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T A Partridge

Publications and source records attributed to T A Partridge.

At least 37 records · Page 2Linked to original sources
PubMed1997

Viral gene delivery to skeletal muscle: insights on maturation-dependent loss of fiber infectivity for adenovirus and herpes simplex type 1 viral vectors.

The mechanisms causing age-dependent loss of muscle fiber infectivity observed in vivo for both adenoviral (Ad) and herpes simplex virus type 1 (HSV-1) gene delivery vectors remain poorly understood. Here we investigate the possible bases for this phenomenon using the novel application of enzymatically isolated, viable, single muscle fibers. We show that maturation-dependent loss of fiber infectivity is recapitulated in single fibers, and, thus, is not solely due to host immune response. Using localized irradiation of muscle in vivo, we show data suggesting that Ad infectivity of differentiated myofibers depends, at least in part, on myoblasts to mediate fiber transduction. On the other hand, infection of single fibers by HSV-1 is not affected by irradiation. Using confocal microscopy, we show that the basal lamina of myogenic cells efficiently infected by HSV-1 is structurally less organized than that of fibers resistant to infection by HSV-1. As well, we show that single myofibers isolated from adult, basal lamina-defective mice (merosin-deficient, dy/dy) are at least 10-fold more susceptible to infection by HSV-1 than are myofibers isolated from control mice. Together, these observations support the hypothesis that the basal lamina acts as a physical barrier to HSV-1 infection of mature muscle.

Adenoviridae↗
PubMed1997

Selection and use of ligands for receptor-mediated gene delivery to myogenic cells.

Identification of myogenic cell targeting ligands is a critical step in the development of synthetic vectors for gene delivery to skeletal muscle. Here we describe the screening of six potential targeting ligands (insulin, insulin-like growth factor I, iron transferrin, gallium transferrin, alpha-bungarotoxin and carnitine) for their ability to bind dystrophin-deficient myotubes in vitro. Those ligands showing high levels of binding to myotubes were then tested on fully differentiated, isolated, viable myofibers. Of the ligands tested, transferrin showed the most promise based on high levels of binding to myogenic cells, high levels of receptor observed in regenerating fibers of patients with Duchenne muscular dystrophy and the ability to direct a large enzyme conjugate to the cytoplasm of myotubes. Finally, we show that incorporation of transferrin into an artificial virus consisting of poly-L-lysine-condensed DNA coated with a lipid shell (LPDII formulation) results in ligand-directed delivery of DNA to myogenic cells. This is the first report of gene transfer to myogenic cells using a ligand-directed synthetic vector. These results suggest that rational design of ligand-directed, fully synthetic, gene delivery vehicles is a viable approach to skeletal muscle vector development.

Animals↗
PubMed1996

Yields of muscle from myogenic cells implanted into young and old mdx hosts.

Implantation of normal muscle precursor cells (mpc) for treatment for inherited myopathies such as Duchenne muscular dystrophy is in clear need of improvement to become practicable, but few variables have been studied comparatively. Here, we report the first quantitative estimate of the effectiveness of implanting mpc into preirradiated muscles of young and old mice and into preirradiated and nonirradiated old muscles. Estimates were made of the amount of muscle formed by injection of 5 x 10(5) cells dissociated from neonatal normal mouse muscle into tibialis anterior muscles of the dystrophin-deficient mdx mouse. We show that normal mpc are incorporated slightly more efficiently into muscles of young than old host mice, to form some 10 mg of dystrophin-positive fibers. In older muscles, prior irradiation has little effect on the total yield of new muscle.

Animals↗
PubMed1996

Phenotype of adult mouse muscle myoblasts reflects their fiber type of origin.

Phenotypic diversity among mature skeletal muscle fibers originates from muscle progenitor cells, primary and secondary myoblasts, each of which is intrinsically committed to express a characteristic complement of developmentally regulated myosin heavy chain genes when differentiated. Similarly, postnatal muscle myoblasts, the satellite cells nestling beneath basement membranes of mature skeletal muscle fibers, have been shown to exhibit diversity, related to whether the muscle in which they reside is of a slow, fast or superfast type. Here we analyzed this association in more detail, evaluating the myosin heavy chain gene expression in immature muscle fibers (myotubes) formed in vitro from satellite cells extracted from isolated, living, single muscle-fibers of mature murine muscle. We identified a population of satellite cells that form myotubes expressing type I (slow) myosin heavy chain and found this population to be preferentially associated with individual slow muscle-fibers. These results not only confirm diversity among mammalian satellite cells, but also demonstrate that the phenotype of satellite cells is indicative of the type of fiber from which they derive.

