Inhibition of Rev-erbα ameliorates muscular dystrophy.

Duchene muscular dystrophy leads to progressive muscle structural and functional decline due to chronic degenerative-regenerative cycles. Enhancing the regenerative capacity of dystrophic muscle provides potential therapeutic options. We previously demonstrated that the circadian clock repressor Rev-erbα inhibited myogenesis and Rev-erbα ablation enhanced muscle regeneration. Here we show that Rev-erbα deficiency in the dystrophin-deficient mdx mice promotes regenerative myogenic response to ameliorate muscle damage. Loss of Rev-erbα in mdx mice improved dystrophic pathology and muscle wasting. Rev-erbα-deficient dystrophic muscle exhibit augmented myogenic response, enhanced neo-myofiber formation and attenuated inflammatory response. In mdx myoblasts devoid of Rev-erbα, myogenic differentiation was augmented together with up-regulation of Wnt signaling and proliferative pathways, suggesting that loss of Rev-erbα inhibition of these processes contributed to the improvement in regenerative myogenesis. Collectively, our findings revealed that the loss of Rev-erbα function protects dystrophic muscle from injury by promoting myogenic repair, and inhibition of its activity may have therapeutic utilities for muscular dystrophy.

PDE10A Inhibition Reduces the Manifestation of Pathology in DMD Zebrafish and Represses the Genetic Modifier PITPNA.

Duchenne muscular dystrophy (DMD) is a severe genetic disorder caused by mutations in the DMD gene. Absence of dystrophin protein leads to progressive degradation of skeletal and cardiac function and leads to premature death. Over the years, zebrafish have been increasingly used for studying DMD and are a powerful tool for drug discovery and therapeutic development. In our study, a birefringence screening assay led to identification of phosphodiesterase 10A (PDE10A) inhibitors that reduced the manifestation of dystrophic muscle phenotype in dystrophin-deficient sapje-like zebrafish larvae. PDE10A has been validated as a therapeutic target by pde10a morpholino-mediated reduction in muscle pathology and improvement in locomotion, muscle, and vascular function as well as long-term survival in sapje-like larvae. PDE10A inhibition in zebrafish and DMD patient-derived myoblasts were also associated with reduction of PITPNA expression that has been previously identified as a protective genetic modifier in two exceptional dystrophin-deficient golden retriever muscular dystrophy (GRMD) dogs that escaped the dystrophic phenotype. The combination of a phenotypic assay and relevant functional assessments in the sapje-like zebrafish enhances the potential for the prospective discovery of DMD therapeutics. Indeed, our results suggest a new application for a PDE10A inhibitor as a potential DMD therapeutic to be investigated in a mouse model of DMD.

Dystrophin Gene-Editing Stability Is Dependent on Dystrophin Levels in Skeletal but Not Cardiac Muscles.

Gene editing is often touted as a permanent method for correcting mutations, but its long-term benefits in Duchenne muscular dystrophy (DMD) may depend on sufficiently high editing efficiencies to halt muscle degeneration. Here, we explored the persistence of dystrophin expression following recombinant adeno-associated virus serotype 6 (rAAV6):CRISPR-Cas9-mediated multi-exon deletion/reframing in systemically injected 2- and 11-week-old dystrophic mice and show that induction of low dystrophin levels persists for several months in cardiomyocytes but not in skeletal muscles, where myofibers remain susceptible to necrosis and regeneration. Whereas gene-correction efficiency in both muscle types was enhanced with increased ratios of guide RNA (gRNA)-to-nuclease vectors, obtaining high dystrophin levels in skeletal muscles via multi-exon deletion remained challenging. In contrast, when AAV-microdystrophin was codelivered with editing components, long-term gene-edited dystrophins persisted in both muscle types. These results suggest that the high rate of necrosis and regeneration in skeletal muscles, compared with the relative stability of dystrophic cardiomyocytes, caused the rapid loss of edited genomes. Consequently, stable dystrophin expression in DMD skeletal muscles will require either highly efficient gene editing or the use of cotreatments that decrease skeletal muscle degeneration.

The clock regulator Bmal1 protects against muscular dystrophy.

