Nicholas P. Evans

Endurance capacity in maturing mdx mice is markedly enhanced by combined voluntary wheel running and green tea extract

Duchenne muscular dystrophy is characterized by the absence of dystrophin from muscle cells. Dystrophic muscle cells are susceptible to oxidative stress. We tested the hypothesis that 3 wk of endurance exercise starting at age 21 days in young male mdx mice would blunt oxidative stress and improve dystrophic skeletal muscle function, and these effects would be enhanced by the antioxidant green tea extract (GTE). In mice fed normal diet, average daily running distance increased 300% from week 1 to week 3, and total distance over 3 wk was improved by 128% in mice fed GTE. Running, independent of diet, increased serum antioxidant capacity, extensor digitorum longus tetanic stress, and total contractile protein content, heart citrate synthase, and heart and quadriceps beta-hydroxyacyl-CoA dehydrogenase activities. GTE, independent of running, decreased serum creatine kinase and heart and gastrocnemius lipid peroxidation and increased gastrocnemius citrate synthase activity. These data suggest that both endurance exercise and GTE may be beneficial as therapeutic strategies to improve muscle function in mdx mice.

Conjugated Linoleic Acid Ameliorates Inflammation-Induced Colorectal Cancer in Mice through Activation of PPARγ

Conjugated linoleic acid (CLA) exerts a protective effect on experimental inflammatory bowel disease and shows promise as a chemopreventive agent against colorectal cancer (CRC) in mice, although the mechanisms by which it exerts its beneficial effects against malignancies in the gut are not completely understood. Mice lacking PPARgamma in immune and epithelial cells and PPARgamma-expressing littermates were fed either control or CLA-supplemented (1 g CLA/100 g) diets to determine the role of PPARgamma in inflammation-induced CRC. To induce tumor formation and colitis, mice were treated with azoxymethane and then challenged with 2% dextran sodium sulfate, respectively. Dietary CLA ameliorated disease activity, decreased colitis, and prevented adenocarcinoma formation in the PPARgamma-expressing floxed mice but not in the tissue-specific PPARgamma-null mice. Dietary CLA supplementation significantly decreased the percentages of macrophages in the mesenteric lymph nodes (MLN) regardless of the genotype and increased regulatory T cell numbers in MLN of PPARgamma-expressing, but not in the tissue-specific, PPARgamma-null mice. Colonic tumor necrosis factor-alpha mRNA expression was significantly suppressed in CLA-fed, PPARgamma-expressing mice. This study suggests CLA ameliorates colitis and prevents tumor formation in part through a PPARgamma-dependent mechanism.

Immune-Mediated Mechanisms Potentially Regulate the Disease Time-Course of Duchenne Muscular Dystrophy and Provide Targets for Therapeutic Intervention

Duchenne muscular dystrophy is a lethal muscle‐wasting disease that affects boys. Mutations in the dystrophin gene result in the absence of the dystrophin glycoprotein complex (DGC) from muscle plasma membranes. In healthy muscle fibers, the DGC forms a link between the extracellular matrix and the cytoskeleton to protect against contraction‐induced membrane lesions and to regulate cell signaling. The absence of the DGC results in aberrant regulation of inflammatory signaling cascades. Inflammation is a key pathological characteristic of dystrophic muscle lesion formation. However, the role and regulation of this process in the disease time‐course has not been sufficiently examined. The transcription factor nuclear factor‐κB has been shown to contribute to the disease process and is likely involved with increased inflammatory gene expression, including cytokines and chemokines, found in dystrophic muscle. These aberrant signaling processes may regulate the early time‐course of inflammatory events that contribute to the onset of disease. This review critically evaluates the possibility that dystrophic muscle lesions in both patients with Duchenne muscular dystrophy and mdx mice are the result of immune‐mediated mechanisms that are regulated by inflammatory signaling and also highlights new therapeutic directions.

Dysregulated Intracellular Signaling and Inflammatory Gene Expression During Initial Disease Onset in Duchenne Muscular Dystrophy

Evans NP, Misyak SA, Robertson JL, Bassaganya-Riera J, Grange RW: Dysregulated intracellular signaling and inflammatory gene expression during initial disease onset in Duchenne muscular dystrophy. Duchenne muscular dystrophy is a debilitating genetic disorder characterized by severe muscle wasting and early death in affected boys. The primary cause of this disease is mutations in the dystrophin gene that result in the absence of the protein dystrophin and the associated dystrophin-glycoprotein complex in the plasma membrane of muscle fibers. In normal muscle, this complex forms a link between the extracellular matrix and the cytoskeleton that is thought to protect muscle fibers from contraction-induced membrane lesions and to regulate cell signaling cascades. Although the primary defect is known, the mechanisms that initiate disease onset have not been characterized. Data collected during early maturation suggest that inflammatory and immune responses are key contributors to disease pathogenesis and may be initiated by aberrant signaling in dystrophic muscle. However, detailed time course studies of the inflammatory and immune processes are incomplete and need to be characterized further to understand the disease progression. The purposes of this review are to examine the possibility that initial disease onset in dystrophin-deficient muscle results from aberrant inflammatory signaling pathways and to highlight the potential clinical relevance of targeting these pathways to treat Duchenne muscular dystrophy.