Animals↗
PubMed1995

Culturing satellite cells from living single muscle fiber explants.

Conventional methods for isolating myogenic (satellite) cells are inadequate when only small quantities of muscle, the tissue in which satellite cells reside, are available. We have developed a tissue culture system that reliably permits isolation of intact, living, single muscle fibers with associated satellite cells from predominantly fast and slow muscles of rat and mouse; maintenance of the isolated fibers in vitro; dissociation, proliferation, and differentiation of satellite cells from each fiber; and removal of the fiber from culture for analysis.

Animals↗
PubMed1995

Myoblast-based gene therapies.

Recent identification of the genetic causes of several neuromuscular disorders has aroused interest in gene therapy in skeletal muscle. The genetic constitution of skeletal muscle can be altered by a number of means. Myoblasts can be used to introduce new genes, endogenous or exogenous, into muscle fibres during growth and repair. DNA expression-plasmids can be directly transfected into a small proportion of muscle fibres, showing persistent expression despite their lack of genomic integration. Recombinant replication deficient adenoviruses are efficient vectors into myoblasts and developing muscle fibres; again, the introduced constructs show long-term episomal persistence and expression. By contrast, recombinant replication deficient retroviruses efficiently introduce constructs into the genomes of dividing myoblasts which subsequently fuse into muscle fibres. None of the available methods provides a practical solution for therapy of genetic muscle diseases but might be useful for inducing synthesis of therapeutic non-muscle proteins by skeletal muscle.

Genetic Therapy↗
PubMed1994

Myogenic cell lines derived from transgenic mice carrying a thermolabile T antigen: a model system for the derivation of tissue-specific and mutation-specific cell lines.

Skeletal myoblasts cloned from limb muscles of H-2Kb-tsA58 transgenic mice remained proliferative through at least 80 generations under conditions permissive for expression and function of the tsA58 gene product. When switched to nonpermissive conditions or implanted into muscles of nude mdx mice they underwent differentiation but, in one clonal cell line, a small proportion appeared to become quiescent muscle precursors in vivo. H-2Kb-tsA58 X mdx/mdx F1 male mice yielded dystrophin-deficient myoblasts. By such simple genetic crosses, H-2Kb-tsA58 transgenic mice provide a valuable tool for the rapid isolation of cell lines, myogenic or otherwise, bearing mutations of interest.

Animals↗
PubMed1993

Age-related changes in replication of myogenic cells in mdx mice: quantitative autoradiographic studies.

Cell replication in muscle was measured by tritiated thymidine (3H-TdR) incorporation and autoradiography, in mdx mice from 2-44 weeks of age. Pre-mitotic labelling (within 1 h of 3H-TdR injection) was determined in 16 mice aged from 15 to 300 days. In 30 further mdx mice, one leg was irradiated 1 h after 3H-TdR injection to block DNA synthesis. Post-mitotic labelling was measured in both legs 10-15 days later. Between 20 and 60 days of age a very high proportion (up to 2%) of muscle (satellite cell) nuclei were replicating pre-mitotically; from 80-300 days cell replication was detectable but at much lower levels. Centrally placed nuclei within muscle fibres appeared at 24 days, increased rapidly to 50% by 50-100 days, declining thereafter to 25% at 300 days. In post-mitotic samples, labelled myotubes and labelled peripheral muscle nuclei (satellite cell nuclei and myonuclei) appeared at 28 days and were present in the mdx muscles through to 310 days, indicating continued cell replication and muscle regeneration. Myogenic cell replication was both retarded and inhibited by irradiation. These data demonstrate that muscle cell replication in mdx mice commences at about 3 weeks of age, is maximal at 4-8 weeks, but continues at lower levels until at least 44 weeks.

Aging↗
PubMed1993

Long-term persistence and migration of myogenic cells injected into pre-irradiated muscles of mdx mice.