The muscle-intrinsic clock machinery is required for the maintenance of muscle growth, remodeling and function. Our previous studies demonstrated that the essential transcription activator of the molecular clock feed-back loop, Brain and Muscle Arnt-Like 1(Bmal1), plays a critical role in myogenic progenitor behavior to promote and regenerative myogenesis. Using genetic approaches targeting Bmal1 in the DMDmdx (mdx) dystrophic mouse model, here we report that the loss of Bmal1 function significantly accelerated dystrophic disease progression. In contrast to the mild dystrophic changes in mdx mice, the genetic loss-of-function of Bmal1 aggravated muscle damage in this dystrophic disease background, as indicated by persistently elevated creatine kinase levels, increased injury area and reduced muscle grip strength. Mechanistic studies revealed that markedly impaired myogenic progenitor proliferation and myogenic response underlie the defective new myofiber formation in the chronic dystrophic milieu. Taken together, our study identified the function of pro-myogenic clock gene Bmal1 in protecting against dystrophic damage, suggesting the potential for augmenting Bmal1 function to ameliorate dystrophic or degenerative muscle diseases.

Comprehensive genetic analysis of 961 unrelated Duchenne Muscular Dystrophy patients: Focus on diagnosis, prevention and therapeutic possibilities.

Recently DNA sequencing analysis has played a vital role in the unambiguous diagnosis of clinically suspected patients with Duchenne Muscular Dystrophy (DMD). DMD is a monogenic, X-linked, recessive, degenerative pediatric neuromuscular disorder affecting males, invariably leading to fatal cardiopulmonary failure. Early and precise diagnosis of the disease is an essential part of an effective disease management strategy as care guidelines and prevention through counseling need to be initiated at the earliest particularly since therapies are now available for a subset of patients. In this manuscript we report the DMD gene mutational profiles of 961 clinically suspected male DMD patients, 99% of whom were unrelated. We utilized a molecular diagnostic approach which is cost-effective for most patients and follows a systematic process that sequentially involves identification of hotspot deletions using mPCR, large deletions and duplications using MLPA and small insertions/ deletions and point mutations using an NGS muscular dystrophy gene panel. Pathogenic DMD gene mutations were identified in 84% of patients. Our data compared well with the frequencies and distribution of deletions and duplications reported in the DMD gene in other published studies. We also describe a number of rare in-frame mutations, which appeared to be enriched in the 5' proximal hotspot region of the DMD gene. Furthermore, we identified a family with a rare non-contiguous deletion mutation in the DMD gene where three males were affected and two females were deemed carriers. A subset of patients with mutations in the DMD gene who are likely to benefit therapeutically from new FDA and EMA approved drugs were found in our cohort. Given that the burden of care for DMD patients invariably falls on the mothers, particularly in rural India, effective genetic counseling followed by carrier screening is crucial for prevention of this disorder. We analyzed the carrier status of consented female relatives of 463 probands to gauge the percentage of patients with familial disease. Our analysis revealed 43.7% of mothers with DMD gene mutations. Our comprehensive efforts, involving complete genetic testing coupled with compassionate genetic counseling provided to DMD patients and their families, are intended to improve the quality of life of DMD patients and to empower carrier females to make informed reproductive choices to impede the propagation of this deadly disease.

Elevated MMP2 abundance and activity in mdx mice are alleviated by prenatal taurine supplementation.

Duchenne muscular dystrophy (DMD) is a severe, progressive muscle-wasting disorder that leads to early death. The mdx mouse is a naturally occurring mutant model for DMD. It lacks dystrophin and displays peak muscle cell necrosis at ~28 days (D28), but in contrast to DMD, mdx mice experience muscle regeneration by D70. We hypothesized that matrix metalloproteinase-2 (MMP2) and/or MMP9 play key roles in the degeneration/regeneration phases in mdx mice. MMP2 abundance in muscle homogenates, measured by calibrated Western blotting, and activity, measured by zymogram, were lower at D70 compared with D28 in both mdx and wild-type (WT) mice. Importantly, MMP2 abundance was higher in both D28 and D70 mdx mice than in age-matched WT mice. The higher MMP2 abundance was not due to infiltrating macrophages, because MMP2 content was still higher in isolated muscle fibers where most macrophages had been removed. Prenatal supplementation with the amino acid taurine, which improved muscle strength in D28 mdx mice, produced approximately twofold lower MMP2 activity, indicating that increased MMP2 abundance is not required when muscle damage is attenuated. There was no difference in MMP9 abundance between age-matched WT and mdx mice (P > 0.05). WT mice displayed decreased MMP9 abundance as they aged. While MMP9 may have a role during age-related skeletal muscle growth, it does not appear essential for degeneration/regeneration cycles in the mdx mouse. Our findings indicate that MMP2 plays a more active role than MMP9 in the degenerative phases of muscle fibers in D28 mdx mice.