Passive mechanical properties of maturing extensor digitorum longus are not affected by lack of dystrophin

Mechanical weakness of skeletal muscle is thought to contribute to onset and early progression of Duchenne muscular dystrophy, but this has not been systematically assessed. The purpose of this study was to determine in mice: (1) whether the passive mechanical properties of maturing dystrophic (mdx) muscles were different from control; and (2) if different, the time during maturation when these properties change. Prior to and following the overt onset of the dystrophic process (14–35 days), control and dystrophic extensor digitorum longus (EDL) muscles were subjected to two passive stretch protocols in vitro (5% strain at instantaneous and 1.5 L0/s strain rates). Force profiles were fit to a viscoelastic muscle model to determine stiffness and damping. The mdx and control EDL muscles exhibited similar passive mechanical properties at each age, suggesting a functional threshold for dystrophic muscle below which damage may be minimized. Determining this threshold may have important clinical implications for treatments of muscular dystrophy involving physical activity. Muscle Nerve, 2006

Green tea extract decreases muscle pathology and NF-κB immunostaining in regenerating muscle fibers of mdx mice

BACKGROUND & AIMS Duchenne muscular dystrophy is a debilitating genetic disorder characterized by severe muscle wasting and early death in afflicted boys. The primary cause of this disease is mutations in the dystrophin gene resulting in massive muscle degeneration and inflammation. The purpose of this study was to determine if dystrophic muscle pathology and inflammation were decreased by pre-natal and early dietary intervention with green tea extract. METHODS Mdx breeder mice and pups were fed diets containing 0.25% or 0.5% green tea extract and compared to untreated mdx and C57BL/6J mice. Serum creatine kinase was assessed as a systemic indicator of muscle damage. Quantitative histopathological and immunohistochemical techniques were used to determine muscle pathology, macrophage infiltration, and NF-kappaB localization. RESULTS Early treatment of mdx mice with green tea extract significantly decreased serum creatine kinase by approximately 85% at age 42 days (P< or =0.05). In these mice, the area of normal fiber morphology was increased by as much as approximately 32% (P< or =0.05). The primary histopathological change was a approximately 21% decrease in the area of regenerating fibers (P< or =0.05). NF-kappaB staining in regenerating muscle fibers was also significantly decreased in green tea extract-treated mdx mice when compared to untreated mdx mice (P< or =0.05). CONCLUSION Early treatment with green tea extract decreases dystrophic muscle pathology potentially by regulating NF-kappaB activity in regenerating muscle fibers.

Synergy in Polymicrobial Infections in a Mouse Model of Type 2 Diabetes

ABSTRACT Human diabetics frequently suffer delayed wound healing, increased susceptibility to localized and systemic infections, and limb amputations as a consequence of the disease. Lower-limb infections in diabetic patients are most often polymicrobial, involving mixtures of aerobic, facultative anaerobic, and anaerobic bacteria. The purpose of this study is to determine if these organisms contribute to synergy in polymicrobial infections by using diabetic mice as an in vivo model. The model was the obese diabetic mouse strain BKS.Cg-m +/+ Leprdb/J, a model of human type 2 diabetes. Young (5- to 6-week-old) prediabetic mice and aged (23- to 24-week-old) diabetic mice were compared. The mice were injected subcutaneously with mixed cultures containing Escherichia coli, Bacteroides fragilis, and Clostridium perfringens. Progression of the infection (usually abscess formation) was monitored by examining mice for bacterial populations and numbers of white blood cells at 1, 8, and 22 days postinfection. Synergy in the mixed infections was defined as a statistically significant increase in the number of bacteria at the site of injection when coinfected with a second bacterium, compared to when the bacterium was inoculated alone. E. coli provided strong synergy to B. fragilis but not to C. perfringens. C. perfringens and B. fragilis provided moderate synergy to each other but only in young mice. B. fragilis was anergistic (antagonistic) to E. coli in coinfections in young mice at 22 days postinfection. When age-matched nondiabetic mice (C57BLKS/J) were used as controls, the diabetic mice exhibited 5 to 35 times the number of CFU as did the nondiabetic mice, indicating that diabetes was a significant factor in the severity of the polymicrobial infections.

