Macrophage function in the elderly and impact on injury repair and cancer.

Older age is associated with deteriorating health, including escalating risk of diseases such as cancer, and a diminished ability to repair following injury. This rise in age-related diseases/co-morbidities is associated with changes to immune function, including in myeloid cells, and is related to immunosenescence. Immunosenescence reflects age-related changes associated with immune dysfunction and is accompanied by low-grade chronic inflammation or inflammageing. This is characterised by increased levels of circulating pro-inflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1ß and IL-6. However, in healthy ageing, there is a concomitant age-related escalation in anti-inflammatory cytokines such as transforming growth factor-ß1 (TGF-ß1) and IL-10, which may overcompensate to regulate the pro-inflammatory state. Key inflammatory cells, macrophages, play a role in cancer development and injury repair in young hosts, and we propose that their role in ageing in these scenarios may be more profound. Imbalanced pro- and anti-inflammatory factors during ageing may also have a significant influence on macrophage function and further impact the severity of age-related diseases in which macrophages are known to play a key role. In this brief review we summarise studies describing changes to inflammatory function of macrophages (from various tissues and across sexes) during healthy ageing. We also describe age-related diseases/co-morbidities where macrophages are known to play a key role, focussed on injury repair processes and cancer, plus comment briefly on strategies to correct for these age-related changes.

Effects of loaded voluntary wheel exercise on performance and muscle hypertrophy in young and old male C57Bl/6J mice.

This study compared the capacity of young and old male C57Bl/6J mice to exercise with increasing resistance over 10 weeks, and its impact on muscle mass. Young mice (aged 15-25 weeks) were subjected to low (LR) and high (HR) resistance exercise, whereas only LR was used for old mice (107-117 weeks). Weekly patterns of voluntary wheel activity, food consumption and body weights were measured. Running patterns changed over time and with age, with two peaks of activity detected for young, but only one for old mice: speed and distance run was also less for old mice. The mass for six limb muscles was measured at the end of the experiment. The most pronounced increase in mass in response to exercise was for the soleus in young and old mice, and also quadriceps and gastrocnemius in young mice. Soleus and quadriceps muscles were analyzed histologically for myofiber number and size. A striking feature was the many small myofibers in response to exercise in young (but not old) soleus, whereas these were not present after exercise in young or old quadriceps. Overall, there was a striking difference in response to exercise between muscles and this was influenced by age.

Lifelong exercise and locally produced insulin-like growth factor-1 (IGF-1) have a modest influence on reducing age-related muscle wasting in mice.

The age-related loss of skeletal muscle mass and function is termed sarcopenia and has been attributed to a decline in concentrations of insulin-like growth factor-1 (IGF-1). We hypothesized that constitutively expressed IGF-1 within skeletal muscles with or without exercise would prevent sarcopenia. Male transgenic mice that overexpress IGF-1 Ea in skeletal muscles were compared with wild-type littermates. Four-month-old mice were assigned to be sedentary, or had access to free-running wheels, until 18 or 28 months of age. In wild-type mice, the mass of the quadriceps muscles was reduced at 28 months and exercise prevented such loss, without affecting the diameter of myofibers. Conversely, increased IGF-1 alone was ineffective, whereas the combination of exercise and IGF-1 was additive in maintaining the diameter of myofibers in the quadriceps muscles. For other muscles, the combination of IGF-1 and exercise was variable and either increased or decreased the mass at 18 months of age, but was ineffective thereafter. Despite an increase in the diameter of myofibers, grip strength was not improved. In conclusion, our data show that exercise and IGF-1 have a modest effect on reducing aged-related wasting of skeletal muscle, but that there is no improvement in muscle function when assessed by grip strength.

Impact of fasting on the rhythmic expression of myogenic and metabolic factors in skeletal muscle of adult mice.