Papel de las poliaminas en la transducción de señales intracelulares: posibles proyec­ciones de uso clínico en el manejo de las distrofias musculares por medio de su inhibi­ción con difluorometilornitina / Polyamines role in intracellular signals transduction: possible projections for clinical use in muscular dy strophies ' management through its inhibition with difluoromethylornithine

Las distrofias musculares de origen genético son muy diversas y, tanto su diagnóstico preciso como su manejo, suponen un reto importante. En cuanto a este último aspecto, no obstante el desarrollo en proceso de nuevas estrategias a nivel molecular para su tratamiento, las herramientas con que se cuenta para este propósito son limitadas, y pocas veces pueden influir de manera efectiva para evitar el deterioro progresivo que muchos de estos pacientes experimentan. Además, las terapias de última generación no abarcan la gran diversidad de estas patologías y no se espera que estén disponibles a corto plazo para la mayoría de los pacientes. El propósito del artículo es mostrar el papel de las poliaminas, actores ubicuos en el metabolismo in­ tracelular tal vez poco conocidos; cómo están involucrados en los procesos fisiológicos y patológicos, y cómo también pudiesen estar involucrados en la fisiopatología de las distrofias musculares. Su inhi­bición controlada, mediante Difluorometilornitina (DFMO), pudiese constituir un mecanismo para en­ lentecer o eliminar el deterioro muscular de estos pacientes, al utilizarse como una herramienta dentro del arsenal de las ya existentes

Muscular dystrophies of genetic origin are very diverse and, both their precise diagnosis and their management represent an important challenge. Regarding this last aspect, despite the development in process of new strategies at the molecular level for its treatment, the tools available for this pur­ pose are limited, and can rarely influence effectively to avoid the progressive deterioration that many of these patients experience. In addition, the lates t­generation therapies do not cover the great diversity of these pathologies and are not expected to be available in the short term for most patients. The purpose of the article is to show the role of polyamines, ubiquitous actors in intracellular meta­ bolism, perhaps little known; how they are involved in physiological and pathological processes, and how they could also be involved in the physiopathology of muscular dystrophies. Its controlled inhi­ bition, by difluoromethylilitin (DFMO), could be a mechanism to slow or eliminate the muscle deterio­ ration of these patients, by being used as a tool within the arsenal of those already existing.

Molecular hydrogen alleviates motor deficits and muscle degeneration in mdx mice.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by a mutation in DMD encoding dystrophin. Oxidative stress accounts for dystrophic muscle pathologies in DMD. We examined the effects of molecular hydrogen in mdx mice, a model animal for DMD. METHODS: The pregnant mother started to take supersaturated hydrogen water (>5 ppm) ad libitum from E15.5 up to weaning of the offspring. The mdx mice took supersaturated hydrogen water from weaning until age 10 or 24 weeks when they were sacrificed. RESULTS: Hydrogen water prevented abnormal body mass gain that is commonly observed in mdx mice. Hydrogen improved the spontaneous running distance that was estimated by a counter-equipped running-wheel, and extended the duration on the rota-rod. Plasma creatine kinase activities were decreased by hydrogen at ages 10 and 24 weeks. Hydrogen also decreased the number of central nuclei of muscle fibers at ages 10 and 24 weeks, and immunostaining for nitrotyrosine in gastrocnemius muscle at age 24 weeks. Additionally, hydrogen tended to increase protein expressions of antioxidant glutathione peroxidase 1, as well as anti-apoptotic Bcl-2, in skeletal muscle at age 10 weeks. DISCUSSION: Although molecular mechanisms of the diverse effects of hydrogen remain to be elucidated, hydrogen potentially improves muscular dystrophy in DMD patients.

Knowledge of carrier status and barriers to testing among mothers of sons with Duchenne or Becker muscular dystrophy.

