Identification of plasma interleukins as biomarkers for deflazacort and omega-3 based Duchenne muscular dystrophy therapy.

Duchenne muscular dystrophy (DMD) is a progressive and fatal disease, characterized by the absence of dystrophin, muscle degeneration and cardiorespiratory failure. Creatine kinase is the classic marker to screen for DMD. However, other markers are needed to follow disease progression and to evaluate the response to therapy over longer periods. In the present study, we aim to identify interleukins in the plasma of the mdx mice model of DMD that could serve as biomarkers to monitor dystrophy progression, at distinct stages of the disease (1, 3 and 8 months of age). We used deflazacort and omega-3 therapies to validate the biomarkers studied. Plasma levels of TNF-α and TGF-ß were increased in mdx mice in relation to control, at all times studied. Differences in IFN-γ and IL-10 contents, comparing mdx x CTRL, were detected only at the early stage (1 month). IL-6 decreased at 3 and 8 months and IL-13 increased at 8 months in the mdx compared to control. Deflazacort and omega-3 reduced the plasma levels of the pro-inflammatory (TNF-α, INF-γ, IL-6) and pro-fibrotic (IL-13 and TGF-ß) interleukins and increased the plasma levels of IL-10. It is suggested that TNF-α and TGF-ß in plasma would be the best markers to follow disease progression. IL-6, INF-γ and IL-10 would be suitable markers to the earlier stages of dystrophy and IL-13 a suitable marker to the later stages of dystrophy.

Comparative study of inorganic elements determined in whole blood from Dmd(mdx)/J mice strain by EDXRF and NAA analytical techniques.

Several diseases can be diagnosed observing the variation of specific elements concentration in body fluids. In this study the concentration of inorganic elements in blood samples of dystrophic (Dmd(mdx)/J) and C57BL/6J (control group) mice strain were determined. The results obtained from Energy Dispersive X-ray Fluorescence (EDXRF) were compared with Neutron Activation Analysis (NAA) technique. Both analytical techniques showed to be appropriate and complementary offering a new contribution for veterinary medicine as well as detailed knowledge of this pathology.

The L-arginine/NO pathway and homoarginine are altered in Duchenne muscular dystrophy and improved by glucocorticoids.

The L-arginine/nitric oxide (L-Arg/NO) pathway regulates endothelial function and may play an important role in the pathogenesis of Duchenne muscular dystrophy (DMD). Yet, this pathway is poorly investigated in children suffering from DMD. Endothelial dysfunction can affect the perfusion of contracting muscles, thus leading to ischemia and hypoxia. In the present study, we tested the hypothesis that reduced NO production due to elevated synthesis of N (G),N (G)-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of NO synthesis, is a possible pathophysiological mechanism for progressive intramuscular muscle ischemia and disturbed endothelial function in children with DMD. Given the possible antagonistic action of homoarginine (hArg) on ADMA, we also analyzed this amino acid. We investigated 55 male patients with DMD and 54 healthy male controls (HC; aged 11.9 ± 4.8 vs. 11.1 ± 4.9 years, mean ± SD). Urinary creatinine and metabolites of the L-Arg/NO pathway were measured in plasma and urine by GC-MS or GC-MS/MS. Urine levels of ADMA and its major urinary metabolite dimethylamine (DMA), nitrite and nitrate (P < 0.001 for all) and hArg (P = 0.002) were significantly higher in DMD patients compared to HC, while the urinary DMA/ADMA molar ratio was lower (P = 0.002). In plasma, nitrate (P < 0.001), hArg (P = 0.002) and the hArg/ADMA ratio (P < 0.001) were lower in DMD than in HC. In plasma, ADMA (631 ± 119 vs. 595 ± 129 nM, P = 0.149), arginine and nitrite did not differ between DMD and HC. In DMD, positive correlations between ADMA, DMA or nitrate excretion and the stage of disease (according to Vignos and Thompson) were found. In DMD patients on steroid medication, lower concentrations of ADMA in plasma, and of DMA, ADMA, nitrate and hArg in urine were observed compared to non-treated patients. The L-Arg/NO pathway is impaired in DMD patients, with the disease progression being clinically negatively correlated with the extent of impairment. One of the underlying mechanisms in DMD may involve insufficient antagonism of ADMA by hArg. Steroids, but not creatine supplementation, seems to improve the L-Arg/NO pathway in DMD.

Pre-clinical study of 21 approved drugs in the mdx mouse.

Duchenne muscular dystrophy, a genetic disease caused by the absence of functional dystrophin, remains without adequate treatment. Although great hopes are attached to gene and cell therapies, identification of active small molecules remains a valid option for new treatments. We have studied the effect of 20 approved pharmaceutical compounds on the muscles of dystrophin-deficient mdx5Cv mice. These compounds were selected as the result of a prior screen of 800 approved molecules on a dystrophin mutant of the invertebrate animal model Cænorhabditis elegans. Drugs were administered to the mice through maternal feeding since 2weeks of life and mixed in their food after the 3rd week of life. The effects of the drugs on mice were evaluated both at 6weeks and 16weeks. Each drug was tested at two concentrations. Prednisone was added to the molecule list as a positive control. To investigate treatment efficiency, more than 30 histological, biochemical and functional parameters were recorded. This extensive study reveals that tricyclics (Imipramine and Amitriptyline) are beneficial to the fast muscles of mdx mice. It also highlights a great variability of responses according to time, muscles and assays.

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.