Dystrophin acts as a transplantation rejection antigen in dystrophin-deficient mice: implication for gene therapy

Duchenne muscular dystrophy is a lethal and common X-linked recessive disease caused by a defect in dystrophin. Normal myoblast transplantation and dystrophin gene transfer have been expected to correct the deficiency in the muscles, but their clinical application has been hampered by the limited preservation of dystrophin-positive myofibers. In this study we investigated the mechanism for immunologic rejection of normal C57BL/10 (B10) myoblasts transplanted into dystrophin-deficient mdx mice, an animal model of Duchenne muscular dystrophy. We found that mdx mice develop CTL specific for dystrophin itself, which were CD8 dominant and restricted by H-2Kb. We identified several antigenic peptides derived from dystrophin that bind to H-2Kb and are recognized by the mdx anti-B10 CTL. Immunologic tolerance against dystrophin was successfully induced by i.v. injection of these peptides before B10 myoblast transplantation, which resulted in sustained preservation of dystrophin-expressing myofibers in mdx mice. These results demonstrate that dystrophin is antigenic in dystrophin-deficient mice and that immunologic regimen would be necessary to achieve the persistent expression of introduced dystrophin in the muscles of dystrophin-deficient individuals.     

Factors inducing mast cell accumulation in skeletal muscle

It has been suggested that mast cells contribute to the phenotype of dystrophinopathies, but the mechanisms of their recruitment into the skeletal muscle remain hypothetical. The aim of this study is to quantify the presence of mast cells in muscle during the cellular events of myofibre degeneration and regeneration. For this purpose, we compare the mast cell profile in dystrophin-deficient mdx mice in which muscles exhibit spontaneous cycles of degeneration-regeneration from 3 weeks of age, with that in Swiss mice in which muscles were injured either by ischaemia or by notexin injection. Notexin is an A2-type phospholipase that rapidly disrupts myofibre plasma membranes, while ischaemia results in a slower process of degeneration. Both lesions are followed by a successful regeneration. In intact muscles, mast cell counts (mean +/- SEM/mm2) range from 1.8 +/- 1 to 4.3 +/- 1.6. The injection of notexin is far more potent in recruiting mast cells into damaged muscle than is ischaemia (118.5 +/- 13.0 vs 12.3 +/- 1.8/mm2). Thus we conclude that the early disruption of the myofibre membrane could elicit mast cell accumulation in skeletal muscle. This may explain the elevated number of mast cells observed in mdx muscles, as dystrophin deficiency is though to induce myofibre membrane leakage. On the other hand, mast cells are more numerous in muscles of young and adult mdx mice that are allowed to regenerate, than in muscles of older animals in which there is little regeneration and fibrosis develops. In injured muscles, the peak of mast cell number is at the onset of regeneration (by day 3 after notexin injection, and by day 11 after ischaemia), rather than during the phase of myofibre necrosis. Therefore, we suggest that the mast cells, through the effects of released mediators, could contribute to muscle regeneration.     

Mechanism of increasing dystrophin-positive myofibers by myoblast transplantation: study using mdx/beta-galactosidase transgenic mice

Female mdx/mdx mice were crossed with non-dystrophic transgenic males expressing the beta-galactosidase (beta-gal) gene under a muscle-specific promoter (TnILacZ1/29). All male offspring were mdx mice and about 50% of them also expressed the beta-gal gene. The beta-gal/mdx mice were selected as recipients for the transplantation of myoblasts from non-transgenic normal BALB/c mice. When host muscles were not irradiated before myoblast transplantation, 4.6% of the muscle fibers in host muscles were dystrophin positive 1 month after transplantation. Most of these dystrophin-positive muscle fibers were also beta-gal positive. About one quarter of these fibers are the result of reverse mutations; most of them have, however, been produced by fusion of donor myoblasts with host muscle fibers or with host myoblasts. The virtual absence of beta-gal-negative fibers indicates that there were no exclusively donor-donor fusions. When host muscles were irradiated before myoblast transplantation, roughly the same percentage (5.5%) of dystrophin-positive fibers were formed in the injected muscle, but 42% of them were beta-gal negative. These beta-gal-negative dystrophin-positive muscle fibers were formed by the exclusive fusion of donor myoblasts with one another rather than with host cells. This clearly indicates that myoblast transplantation can form completely new muscle fibers or muscle fiber segments when host satellite cell proliferation is reduced by irradiation. These newly formed muscle fibers had, however, a small diameter and additional myoblast transplantation may be required to increase their size. This situation has some similarities with findings in Duchenne muscular dystrophy patients of more than 6 years of age, who also have a limited proliferation capacity of their satellite cells.     