Our study objective was to survey female carriers for Duchenne and Becker muscular dystrophy to identify barriers to carrier testing and the impact of carrier risk knowledge on cardiac and reproductive health management. We surveyed women who have or had biological sons with Duchenne or Becker muscular dystrophy and were enrolled in the US DuchenneConnect patient registry, with questions assessing knowledge of carrier status and recurrence risk, knowledge of care standards for carriers, and barriers to testing. Of the 182 eligible respondents, 25% did not know their carrier status and 14% incorrectly classified themselves as not at risk. Cost of testing was the most commonly identified barrier to testing. Women reporting unknown carrier status were 13 times as likely to express uncertainty regarding their recurrence risk compared to women reporting positive carrier status. 37% of women at an increased risk for cardiomyopathy had never had an echocardiogram. Women who were certain of their positive carrier status were twice as likely to have had an echocardiogram in the last five years compared to women with unknown carrier status. Future research on reducing barriers to counseling and carrier testing, such as cost, may improve care standard adherence.

Prophylactic oral bisphosphonate therapy in duchenne muscular dystrophy.

INTRODUCTION: We assessed prophylactic use of bisphosphonate (BP) in Duchenne muscular dystrophy (DMD) patients on glucocorticoid (GC) therapy. METHODS: Fifty-two DMD patients on daily GC were offered BP (oral risedronate). Patients were reviewed for tolerability, side effects, bone pain, and fracture frequency. Bone mineral density (BMD) was determined by annual dual energy X-ray absorptiometry. BP-treated patients were compared with 15 BP-naïve patients (untreated cohort). RESULTS: Side effects occurred in 9 patients. Thirty-six patients continued BP therapy for over 12 months (mean, 3.6 years). Five treated patients reported bone pain. Three treated patients suffered a vertebral fracture, significantly less than in the untreated cohort (5/15). Lumbar spine adjusted BMD Z-scores remained unchanged in treated patients and were significantly greater than in the untreated cohort. CONCLUSIONS: Prophylactic oral risedronate therapy was tolerated by most DMD patients. It appears to maintain BMD and may reduce fracture rate in DMD patients on GC. Muscle Nerve 54: 79-85, 2016.

Enhancement of Muscle T Regulatory Cells and Improvement of Muscular Dystrophic Process in mdx Mice by Blockade of Extracellular ATP/P2X Axis.

Infiltration of immune cells and chronic inflammation substantially affect skeletal and cardiac muscle degeneration in Duchenne muscular dystrophy. In the immune system, extracellular adenosine triphosphate (ATP) released by dying cells is sensed as a danger associated molecular pattern through P2 purinergic receptors. Specifically, the P2X7 subtype has a prominent role in regulating immune system physiology and contributes to inflammasome activation also in muscle cells. Here, we show that in vivo blockade of the extracellular ATP/P2X purinergic signaling pathway by periodate-oxidized ATP delayed the progression of the dystrophic phenotype and dampened the local inflammatory response in mdx mice, a spontaneous mouse model of dystrophin deficiency. Reduced infiltration of leukocytes and macrophages and decreased expression of IL-6 were revealed in the muscles of periodate-oxidized ATP-treated mdx mice. Concomitantly, an increase in Foxp3(+) immunosuppressive regulatory T cells was observed and correlated with enhanced myofiber regeneration. Moreover, we detected reduced concentrations of profibrotic cytokines, including transforming growth factor-ß and connective tissue growth factor, in muscles of periodate-oxidized ATP-treated mdx mice. The improvement of inflammatory features was associated with increased strength and reduced necrosis, thus suggesting that pharmacologic purinergic antagonism altering the adaptive immune component in the muscle infiltrates might represent a promising therapeutic approach in Duchenne muscular dystrophy.

Reduction of Oxidative Damage and Inflammatory Response in the Diaphragm Muscle of mdx Mice Using Iron Chelator Deferoxamine.