T cell PPARγ is required for the anti-inflammatory efficacy of abscisic acid against experimental IBD.

The phytohormone abscisic acid (ABA) has been shown to be effective in ameliorating chronic and acute inflammation. The objective of this study was to investigate whether ABAs anti-inflammatory efficacy in the gut is dependent on peroxisome proliferator-activated receptor γ (PPARγ) in T cells. PPARγ-expressing and T cell-specific PPARγ null mice were fed diets with or without ABA (100 mg/kg) for 35 days prior to challenge with 2.5% dextran sodium sulfate. The severity of clinical disease was assessed daily, and mice were euthanized on Day 7 of the dextran sodium sulfate challenge. Colonic inflammation was assessed through macroscopic and histopathological examination of inflammatory lesions and real-time quantitative RT-PCR-based quantification of inflammatory genes. Flow cytometry was used to phenotypically characterize leukocyte populations in the blood and mesenteric lymph nodes. Colonic sections were stained immunohistochemically to determine the effect of ABA on colonic regulatory T (T(reg)) cells. ABAs beneficial effects on disease activity were completely abrogated in T cell-specific PPARγ null mice. Additionally, ABA improved colon histopathology, reduced blood F4/80(+)CD11b(+) monocytes, increased the percentage of CD4(+) T cells expressing the inhibitory molecule cytotoxic T lymphocyte antigen 4 in blood and enhanced the number of T(reg) cells in the mesenteric lymph nodes and colons of PPARγ-expressing but not T cell-specific PPARγ null mice. We conclude that dietary ABA ameliorates experimental inflammatory bowel disease by enhancing T(reg) cell accumulation in the colonic lamina propria through a PPARγ-dependent mechanism.

A Mathematical Model of Skeletal Muscle Disease and Immune Response in the mdx Mouse

Duchenne muscular dystrophy (DMD) is a genetic disease that results in the death of affected boys by early adulthood. The genetic defect responsible for DMD has been known for over 25 years, yet at present there is neither cure nor effective treatment for DMD. During early disease onset, the mdx mouse has been validated as an animal model for DMD and use of this model has led to valuable but incomplete insights into the disease process. For example, immune cells are thought to be responsible for a significant portion of muscle cell death in the mdx mouse; however, the role and time course of the immune response in the dystrophic process have not been well described. In this paper we constructed a simple mathematical model to investigate the role of the immune response in muscle degeneration and subsequent regeneration in the mdx mouse model of Duchenne muscular dystrophy. Our model suggests that the immune response contributes substantially to the muscle degeneration and regeneration processes. Furthermore, the analysis of the model predicts that the immune system response oscillates throughout the life of the mice, and the damaged fibers are never completely cleared.

Preharvest Salmonella Detection for Evaluation of Fresh Ground Poultry Product Contamination.

Salmonella is an important economic and public health concern for the poultry industry. Fresh ground product has been linked with multiple salmonellosis outbreaks in humans. Exposure can be controlled by proper handling and preparation by consumers; however, the industry desires to minimize carriage levels in the final product. A substantial obstacle in reducing product contamination stems from limitations in diagnostic methodologies. Detection of Salmonella contamination currently requires extended incubation periods, and by the time test results are available, the fresh product has reached retail shelves. The goal of this study was to develop a preharvest diagnostic protocol for the evaluation of ground product contamination. The turkey processing plant where this research was conducted had previously established Salmonella screening (BAX system) of ground product, thus providing an opportunity for preharvest sample comparison. Drag swabs were collected from live-haul trailers entering the processing plant over a 12-month period. The swabs were added to modified buffered peptone water and incubated at 40°C. After incubation for 6 h or overnight, samples were tested for the presence of Salmonella with the DNAble assay and related to ground turkey samples from corresponding lots. The linear relationship for the percentage of Salmonella-positive live-haul trailers was significant for both the 6-h (slope = 1.02, R(2) = 0.96, and P < 0.0001) and overnight (slope = 0.35, R(2) = 0.93, and P = 0.0015) incubations, with the percentage of Salmonella-positive ground turkey samples. These data indicate that preharvest screening provides a meaningful evaluation of product contamination. Additionally, the 6-h incubation protocol is rapid enough to allow for product mitigation and could potentially aid in the reduction of future salmonellosis outbreaks.