Circadian rhythms and metabolism are tightly integrated, and rhythmic expression of metabolic factors is common in homeostatic processes. We measured the temporal changes in the expression of myogenic regulatory factors and expression and activity level of molecules involved in protein metabolism in skeletal muscles and livers in mice and examined the impact of fasting. Tissues were collected over 24 h (at zeitgeber times ZT1, ZT5, ZT9, ZT13, ZT17, ZT21, and ZT1 the following day) from adult male C57Bl/6J mice that had been either freely fed or fasted for 24 h. In skeletal muscle, there was a robust rise in the mRNA expression of the myogenic regulatory factors MyoD and myogenin during dark hours which was strongly suppressed by fasting. Circadian pattern was observed for mRNA of MuRF1, Akt1, and ribosomal protein S6 in muscles in fed and fasted mice and for Fbxo32 in fed mice. Activity (phosphorylation) levels of Akt(Ser473) displayed temporal regulation in fasted (but not fed) mice and were high at ZT9. Fasting caused significant reductions in phosphorylation for both Akt and S6 in muscles, indicative of inactivation. Hepatic phosphorylated Akt(Ser473) and S6(Ser235/236) proteins did not exhibit daily rhythms. Fasting significantly reduced hepatic Akt(473) phosphorylation compared with fed levels, although (unlike in muscle) it did not affect S6(Ser235/236) phosphorylation. This in vivo circadian study addresses for the first time the signaling activities of key molecules related to protein turnover and their possible cross-regulation of expression of genes related to protein degradation.

Blockade of TNF in vivo using cV1q antibody reduces contractile dysfunction of skeletal muscle in response to eccentric exercise in dystrophic mdx and normal mice.

This study evaluated the contribution of the pro-inflammatory cytokine, tumour necrosis factor (TNF) to the severity of exercise-induced muscle damage and subsequent myofibre necrosis in mdx mice. Adult mdx and non-dystrophic C57 mice were treated with the mouse-specific TNF antibody cV1q before undergoing a damaging eccentric contraction protocol performed in vivo on a custom built mouse dynamometer. Muscle damage was quantified by (i) contractile dysfunction (initial torque deficit) immediately after the protocol, (ii) subsequent myofibre necrosis 48 h later. Blockade of TNF using cV1q significantly reduced contractile dysfunction in mdx and C57 mice compared with mice injected with the negative control antibody (cVaM) and un-treated mice. Furthermore, cV1q treatment significantly reduced myofibre necrosis in mdx mice. This in vivo evidence that cV1q reduces the TNF-mediated adverse response to exercise-induced muscle damage supports the use of targeted anti-TNF treatments to reduce the severity of the functional deficit and dystropathology in DMD.

Two-tiered hypotheses for Duchenne muscular dystrophy.

New approaches to understanding and designing treatments for Duchenne muscular dystrophy (DMD) may emerge from two hypotheses outlined here. The proposal that growing skeletal muscle is more susceptible to necrosis than adult muscle raises the possibility that less intensive treatments may be sufficient to protect muscles during the adult phase. The second proposal is that a different balance of cell and molecular events contributes to acute necrosis (e.g. resulting from exercise) compared with chronic damage of dystrophic muscle. Validation of such differences presents the potential for more specific targeting of drugs or nutritional interventions to events downstream of the dystrophin deficiency. A deeper understanding of the events arising as an early consequence of dystrophin deficiency in these two situations may strengthen approaches to therapy for DMD designed to improve muscle function and the quality of life.

Age influences the early events of skeletal muscle regeneration: studies of whole muscle grafts transplanted between young (8 weeks) and old (13-21 months) mice.