Oxidative stress and inflammatory processes strongly contribute to pathogenesis in Duchenne muscular dystrophy (DMD). Based on evidence that excess iron may increase oxidative stress and contribute to the inflammatory response, we investigated whether deferoxamine (DFX), a potent iron chelating agent, reduces oxidative stress and inflammation in the diaphragm (DIA) muscle of mdx mice (an experimental model of DMD). Fourteen-day-old mdx mice received daily intraperitoneal injections of DFX at a dose of 150 mg/kg body weight, diluted in saline, for 14 days. C57BL/10 and control mdx mice received daily intraperitoneal injections of saline only, for 14 days. Grip strength was evaluated as a functional measure, and blood samples were collected for biochemical assessment of muscle fiber degeneration. In addition, the DIA muscle was removed and processed for histopathology and Western blotting analysis. In mdx mice, DFX reduced muscle damage and loss of muscle strength. DFX treatment also resulted in a significant reduction of dystrophic inflammatory processes, as indicated by decreases in the inflammatory area and in NF-κB levels. DFX significantly decreased oxidative damage, as shown by lower levels of 4-hydroxynonenal and a reduction in dihydroethidium staining in the DIA muscle of mdx mice. The results of the present study suggest that DFX may be useful in therapeutic strategies to ameliorate dystrophic muscle pathology, possibly via mechanisms involving oxidative and inflammatory pathways.

Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA.

Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutations in the gene encoding dystrophin, a protein required for muscle fiber integrity. DMD is characterized by progressive muscle weakness and a shortened life span, and there is no effective treatment. We used clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9)-mediated genome editing to correct the dystrophin gene (Dmd) mutation in the germ line of mdx mice, a model for DMD, and then monitored muscle structure and function. Genome editing produced genetically mosaic animals containing 2 to 100% correction of the Dmd gene. The degree of muscle phenotypic rescue in mosaic mice exceeded the efficiency of gene correction, likely reflecting an advantage of the corrected cells and their contribution to regenerating muscle. With the anticipated technological advances that will facilitate genome editing of postnatal somatic cells, this strategy may one day allow correction of disease-causing mutations in the muscle tissue of patients with DMD.

First successful pregnancy in Switzerland after prospective sex determination of the embryo through the separation of X-chromosome bearing spermatozoa.

QUESTION UNDER STUDY: The feasibility and the potential advantages of separating X-chromosome bearing spermatozoa for the prevention of a severe X-chromosome linked disorder with the use of intracytoplasmic sperm injection are presented. METHOD: A carrier of muscular dystrophy type Becker was treated with intracytoplasmic sperm injection, using spermatozoa previously stained with the Hoechst dye 33342 and sorted with flow cytometry. RESULTS: After transfer of one single blastocyst, an intrauterine pregnancy arose. In the ninth week of gestation, the female sex of the embryo was confirmed with proof of absence of the SRY gene of the Y-chromosome. After normal pregnancy, the patient delivered a healthy daughter. CONCLUSIONS: The staining of spermatozoa with specific markers and sorting with flow cytometry provides a means of preventing significant disease in the offspring and may help in reducing the number of surplus embryos needed for preimplantation genetic diagnosis.

A method of utrophin up-regulation through RNAi-mediated knockdown of the transcription factor EN1.

The aim of this study was to induce up-regulation of the dystrophin-related gene UTRN that encodes the protein utrophin, to determine whether this could compensate for the lack of dystrophin function in Duchenne muscular dystrophy. The human UTRN promoter, which contains two putative binding sites for homeobox protein engrailed-1 (EN1), was analysed. It was found that EN1 binding site 2 in the UTRN gene promoter directly interacted with transcription factor EN1 in vitro. Chromatin immunoprecipitation assays of the EN1-UTRN promoter complex from rhabdomyosarcoma and HeLa cell lines confirmed that endogenous EN1 interacted with this region in vivo. The findings suggest that EN1 directly interacts with the UTRN promoter. Small interfering RNA was used to inhibit EN1 gene expression. Higher utrophin mRNA levels were observed in EN1-inhibited cells compared with controls. The increase in utrophin mRNA in rhabdomyosarcoma cells and HeLa cells may have resulted from inhibition of EN1 expression.