Expression of the human dystrophin gene in mdx mouse muscle fibers after transfection using liposomes and synthetic oligopeptides

The number of dysrophin-positive fibers appearing in the femoral quadriceps muscle of mdx mice after injection of the full-length human dystrophin cDNA within the pHSADy plasmid was examined by means of immunohystochemical techniques. Transfection was carried out using lipofectamine (LFA), or synthetic oligopeptide complexes that provided the condensation of plasmid DNA (K8) and its release from endosomes gopeptide complexes that provided the condensation of plasmid DNA (K8) and its release from endosomes (JTS1). The LFA + pHSADy at a dose of 10 micrograms DNA did not affect the number of dystrophin-positive fibers at the site of injection (0.6-0.8%), whereas it caused a statistically significant increase in the number of these fibers in the same muscle of the contralateral leg (up to 2.3%). Injection of the SO + pHSADy complex resulted in the occurrence of dystrophin-positive muscle fibers characterized by a heterogeneous content and the distribution of dystrophin. The greatest number of dystrophin-positive fibers (about 16%) was observed under a ratio of pHSADy to K8 of 1:3 or 1:4. The observed maximal number of dystrophin-positive fibers after a single injection of SO + pHSADy was 3.8%, and it was 17.7% after three injections. These values were statistically significantly higher compared to intact mice (0.6%), the injection of pure plasmid (2.2%), or the intramuscular injection of sucrose (from 0.7 to 1.3%). A relatively high level of transfection (about 5%) was observed after an intracardiac injection of a large dose of the pHSADy (70 micrograms DNA). The perspectives of the targeted delivery of the dystrophin gene into muscles under conditions of parenteral administration are discussed.     

Back pain following epidural anaesthesia in labour

For palliative therapy of Duchenne muscular dystrophy (DMD), corticosteroids have been tried since 1970. According to recent reports, corticosteroids maintained muscular strength to some extent and prolonged period of ambulation. However, their mechanism of action is mostly unclear. In the present study, mdx mice were injected with 0.6 mg prednisolone 3 times a week for 30 weeks. Serum creatine kinase (CK) values remained 23% of controls. In muscle pathology of the quadriceps muscle, fibers with peripheral nuclei were increased, suggesting reduction of muscle necrosis. In pathological examination of liver, pyknotic cytoplasmic masses and formation of vacuoles were observed. Present study showed that prednisolone might attenuate muscle fiber necrosis at least for 30 weeks. Prednisolone may reduce secondary tissue reactions, which develop more serious muscle damage.     

Apoptotic myonuclei in human Duchenne muscular dystrophy

The current view that apoptosis precedes necrosis in death of dystrophin-deficient muscle fibers of the mdx mouse has been well substantiated. Moreover, apoptotic myonuclei have been reported to increase in dystrophin-deficient mice 2 days after spontaneous exercise. To investigate the role of apoptosis in human muscular dystrophy, muscles from 11 patients of different ages with Duchenne muscular dystrophy were analyzed for apoptosis. The amount of apoptosis was assessed by terminal deoxynucleotidyl transferase assay, and the expression of bcl-2 and bax was examined by immunohistochemistry. Although very rare in normal muscles (less than 0.1%), apoptotic nuclei were detected in dystrophic muscles, particularly at the interstitial level. Nevertheless, few dystrophin-deficient myofibers with centrally located nuclei showed a positive reaction for DNA fragmentation. A mosaic pattern of bcl-2/bax-positive myofibers characterized dystrophic muscles, thus the relative proportion of pro- and antiapoptotic proteins differs among muscle fibers in correlation with the presence of apoptotic myonuclei. In the interstitium, apoptotic cells were identified as macrophages and activated satellite cells. This is the first study to show an apoptotic process in adult muscle fibers of patients with Duchenne muscular dystrophy, thereby shedding new light on muscle damage and its progression in dystrophinopathies.     