Injured skeletal muscle generally regenerates less efficiently with age, but little is understood about the effects of ageing on the very early inflammatory and neovascular events in the muscle repair process. This study used a total of 174 whole muscle grafts transplanted within and between young and old mice to analyse the effects of ageing on the early inflammatory response in two strains of mice (BALB/c and SJL/J). There was a very slight delay in the early inflammatory response, and in the appearance of myotubes at day 4 in BALB/c muscle grafted into an old host environment (implicating systemic events). In SJL/J mice, the initial speed of the inflammatory response was slightly delayed with old muscle grafts regardless of host age (implicating muscle-derived factors), while an old host environment transiently affected myogenesis (myotube formation). The slight delays in inflammatory and neovascular responses in old mice did not dramatically impact on the overall formation of new muscle. The neovascular response to injured young and old muscle tissue was further analysed using the corneal micropocket assay. This showed a very clear 1-2 day delay in angiogenesis induced by old versus young BALB/c muscle tissue implanted into the young rat cornea, indicating that new blood vessel formation is at least partly determined by muscle-derived factors. Taken together these results indicate that, while there are slight age-associated delays in inflammation and neovascularisation in response to injured muscle, there is no detrimental effect on myogenesis in the mouse model used in this study.

Duchenne muscular dystrophy: focus on pharmaceutical and nutritional interventions.

Duchenne muscular dystrophy is a lethal X-linked muscle disease resulting from a defect in the muscle membrane protein dystrophin. The absence of dystrophin leads to muscle membrane fragility, muscle death (necrosis) and eventual replacement of skeletal muscle by fat and fibrous connective tissue. Extensive muscle wasting and respiratory failure results in premature death often by the early 20s. This short review evaluates drug and nutritional interventions designed to reduce the severity of muscular dystrophy, while awaiting the outcome of research into therapies to correct the fundamental gene defect. Combinations of dietary supplementation with amino-acids such as creatine, specific anti-inflammatory drugs and perhaps drugs that target ion channels might have immediate realistic clinical benefits although rigorous research is required to determine optimal combinations of such interventions.

Rskalpha-actin/hIGF-1 transgenic mice with increased IGF-I in skeletal muscle and blood: impact on regeneration, denervation and muscular dystrophy.

Human IGF-I was over-expressed in skeletal muscles of C57/BL6xCBA mice under the control of the rat skeletal alpha-actin gene promoter. RT-PCR verified expression of the transgene in skeletal muscle but not in the liver of 1- and 21-day old heterozygote transgenic mice. The concentration of endogenous mouse IGF-I, measured by an immunoassay which does not detect human IGF-I, was not significantly different between transgenic mice and wild-type littermates (9.5 +/- 0.8 and 13.3 +/- 1.9 ng/g in muscle; 158.3 +/- 18.6 and 132.9 +/- 33.1 ng/ml in plasma, respectively). In contrast, quantitation with antibodies to human IGF-I showed an increase in IGF-I of about 100 ng/ml in plasma and 150 ng/g in muscle of transgenic mice at 6 months of age. Transgenic males, compared to their age matched wild-type littermates, had a significantly higher body weight (38.6 +/- 0.53 g vs. 35.8 +/- 0.64 g at 6 months of age; P < 0.001), dry fat-free carcass mass (5.51 +/- 0.085 vs. 5.08 +/- 0.092 g; P < 0.001) and myofibrillar protein mass (1.62 +/- 0.045 vs. 1.49 +/- 0.048 g; P < 0.05), although the fractional content of fat in the carcass was lower (167 +/- 7.0 vs. 197 +/- 7.7 g/kg wet weight) in transgenic animals. There was no evidence of muscle hypertrophy and no change in the proportion of slow type I myofibres in the limb muscles of Rskalpha-actin/hIGF-I transgenic mice at 3 or 6 months of age. Phenotypic changes in Rskalpha-actin/hIGF-I mice are likely to be due to systemic as well as autocrine/paracrine effects of overproduction of IGF-I due to expression of the human IGF-I transgene. The effect of muscle specific over-expression of Rskalpha-actin/hIGF-I transgene was tested on: (i) muscle regeneration in auto-transplanted whole muscle grafts; (ii) myofibre atrophy following sciatic nerve transection; and (iii) sarolemmal damage and myofibre necrosis in dystrophic mdx muscle. No beneficial effect of muscle specific over-expression of Rskalpha-actin/hIGF-I transgene was seen in these three experimental models.