Experiments were conducted to study the fate(s) of normal muscle precursor cells (mpc) which had been injected into the muscles of mdx mice. Right legs of mdx nu/nu mice were X-irradiated (18 Gray), to inhibit the proliferation of host mpc. Normal mpc were injected into the tibialis anterior (TA) muscles of these legs and the non-irradiated, contralateral legs. In pre-irradiated legs injected with normal mpc, the number of dystrophin-positive fibres was similar at 35, 49 and at 250 days after injection, but the number of dystrophin-negative fibres was much less at the latter time point, indicating prolonged survival of dystrophin-positive muscle fibres. Non-injected muscles neighbouring the injected TA muscle rarely contained muscle of donor origin 49 days after injection, but frequently did so 250 days after injection. This indicates that some of the injected mpc must have retained the ability to proliferate, to migrate into a neighbouring muscle and to differentiate into new muscle for a considerable period after the original cell implant. In non-irradiated legs, the implanted normal mpc formed markedly fewer dystrophin-positive fibres than in the contralateral, irradiated muscle, and undertook little or no migration to adjacent muscles.

Animals↗
PubMed1992

Cell transplantation and gene therapy in muscular dystrophy.

Duchenne's muscular dystrophy (DMD), which affects 1/3500 live male births, involves a progressive degeneration of skeletal and cardiac muscle, leading to early death. The protein dystrophin is lacking in DMD and present, but defective, in the allelic, less severe, Becker muscular dystrophy and is also missing in the mdx mouse. Experiments on the mdx mouse have suggested two possible therapies for these myopathies. Implantation of normal muscle precursor cells (mpc) into mdx skeletal muscle leads to the conversion of dystrophin-negative fibres to -positive, with consequent improvement in muscle histology. Direct injection of dystrophin cDNA into skeletal or cardiac muscle also gives rise to dystrophin-positive fibres. Although both appear promising, there are a number of questions to be answered and refinements to be made before either technique could be considered possible as treatments for myopathies in man.

Animals↗
PubMed1992

Regeneration after free muscle grafting in normal and dystrophic (mdx) mice.

Soleus muscles from C57BL/10 and mdx mice were isotransplanted to induce a cycle of degeneration/regeneration. Sixty days post-surgery, transplanted and contralateral soleus muscles were removed for mechanical and biochemical analyses. The regeneration which occurs after transplantation, induces in both mdx and C57BL/10 soleus muscles a decrease in maximal isometric force, together with an increase of the velocity of contraction. This increase in velocity is accompanied by the expression of typically fast-type myosin heavy chains. Thus degeneration/regeneration of both mdx and normal mice are very similar, causing a shift towards physiologically 'faster' muscle. Previous physiological and biochemical studies of mdx muscles have shown that mdx muscle is shifted towards 'slower' muscle compared to normal mice. One explanation of these findings was that the degeneration/regeneration cycles inherent in dystrophin-deficient mdx muscle causes a shift towards 'slow'. Our results argue against this hypothesis: degeneration/regeneration in both normal and mdx mice causes a shift towards 'fast'.

Animals↗
PubMed1992

Formation of skeletal muscle in vivo from the mouse C2 cell line.

The C2 muscle cell line is myogenic in vitro and has been extensively used in studies of muscle cell differentiation. Here, we have investigated the myogenicity in vivo of C2 cells implanted into suitable sites in the mouse. Large amounts of new muscle were formed when C2 cells were implanted into sites in nude mice which were undergoing regeneration following whole muscle grafting and in scaffolding of freeze-killed muscle or vicryl suture in the anterior tibial compartment. When implanted into regenerating muscle, C2 cells fused with the host muscle to form mosaic fibres; when implanted into inert sites, they formed muscle of largely donor origin. C2-derived muscle fibres appeared to become innervated, but the progression of N-CAM (neural cell adhesion molecule) isoform changes in such regenerates indicated that they did not become fully mature. Proliferating, undifferentiated cells of C2 origin form tumours in older grafts; however, this was more pronounced in the absence of competition from host muscle cells. In the short term, C2 cells can form large amounts of muscle in vivo for biochemical analysis. In addition, C2 cells are easily manipulable in vitro; genes of interest may be transfected into them prior to implantation of the cells into skeletal muscle and the effects of these genes in vivo may thus be examined.