Melatonin chimeras alter reproductive development and photorefractoriness in Siberian hamsters

Apoptosis is well accepted as a type of cell death occurring in the development of mammalian muscles, but the death of adult myofibres in neuromuscular disorders and exercise-induced muscle damage is usually explained in terms of muscle necrosis. The current view that apoptosis precedes necrosis in death of dystrophin-deficient muscle fibres of mdx mouse has been well substantiated. Moreover, apoptotic myonuclei have been reported to increase in mdx mice 2 days after spontaneous exercise. To investigate the contribution of apoptosis to exercise-induced damage of normal muscle fibre a time-course analysis has been performed in adult C57BL/6 mice. Groups of five mice were sacrificed immediately after the end of the exercise, and after a rest period of 6 or 96 h. The amount of apoptosis in leg muscles was assessed by electron microscopy, by the terminal deoxynucleotidyl transferase assay and by electrophoretic detection of fragmented DNA; the expression of Bcl-2, Bax, Fas, ICE, p53 and ubiquitin was examined by immunohistochemistry and Western blot. Absent in muscles of normal 'sedentary' mice, apoptotic myonuclei peak in muscles of normal mice after a night of spontaneous wheel-running (4% +/- 3.5, immediately and 2.5% +/- 1.8 after 6 h rest, P < 0.05 vs non-runner mice); they then decrease but are present 4 days later (0.8% +/- 1.5). Satellite cells are also involved in the apoptotic process. Myofibre content of Bcl-2 decreases whereas Bax, Fas, ICE and ubiquitin modify their pattern of expression in correlation with the changes in apoptotic myonuclei. Apoptosis of endothelial cells is present after the night of wheel-running and with a twofold increase 4 days later (1.5 +/- 2.3 and 4.8 +/- 4.4 P < 0.05, respectively). Satellite cells are also involved in the apoptotic process. Thus, spontaneous running in unaccustomed mice increases the number of apoptotic nuclei in adult muscle fibres and in endothelial cells. It remains to be established whether muscle apoptosis is restricted to the repair mechanisms, as often suggested in many pathologic processes, or it is also part of pathogenesis of muscle damage. Regardless of whether these results are extended to human dystrophies, the clinical implications in terms of secondary pathogenetic mechanisms and muscle training are obvious.     

Consequences of the combined deficiency in dystrophin and utrophin on the mechanical properties and myosin composition of some limb and respiratory muscles of the mouse

The mechanical properties and the myosin isoform composition were studied in three isolated muscles (EDL, soleus, diaphragm) of mutant mice lacking both dystrophin and utrophin (dko). They were compared with the corresponding muscles of the normal and the dystrophin-deficient (mdx) and the utrophin-deficient (uko) mice. In comparison with mdx muscles, dko muscles show a significant reduction of the normalized isometric force, confirmed by the reduced muscular activity of the whole animal. Kinetics parameters (twitch time-to-peak and half-relaxation time) were slightly reduced, and the maximal speed of shortening of soleus, Vmax, was reduced by 30%. The maximal power output (muW/mm3) was reduced by 50% in dko soleus. In the three muscles studied, the relative myosin heavy chains (MHC) composition showed a shift towards slower isoforms. dko EDL presented a dramatic decrease of the resistance ot tetanic contraction with forced lengthenings (eccentric contractions), while muscle lacking only utrophin (uko mutants) display a normal resistance to this exacting mechanical challenge. These experiments suggest that lack of both dystrophin and utrophin is very detrimental to the mice and that mechanical properties of the muscles may explain the overall phenotype. Moreover these results bring some support to the idea that the expression of utrophin in mdx muscle compensates, to some extent, for the lack of dystrophin.     