Strength at the extracellular matrix-muscle interface.

Mechanical force is generated within skeletal muscle cells by contraction of specialized myofibrillar proteins. This paper explores how the contractile force generated at the sarcomeres within an individual muscle fiber is transferred through the connective tissue to move the bones. The initial key point for transfer of the contractile force is the muscle cell membrane (sarcolemma) where force is transferred laterally to the basement membrane (specialized extracellular matrix rich in laminins) to be integrated within the connective tissue (rich in collagens) before transmission to the tendons. Connections between (1) key molecules outside the myofiber in the basement membrane to (2) molecules within the sarcolemma of the myofiber and (3) the internal cytoplasmic structures of the cytoskeleton and sarcomeres are evaluated. Disturbances to many components of this complex interactive system adversely affect skeletal muscle strength and integrity, and can result in severe muscle diseases. The mechanical aspects of these crucial linkages are discussed, with particular reference to defects in laminin-alpha2 and integrin-alpha7. Novel interventions to potentially increase muscle strength and reduce myofiber damage are mentioned, and these are also highly relevant to muscle diseases and aging muscle.

A novel role for non-muscle gamma-actin in skeletal muscle sarcomere assembly.

Existing models describing sarcomere assembly have arisen primarily from studies using cardiac muscle. In contrast to cardiac muscle, skeletal muscle differentiation is characterised by dramatic changes in protein expression, from non-muscle to muscle-specific isoforms before organisation of the sarcomeres. Consequently, little is understood of the potential influence of non-muscle cytoskeletal proteins on skeletal sarcomere assembly. To address this issue, transfectant (gamma33-B1) and control mouse C2 myoblasts were differentiated to form myotubes, and various stages of skeletal sarcomere assembly were studied. Organisation of non-muscle gamma-actin and co-localisation with sarcomeric alpha-actinin, an early marker of sarcomere assembly and a major component of Z lines, was noted. gamma-Actin was also identified in young myotubes with developing sarcomeric myofibrils in regenerating adult mouse muscle. Localisation of gamma-actin in a different area of the myotube to the muscle-specific sarcomeric alpha-actin also indicated a distinct role for gamma-actin. The effects of aberrant gamma-actin expression in other myoblast lines, further suggested a sequestering role for gamma-actin. These observations make the novel suggestion that non-muscle gamma-actin plays a role in skeletal sarcomere assembly both in vitro and in vivo. Consequently, a modified model is proposed which describes the role of gamma-actin in skeletal sarcomere assembly.

Evans Blue Dye as an in vivo marker of myofibre damage: optimising parameters for detecting initial myofibre membrane permeability.

Evans Blue Dye (EBD) is widely used to study cellular membrane permeability and has recently been utilised in mdx mice to identify permeable skeletal myofibres that have become damaged as a result of muscular dystrophy. EBD has the potential to be a useful vital stain of myofibre permeability in other models of skeletal muscle injury and membrane-associated fragility. The parameters for its use for such purposes were optimised in the present study, of particular interest is the use of EBD to identify the onset of muscle damage. This study compared intravenous vs. intraperitoneal injection; tissue fixation; volume of EBD; time of availability in tissue; and persistence after injection in mdx mice (with endogenous muscle damage) and control mice. Satisfactory labelling of permeable myofibres was seen in frozen sections viewed with fluorescence microscopy when intraperitoneal injection of a 1% EBD solution injected at 1% volume relative to body mass was administered between 16 and 24 h prior to tissue sampling. EBD labelling was then assessed in three mouse models of experimental injury and repair-cut injury, whole muscle grafts, and exercise-induced muscle damage. These experiments demonstrated that (i) following a cut injury across myofibres, EBD penetrated up to 150 microm from the injury site over a 20-h period; (ii) EBD was present throughout myofibres of avascular whole muscle graft by one day after transplantation; and (iii) damaged myofibres were detected within 20 min after controlled lengthening-contraction exercise. This simple and inexpensive technique has sensitivity for the detection of increased myofibre permeability and/or sublethal damage that has advantages over other traditional histological techniques at the light microscopy level.