Rapamycin ameliorates dystrophic phenotype in mdx mouse skeletal muscle.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal, degenerative disease that results from mutations in the dystrophin gene, causing necrosis and inflammation in skeletal muscle tissue. Treatments that reduce muscle fiber destruction and immune cell infiltration can ameliorate DMD pathology. We treated the mdx mouse, a model for DMD, with the immunosuppressant drug rapamycin (RAPA) both locally and systemically to examine its effects on dystrophic mdx muscles. We observed a significant reduction of muscle fiber necrosis in treated mdx mouse tibialis anterior (TA) and diaphragm (Dia) muscles 6 wks post-treatment. This effect was associated with a significant reduction in infiltration of effector CD4(+) and CD8(+) T cells in skeletal muscle tissue, while Foxp3(+) regulatory T cells were preserved. Because RAPA exerts its effects through the mammalian target of RAPA (mTOR), we studied the activation of mTOR in mdx TA and Dia with and without RAPA treatment. Surprisingly, mTOR activation levels in mdx TA were not different from control C57BL/10 (B10). However, mTOR activation was different in Dia between mdx and B10; mTOR activation levels did not rise between 6 and 12 wks of age in mdx Dia muscle, whereas a rise in mTOR activation level was observed in B10 Dia muscle. Furthermore, mdx Dia, but not TA, muscle mTOR activation was responsive to RAPA treatment.

Long-term blocking of calcium channels in mdx mice results in differential effects on heart and skeletal muscle.

The disease mechanisms underlying dystrophin-deficient muscular dystrophy are complex, involving not only muscle membrane fragility, but also dysregulated calcium homeostasis. Specifically, it has been proposed that calcium channels directly initiate a cascade of pathological events by allowing calcium ions to enter the cell. The objective of this study was to investigate the effect of chronically blocking calcium channels with the aminoglycoside antibiotic streptomycin from onset of disease in the mdx mouse model of Duchenne muscular dystrophy (DMD). Treatment in utero onwards delayed onset of dystrophic symptoms in the limb muscle of young mdx mice, but did not prevent degeneration and regeneration events occurring later in the disease course. Long-term treatment had a positive effect on limb muscle pathology, reduced fibrosis, increased sarcolemmal stability, and promoted muscle regeneration in older mice. However, streptomycin treatment did not show positive effects in diaphragm or heart muscle, and heart pathology was worsened. Thus, blocking calcium channels even before disease onset does not prevent dystrophy, making this an unlikely treatment for DMD. These findings highlight the importance of analyzing several time points throughout the life of the treated mice, as well as analyzing many tissues, to get a complete picture of treatment efficacy.

Read-through strategies for suppression of nonsense mutations in Duchenne/ Becker muscular dystrophy: aminoglycosides and ataluren (PTC124).

Nucleotide changes within an exon can alter the trinucleotide normally encoding a particular amino acid, such that a new ''stop'' signal is transcribed into the mRNA open reading frame. This causes the ribosome to prematurely terminate its reading of the mRNA, leading to nonsense-mediated decay of the transcript and lack of production of a normal full-length protein. Such premature termination codon mutations occur in an estimated 10% to 15% of many genetically based disorders, including Duchenne/Becker muscular dystrophy. Therapeutic strategies have been developed to induce ribosomal read-through of nonsense mutations in mRNA and allow production of a full-length functional protein. Small-molecule drugs (aminoglycosides and ataluren [PTC124]) have been developed and are in clinical testing in patients with nonsense mutations within the dystrophin gene. Use of nonsense mutation suppression in Duchenne/Becker muscular dystrophy may offer the prospect of targeting the specific mutation causing the disease and correcting the fundamental pathophysiology.

Rescue of developmental defects by blastocyst stem cell injection: towards elucidation of neomorphic corrective pathways.

Stem cell-based therapy is an exciting area of high potential for regenerative medicine. To study disease prevention, we inject mouse embryonic stem cells (ESCs) into a variety of mouse blastocysts, most of which harbor mutations. Mice derived from these mutant blastocysts develop human-like diseases, either at developmental stages or in the adult, but blastocyst injection of ESCs prevents disease from occurring. Rather than entirely repopulating the affected organs, with just 20% of chimerism, the ESCs replenish protein levels that are absent in mutant mice, and induce novel or "neomorphic" signals that help circumvent the requirements for the mutations. We also show data indicating that the "neomorphic" mechanisms arise as a result of blastocyst injection of ESCs, regardless of the nature of the host blastocyst (mutant or wild-type). Thus, blastocyst injection of ESCs not only allows the study of disease prevention, but also unveils novel pathways whose activation may aid in the correction of congenital or acquired disease.

Green tea extract decreases muscle pathology and NF-kappaB 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.