ABSTRACT
BACKGROUND: Nitric oxide (NO) is an inorganic gas produced by a family of NO synthase (NOS) proteins. The presence and the distribution of inducible-NOS (NOS II or iNOS), and NADPH-diaphorase (NADPH-d), a marker for NOS catalytic activity, were determined in muscle sections from control, DMD, and BMD patients. MATERIALS AND METHODS: NADPH-d reactivity, iNOS- and nNOS (NOS I)-immunolocalization were studied in muscles from mdx mice before and after somatic gene transfer of dystrophin or utrophin. RESULTS: In control patients, few fibers (<2%) demonstrated focal accumulation of iNOS in sarcolemma. In DMD patients, a strong iNOS immunoreactivity was observed in some necrotic muscle fibers as well as in some mononuclear cells, and regenerating muscle fibers had diffusely positive iNOS immunoreactivity. In DMD patients, NADPH-d reactivity was increased and mainly localized in regenerating muscle fibers. In mdx mice quadriceps, iNOS expression was mainly observed in regenerating muscle fibers, but not prior to 4 weeks postnatal, and was still present 8 weeks after birth. The expression of dystrophin and the overexpression of utrophin using adenovirus-mediated constructs reduced the number of iNOS-positive fibers in mdx quadriceps muscles. The correction of some pathology in mdx by dystrophin expression or utrophin overexpression was independent of the presence of nNOS. CONCLUSIONS: These results suggest that iNOS could play a role in the physiopathology of DMD and that the abnormal expression of iNOS could be corrected by gene therapy.
Subject(s)
Cytoskeletal Proteins/genetics , Dystrophin/genetics , Genetic Therapy , Membrane Proteins/genetics , Muscle, Skeletal/enzymology , Muscular Dystrophy, Animal/therapy , Muscular Dystrophy, Duchenne/enzymology , Nitric Oxide Synthase/metabolism , Adenoviridae/genetics , Animals , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Gene Transfer Techniques , Genetic Vectors , Humans , Immunohistochemistry , Mice , Mice, Inbred mdx , Muscle, Skeletal/pathology , Muscular Dystrophy, Animal/genetics , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/therapy , NADPH Dehydrogenase/analysis , NADPH Dehydrogenase/immunology , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type II , Promoter Regions, Genetic , Time Factors , UtrophinABSTRACT
The presence and the distribution of tumor necrosis factor-alpha, interferon-gamma, and p65 subunit of nuclear factor-kappaB, molecules known to induce synergistically and to mediate major histocompatibility complex (MHC) class I expression, were determined in muscle sections from control and X-linked vacuolated myopathy patients. MHC class I colocalized with tumor necrosis factor-alpha and interferon-gamma, as well as with p65, in most of the membrane attack complex- and/or calcium-positive muscle fibers in X-linked vacuolated myopathy. These results suggest that the expression of MHC class I in X-linked vacuolated myopathy could be induced by tumor necrosis factor-alpha and interferon-gamma and partly mediated by nuclear factor-kappaB.
Subject(s)
Muscular Diseases/pathology , X Chromosome/genetics , Gene Expression Regulation , Genetic Linkage , Histocompatibility Antigens Class I/analysis , Histocompatibility Antigens Class I/genetics , Humans , Immunohistochemistry , Interferon-gamma/analysis , Interferon-gamma/genetics , Muscle Fibers, Skeletal/chemistry , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Muscular Diseases/genetics , Muscular Diseases/metabolism , NF-kappa B/analysis , NF-kappa B/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Necrosis Factor-alpha/analysis , Tumor Necrosis Factor-alpha/geneticsABSTRACT
We have studied the expression of S-protein on the muscle from patients with X-linked vacuolated myopathy [characterized by the deposition of the complement C5b-9 membrane attack complex (MAC) over abnormal muscle fibers] and controls by immunocytochemistry and immunoblotting. No expression was detected on muscle from controls and patients with X-linked vacuolated myopathy. These findings suggest that S-protein does not render the MAC inactive in X-linked vacuolated myopathy. This situation may be due to the fact that the pathways of MAC activation and the expression of S-protein in X-linked vacuolated myopathy are different from the ones observed in ischemic and/or necrotic, or immune diseases. These results emphasize the role of the membrane complement regulatory proteins (i.e., CD59) in X-linked vacuolated myopathy.