The absence of MyoD in regenerating skeletal muscle affects the expression pattern of basement membrane, interstitial matrix and integrin molecules that is consistent with delayed myotube formation.

MyoD is a member of a skeletal muscle specific family of transcription factors which directs the events of myogenesis during development and regeneration. Muscle cells that lack MyoD show delayed fusion in vivo and in vitro and defects have been observed in vitro in the attachment of MyoD(-/-) myoblasts to complex substrates such as Matrigel. Since interactions with the extracellular matrix (ECM) are important during myoblast fusion (i. e. myotube formation), it was hypothesised that expression of ECM molecules or their receptors may be altered in MyoD(-/-) muscle. The production of basement membrane molecules such as collagen type IV and several laminins, the interstitial molecules fibronectin and tenascin-C, and the cell surface molecules integrin alpha5 and alpha6 were quantitated in vitro using ELISA on cultured cells from MyoD(-/-) and wild type mice. Differences were observed in the production of fibronectin, tenascin-C, collagen type IV, laminin-1 and integrin alpha5 between control and MyoD(-/-) myotubes in vitro. This corresponded with delayed fusion of myoblasts in MyoD(-/-) cultures. On the basis of these findings with respect to matrix expression in vitro, fluorescent immunohistochemistry was carried out on adult whole muscle autografts to examine whether the expression of these molecules, as well as integrin alpha7, was altered in the complex in vivo environment. Some minor differences in expression patterns were observed in MyoD(-/-) as compared to normal BALB/c autografts. The overall expression of matrix components was consistent with the delayed onset of myotube formation. These results suggest that the delay in myotube formation in MyoD(-/-) muscle is not a direct result of altered expression of the matrix molecules collagen type IV, laminins, fibronectin, tenascin-C, and integrins alpha5, alpha6 or alpha7.

Leukaemia inhibitory factor increases myoblast replication and survival and affects extracellular matrix production: combined in vivo and in vitro studies in post-natal skeletal muscle.

Leukaemia inhibitory factor (LIF) has been reported to specifically enhance myoblast proliferation in vitro and increase the number and size of myotubes in regenerating skeletal muscle in vivo. The present study specifically tests the effect of LIF on myoblast replication in vivo. Administration of exogenous LIF by slow release alginate gels in vivo sustained the level of myoblast proliferation at 2 days in regenerating crush-injured muscle. Since the extracellular matrix (ECM) plays an important role in regulating the effects of many growth factors, the hypothesis was tested, both in vivo and in vitro, that some of the beneficial effects of LIF are mediated by modulation of the ECM. The effects of LIF in vivo on the amount and localisation of the ECM molecules, fibronectin, tenascin-C, collagen type IV and laminin were assessed by immunohistochemistry on regenerating skeletal muscle but no influence of LIF on ECM composition was observed. In tissue culture, LIF increased BALB/c myoblast proliferation at day 3 on culture dishes coated with Matrigel and also increased the viability in vitro of BALB/c myoblasts grown under suboptimal conditions. Quantitation of the ECM produced by cultures (enzyme-linked immunosorbent assay) showed that LIF affected the amount of fibronectin, tenascin-C, collagen type IV and laminin produced by fusing myoblasts. No significant affect of LIF was seen on myotube formation either in vitro or in vivo. These combined in vitro and in vivo studies show an effect of LIF on ECM production in vitro, on myoblast survival and on in vivo myoblast replication.

The role of tumor necrosis factor-alpha (TNF-alpha) in skeletal muscle regeneration. Studies in TNF-alpha(-/-) and TNF-alpha(-/-)/LT-alpha(-/-) mice.