Neurotransmitter transporters as molecular targets for addictive drugs

The mdx mouse, an animal model of the Duchenne muscular dystrophy, was used for the investigation of changes in mitochondrial function associated with dystrophin deficiency. Enzymatic analysis of skeletal muscle showed an approximately 50% decrease in the activity of all respiratory chain-linked enzymes in musculus quadriceps of adult mdx mice as compared with controls, while in cardiac muscle no difference was observed. The activities of cytosolic and mitochondrial matrix enzymes were not significantly different from the control values in both cardiac and skeletal muscles. In saponin-permeabilized skeletal muscle fibers of mdx mice the maximal rates of mitochondrial respiration were about two times lower than those of controls. These changes were also demonstrated on the level of isolated mitochondria. Mdx muscle mitochondria had only 60% of maximal respiration activities of control mice skeletal muscle mitochondria and contained only about 60% of hemoproteins of mitochondrial inner membrane. Similar findings were observed in a skeletal muscle biopsy of a Duchenne muscular dystrophy patient. These data strongly suggest that a specific decrease in the amount of all mitochondrial inner membrane enzymes, most probably as result of Ca2+ overload of muscle fibers, is the reason for the bioenergetic deficits in dystrophin-deficient skeletal muscle.     

Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy

The absence of dystrophin at the muscle membrane leads to Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease that is inevitably fatal in early adulthood. In contrast, dystrophin-deficient mdx mice appear physically normal despite their underlying muscle pathology. We describe mice deficient for both dystrophin and the dystrophin-related protein utrophin. These mice show many signs typical of DMD in humans: they show severe progressive muscular dystrophy that results in premature death, they have ultrastructural neuromuscular and myotendinous junction abnormalities, and they aberrantly coexpress myosin heavy chain isoforms within a fiber. The data suggest that utrophin and dystrophin have complementing roles in normal functional or developmental pathways in muscle. Detailed study of these mice should provide novel insights into the pathogenesis of DMD and provide an improved model for rapid evaluation of gene therapy strategies.     

Intramuscular injection of hrRANTES causes mast cell recruitment and increased transcription of histidine decarboxylase in mice: lack of effects in genetically mast cell-deficient W/WV mice

RANTES (regulated upon activation, normal T cell expressed and presumably secreted) and other chemoattractant proteins are members of the intercrine or chemokine family of proinflammatory basic polypeptides. RANTES is a prototype of the C-C chemokine subfamily that acts as a selective chemoattractant for human monocytes and CD4-positive lymphocytes and increases the adherence of monocytes to endothelial cells. However, the role of RANTES in white cells is still unclear. We report here that hrRANTES at 20 ng/50 microl in mice causes mast cell recruitment 4 h after intramuscular injection, an effect inhibited by anti-RANTES, as evidenced by 0.1% Toluidine blue, a specific dye for coloring mast cells. Injections of PBS (50 microl) vehicle (negative control) did not produce any appreciable inflammatory response, whereas injection of lipopolysaccharide 20 ng/50 microl (positive control) generated a marked inflammatory state. When RANTES was injected intramuscularly in genetically mast cell-deficient W/Wv mice, the inflammatory effect was not present. The RANTES injection sites were then excised and studied under an optical and electron microscope. A Northern blot analysis was performed using a probe that was prepared to detect mRNA encoding the histidine decarboxylase (HDC) gene on excised muscle tissue. We found that hrRANTES provoked generation of HDC mRNA from muscle tissue after 4 h. These effects were inhibited by an anti-RANTES antibody and were absent in genetically mast cell-deficient mice. The increasing number of mast cells in the RANTES injection sites led to an augmentation of histamine content compared to controls (PBS). The injection of hrRANTES 20 ng/20 microl into the sole of a rat paw confirmed the inflammatory and the mast cell recruitment potential of this chemokine. In these studies, hrRANTES injections in muscle tissue provided direct in vivo evidence that RANTES has a significant effect on mast cell recruitment and HDC mRNA generation.     

Cleavage of type I procollagen by human mast cell chymase initiates collagen fibril formation and generates a unique carboxyl-terminal propeptide