Animals↗
PubMed1992

Expression and function of heterotypic adhesion molecules during differentiation of human skeletal muscle in culture.

The infiltration of skeletal muscle by leukocytes occurs in a variety of myopathies and frequently accompanies muscle degeneration and regeneration. The latter involves development of new myofibers from precursor myoblasts, and so infiltrating cells may interact with muscle at all stages of differentiation. The authors have investigated the surface expression of ligands for T-cell adhesion during the differentiation of human skeletal muscle in vitro. Myoblasts expressed low levels of ICAM-1 (CD54), which remained constant during muscle cell differentiation and could be induced by cytokines such as gamma-interferon. It is therefore likely that ICAM-1 is involved in the invasive accumulation of lymphocytes during skeletal muscle inflammation. In contrast, LFA-3 (CD58) was expressed at higher levels than ICAM-1 on myoblasts, decreased significantly during myogenesis, and was unaffected by immune mediators. Both ICAM-1 and LFA-3 were able to mediate T cell binding to myoblasts, whereas adhesion to myotubes was independent of the LFA-3 ligand. Although expressed throughout myogenesis, human leukocyte antigen class I and CD44 did not appear to mediate T cell binding. The expression of ligands that facilitate interaction of myogenic cells with lymphocytes may have important implications for myoblast transplantation.

Antibodies, Monoclonal↗
PubMed1991

X-irradiation improves mdx mouse muscle as a model of myofiber loss in DMD.

The mdx mouse, although a genetic and biochemical homologue of human Duchenne muscular dystrophy (DMD), presents a comparatively mild histopathological and clinical phenotype. These differences are partially attributable to the greater efficacy of regeneration in the mdx mouse than in DMD muscle. To lessen this disparity, we have used a single dose of X-irradiation (16 Gy) to inhibit regeneration in one leg of mdx mice. The result is an almost complete block of muscle fiber regeneration leading to progressive loss of muscle fibers and their replacement by loose connective tissue. Surviving fibers are mainly peripherally nucleated and, surprisingly, of large diameter. Thus, X-irradiation converts mdx muscle to a model system in which the degenerative process can be studied in isolation from the complicating effect of myofiber regeneration. This system should be of use for testing methods of alleviating the myofiber degeneration which is common to mdx and DMD.

Animals↗
PubMed1991

Invited review: myoblast transfer: a possible therapy for inherited myopathies?

A potential therapeutic strategy for genetic diseases is to alter the genetic constitution of the affected tissues by means of grafts of normal precursor or stem cells. Over several years, evidence has accumulated to suggest that primary diseases of skeletal muscle, such as Duchenne muscular dystrophy, may be susceptible to this approach. This review makes a critical examination of such background evidence, and also of more recent data directly addressing the concept of therapy by means of grafts of normal myogenic cells. It is concluded that the data establish the principle that such grafts effect an alteration of the genetic constitution and phenotype of skeletal muscle and, therefore, might be used to alleviate recessively inherited myopathies. Several obstacles to the therapeutic application of this method to human disease are also identified; these seem to be problems of a technical nature rather than of basic principle, and none appears insuperable.

Animals↗
PubMed1991

Localization of donor nuclei in skeletal muscle grafts by in situ hybridization to a cDNA probe.

The development of therapies, based upon implantation of normal muscle cell precursors, for the treatment of skeletal muscle diseases such as Duchenne Muscular Dystrophy is in its infancy. Detailed analysis of the genetic and phenotypic contribution made by donor myoblasts to the regenerated muscle is critical. Using non-radioactive in situ hybridization of a Y chromosome-specific DNA probe to sections of muscle, we have localized the position of male donor nuclei within female host muscles after myoblast implantation. These results were compared with the distribution of immunocytochemically-localized dystrophin and the expression of donor-specific glucose phosphate isomerase by isoelectric-focussing. We found consistent male-specific nuclear hybridization and a close spatial relationship between the distribution of male donor nuclei and dystrophin-positive muscle fibres within female, dystrophin-negative host muscles. This approach will be useful in the further analysis of myoblast implantation experiments.

Animals↗