscle.</p> <p>Conclusion</p> <p>The CXMD_Jdiaphragm showed marked changes in fiber type composition unlike TC muscles, suggesting that the affected diaphragm may be effectively adapted toward dystrophic stress by switching to predominantly slow fibers. Furthermore, the MHC expression profile in the CXMD_Jdiaphragm was markedly different from that in <it>mdx </it>mice, indicating that the dystrophic dog is a more appropriate model than a murine one, to investigate the mechanisms of respiratory failure in DMD.</p

Lumican Expression in Diaphragm Induced by Mechanical Ventilation

Diaphragmatic dysfunction found in the patients with acute lung injury required prolonged mechanical ventilation. Mechanical ventilation can induce production of inflammatory cytokines and excess deposition of extracellular matrix proteins via up-regulation of transforming growth factor (TGF)-β1. Lumican is known to participate in TGF-β1 signaling during wound healing. The mechanisms regulating interactions between mechanical ventilation and diaphragmatic injury are unclear. We hypothesized that diaphragmatic damage by short duration of mechanical stretch caused up-regulation of lumican that modulated TGF-β1 signaling.Male C57BL/6 mice, either wild-type or lumican-null, aged 3 months, weighing between 25 and 30 g, were exposed to normal tidal volume (10 ml/kg) or high tidal volume (30 ml/kg) mechanical ventilation with room air for 2 to 8 hours. Nonventilated mice served as control groups.High tidal volume mechanical ventilation induced interfibrillar disassembly of diaphragmatic collagen fiber, lumican activation, type I and III procollagen, fibronectin, and α-smooth muscle actin (α-SMA) mRNA, production of free radical and TGF-β1 protein, and positive staining of lumican in diaphragmatic fiber. Mechanical ventilation of lumican deficient mice attenuated diaphragmatic injury, type I and III procollagen, fibronectin, and α-SMA mRNA, and production of free radical and TGF-β1 protein. No significant diaphragmatic injury was found in mice subjected to normal tidal volume mechanical ventilation.Our data showed that high tidal volume mechanical ventilation induced TGF-β1 production, TGF-β1-inducible genes, e.g., collagen, and diaphragmatic dysfunction through activation of the lumican

16S rRNA Gene-based Analysis of Fecal Microbiota from Preterm Infants with and without Necrotizing Enterocolitis

Neonatal necrotizing enterocolitis (NEC) is an inflammatory intestinal disorder affecting preterm infants. Intestinal bacteria play a key role; however no causative pathogen has been identified. The purpose of this study was to determine if there are differences in microbial patterns which may be critical to the development of this disease. Fecal samples from twenty preterm infants, ten with NEC and ten matched controls (including four twin pairs) were obtained from patients in a single site Level III neonatal intensive care unit. Bacterial DNA from individual fecal samples were PCR amplified and subjected to terminal restriction fragment length polymorphism analysis and library sequencing of the 16S rRNA gene to characterize diversity and structure of the enteric microbiota. The distribution of samples from NEC patients distinctly clustered separately from controls. Intestinal bacterial colonization in all preterm infants was notable for low diversity. Patients with NEC had even less diversity, an increase in abundance of Gammaproteobacteria, a decrease in other bacteria species, and had received a higher mean number of previous days of antibiotics. Our results suggest that NEC is associated with severe lack of microbiota diversity which may accentuate the impact of single dominant microorganisms favored by empiric and wide-spread use of antibiotics

iNOS Ablation Does Not Improve Specific Force of the Extensor Digitorum Longus Muscle in Dystrophin-Deficient mdx4cv Mice

Nitrosative stress compromises force generation in Duchenne muscular dystrophy (DMD). Both inducible nitric oxide synthase (iNOS) and delocalized neuronal NOS (nNOS) have been implicated. We recently demonstrated that genetic elimination of nNOS significantly enhanced specific muscle forces of the extensor digitorum longus (EDL) muscle of dystrophin-null mdx4cv mice (Li D et al J. Path. 223:88–98, 2011). To determine the contribution of iNOS, we generated iNOS deficient mdx4cv mice. Genetic elimination of iNOS did not alter muscle histopathology. Further, the EDL muscle of iNOS/dystrophin DKO mice yielded specific twitch and tetanic forces similar to those of mdx4cv mice. Additional studies suggest iNOS ablation did not augment nNOS expression neither did it result in appreciable change of nitrosative stress markers in muscle. Our results suggest that iNOS may play a minor role in mediating nitrosative stress-associated force reduction in DMD