The ability of human mast cell chymase and tryptase to process procollagen was examined. Purified human intestinal smooth muscle cell procollagen was incubated with human mast cell tryptase or human mast cell chymase. Purified chymase, but not tryptase, exhibited procollagen proteinase activity in the presence of EDTA. Addition of purified porcine heparin over a range of 0.1-100 microg/ml did not affect either the rate or the products of procollagen chymase cleavage. The cleavage site of chymase on the pro-alpha1(I) collagen carboxyl terminus was found to be in the propeptide region at Leu-1248-Ser-1249. Cleavage at this site suggested that the collagen products would form fibrils and confirmed the production of a unique carboxyl-terminal propeptide. Turbidometric fibril formation assay demonstrated de novo formation of chymase-generated collagen fibrils with characteristic lag, growth, and plateau phases. When observed by dark field microscopy, these fibrils were similar to fibrils formed by the action of procollagen proteinases. Thus, mast cell chymase, but not tryptase, exhibits procollagen peptidase-like activity as evidenced by its ability to process procollagen to fibril-forming collagen with concurrent formation of a unique carboxyl-terminal propeptide. These data demonstrate that mast cell chymase has a potential role in the regulation of collagen biosynthesis and in the pathogenesis of fibrosis.     

Human mast cell chymase induces the accumulation of neutrophils, eosinophils and other inflammatory cells in vivo

The roles of chymase in acute allergic responses are not clear, despite the relative abundance of this serine proteinase in the secretory granules of human mast cells. We have isolated chymase to high purity from human skin tissue by heparin-agarose affinity chromatography and Sephacryl S-200 gel filtration procedures, and have investigated the ability of human mast cell chymase to stimulate cell accumulation following injection into laboratory animals. Injection of chymase provoked marked neutrophilia and eosinophilia in the skin of Dunkin Hartley guinea-pigs. Compared with saline injected control animals, there were some 60 fold more neutrophils and 12 fold more eosinophils present at the injection site. Following injection of chymase into the peritoneum of BALB/c mice, there were up to 700 fold more neutrophils. 21 fold more eosinophils, 19 fold more lymphocytes and 7 fold more macrophages recovered than from saline injected controls at 16 h. Doses of chymase as low as 5 ng (1.7 x 10(-13) mole) stimulated an inflammatory infiltrate, and significant neutrophilia was elicited within 3 h. The chymase induced cell accumulation in both the guinea-pig and mouse models was dependent on an intact catalytic site, being reduced by co-injection of proteinase inhibitors or heat inactivation of the enzyme. Co-injection of histamine or heparin significantly reduced the chymase induced neutrophil accumulation, whereas neither histamine nor heparin by themselves had any effect on the accumulation of nucleated cells. No synergistic or antagonist interactions between chymase and tryptase were observed when these two major mast cell proteinases were co-injected into the mouse peritoneum. Our findings suggest that chymase may provide an potent stimulus for inflammatory cell recruitment following mast cell activation.     

The tryptase, mouse mast cell protease 7, exhibits anticoagulant activity in vivo and in vitro due to its ability to degrade fibrinogen in the presence of the diverse array of protease inhibitors in plasma

Mouse mast cell protease (mMCP) 7 is a tryptase of unknown function expressed by a subpopulation of mast cells that reside in numerous connective tissue sites. Because enzymatically active mMCP-7 is selectively released into the plasma of V3 mastocytosis mice undergoing passive systemic anaphylaxis, we used this in vivo model system to identify a physiologic substrate of the tryptase. Plasma samples taken from V3 mastocytosis mice that had been sensitized with immunoglobulin (Ig) E and challenged with antigen were found to contain substantial amounts of four 34-55-kDa peptides, all of which were derived from fibrinogen. To confirm the substrate specificity of mMCP-7, a pseudozymogen form of the recombinant tryptase was generated that could be activated after its purification. The resulting recombinant mMCP-7 exhibited potent anticoagulant activity in the presence of normal plasma and selectively cleaved the alpha-chain of fibrinogen to fragments of similar size as that seen in the plasma of the IgE/antigen-treated V3 mastocytosis mouse. Subsequent analysis of a tryptase-specific, phage display peptide library revealed that recombinant mMCP-7 preferentially cleaves an amino acid sequence that is nearly identical to that in the middle of the alpha-chain of rat fibrinogen. Because fibrinogen is a physiologic substrate of mMCP-7, this tryptase can regulate clot formation and fibrinogen/integrin-dependent cellular responses during mast cell-mediated inflammatory reactions.     

The effect of age and temperature on mdx muscle fatigue

We have studied the in vitro contractile and fatigue characteristics of extensor digitorum longus (EDL) muscles from 8- and 62-week-old dystrophin-deficient (mdx) and control mice at 20 degrees C and 35 degrees C. There were no differences in fatigability at 20 degrees C, but at 35 degrees C the dystrophin-deficient muscles demonstrated increased fatigability compared to controls, with the older mice exhibiting the greatest fatigue. These results suggest a temperature-related mechanism of myofibrillar fatigue in dystrophin-deficient EDL muscles.     

Immunocytochemical localization of chymase to cytoplasmic vesicles after rat peritoneal mast cell stimulation by compound 48/80

The subcellular events responsible for release of mediators by mast cells may help to clarify roles for mast cells in health and disease. In this study we show that the granule-associated protease chymase is also within cytoplasmic vesicles in appropriately stimulated rat peritoneal mast cells. Rat peritoneal mast cells were recovered before or 1-10 sec after exposure to the secretogogue compound 48/80 (10 micrograms/ml) and then were examined by radioimmunoassay to quantify histamine release or were processed, using routine methods for postembedding immunoelectron microscopy, to identify the subcellular localization of chymase. In comparison to unstimulated cells, compound 48/80 stimulated cells in two independent experiments showed an increase (15%, 28%) in the surface area of the cell and a decrease (12%, 6%) in the surface area of the total granule compartment before degranulation channel formation. These global cellular changes occurred in a background of transient but significant (p < 0.01) increases in the area and number of chymase-immunoreactive vesicles per microns2 cytoplasm. These changes were detectable at 5 or 7 sec after stimulation with compound 48/80 but returned to near prestimulation levels by 9 or 10 sec after addition of compound 48/80 (total cumulative histamine release was 28% by 8 sec and 47% by 14 sec). These observations suggest that vesicles participate in the early stages of regulated secretion of chymase from rat peritoneal mast cells.     

Dystrophin-positive muscle fibers following C2 myoblast transplantation into mdx nude mice

To determine when and how the dystrophin-positive muscle fibers are formed after myoblast transplantation into dystrophin-negative muscles, the tibialis anterior (TA) muscle from mdx nude mouse was chronologically examined after C2 myoblast transplantation by immunohistochemical and glucose 6-phosphate isomerase (GPI) isoenzyme analyses. The host TA muscle transplanted with C2 myoblasts became necrotic with accumulation of basic fibroblast growth factor in the necrotic areas. This may stimulate concomitant proliferation of the host satellite cells and C2 myoblasts. Small dystrophin-positive muscle fibers appeared in the necrotic areas 3 days after transplantation. This TA muscle contained two different kinds of homodimer GPI isoenzymes but did not contain the heterodimer, suggesting rare fusion of host and donor cells. The dystrophin-positive muscle fibers in the necrotic areas rapidly increased in number and in size by 7 days, but they were smaller than the original host muscle fibers. They had central nuclei, indicating that they were regenerating fibers. The presence of heterodimer GPI isoenzyme in these muscles indicated that the regenerating fibers were mosaic host/donor muscle fibers. The dystrophin-positive muscle fibers are probably formed first by fusion of donor cells with each other and then later by the fusion of host satellite and donor cells.     

Differential effect of morphine on dopaminergic neurons in frontal cortex and striatum of the rat

Lactate dehydrogenase-5 and creatine kinase from rabbit muscle were labeled by coupling with N-hydroxysuccinimidyl 3-(4'-hydroxy-[3',5'-125I]diiodophenyl)propionate. After purification, the analytical recovery of catalytically-active labeled enzyme averaged 90% for lactate dehydrogenase, 81% for creatine kinase. The labeled enzymes were injected intravenously into rabbits and disappearance from plasma of catalytic activity and radioactivity was measured. The disappearance curves for lactate dehydrogenase-5 differed considerably from those observed with the enzyme labeled by direct iodination. The discrepancy was due to rapid hydrolysis in vivo of the labeled amide-enzyme linkage, because about 50% of the injected radioactivity appeared in the urine as 125I-labeled 3-(4'-hydroxy-3',5'-diiodophenyl)propionic acid within 4-8 h of injection. Similar outputs were observed after administration of this acid to rabbits. The free acid was also detected in the urines of rabbits within 4-8 h of the intravenous injection of creatine kinase labeled similarly. We conclude that this method of labeling is unsuitable for preparing radioactive enzymes for study of their catabolism.