The role of tumor necrosis factor-alpha (TNF-alpha), an important mediator of the inflammatory response after injury, was investigated in regenerating skeletal muscle. The pattern of expression of TNF-alpha during muscle regeneration was examined by immunohistochemistry in tissue sections of crush-injured or transplanted muscle autografts and in primary cultures of adult skeletal muscle. TNF-alpha was highly expressed in injured myofibers, inflammatory cells, endothelial cells, fibroblasts, and mast cells. Myoblasts and myotubes also expressed TNF-alpha in primary muscle cultures and tissue sections. The essential role of TNF-alpha and its homologue lymphotoxin-alpha (LT-alpha) during muscle regeneration was assessed by basic histology in TNF-alpha(-/-) and TNF-alpha(-/-)/LT-alpha(-/-) mice. No difference was apparent in the onset or pattern of muscle regeneration (i.e., inflammatory response, activation and fusion of myoblasts) between the two strains of null mice or between nulls and normal control mice. However, both strains of null mice appeared more prone to bystander damage of host muscle and regeneration distant from the site of injury/transplantation. Although expression of TNF-alpha may play an important role in muscle regeneration, the studies in the null mice show that redundancy within the cytokine system (or some other response) can effectively compensate for the absence of TNF-alpha in vivo.

Absence of MyoD increases donor myoblast migration into host muscle.

Donor myoblast migration is a major limiting factor in the success of myoblast transfer therapy, a potential treatment for Duchenne muscular dystrophy. A possible strategy to promote the migration of donor myoblasts into host muscle is to enhance their proliferation and delay their fusion, two properties that are major characteristics of myoblasts in regenerating skeletal muscle in MyoD null (-/-) mice. Here we investigate whether the migration of MyoD (-/-) donor myoblasts into host muscle is enhanced in vivo. Sliced muscle grafts from male MyoD (-/-) or normal control (Balb/c) mice were transplanted into the muscles of female normal (Balb/c) host mice. Muscles were sampled at 1, 3, and 12 weeks after grafting, and the fate of male donor myoblasts within female host muscles determined by in situ hybridization with the mouse Y-chromosome-specific Y-1 probe. MyoD (-/-) donor myoblasts migrated into host muscle continuously over 1, 3, and 12 weeks after grafting, in contrast with Balb/c donor myoblasts, whose overall numbers and migratory distances did not increase significantly after 1 week. These results strongly support a role for elevated donor myoblast proliferation and/or their delayed fusion in enhancing migration into host muscle in vivo, and endorse the use of either genetically engineered donor myoblasts, or the administration of exogenous myoblast mitogens to improve donor myoblast migration in myoblast transfer therapy.

Absence of desmin slightly prolongs myoblast proliferation and delays fusion in vivo in regenerating grafts of skeletal muscle.

The expression of desmin, a muscle-specific intermediate filament protein, is upregulated during skeletal myogenesis, but its role in the myogenic process is unclear. Postnatal skeletal muscle regeneration occurs to completion in desmin null (-/-) mice, however, only late time points (i.e., days 7 and 21) in the myogenic process have been examined. This study observes the early events in skeletal muscle regeneration (i.e., from 3 days) in desmin (-/-) mice. Whole muscle autografts were performed in desmin (-/-) and control normal (Balb/c) mice. Muscle samples were taken on days 3, 5, 6, 7, 8, 9 and 11 after transplantation, and regeneration was assessed by graft morphology, patterns of cell proliferation and quantitation of myotube numbers. At day 5 myotube formation was delayed in the desmin (-/-) grafts compared to the normal controls. Immunohistochemical analysis of proliferating cell nuclear antigen demonstrated a very high proportion of proliferating cells in the periphery of desmin (-/-) whole muscle grafts at day 5 compared to the controls, where mitosis in this area was negligible. This strongly indicates t hat myoblast proliferation is prolonged during postnatal myogenesis in the absence of desmin. By day 6 there was no marked morphological difference between desmin (-/-) and normal control whole muscle grafts, although the zonal pattern of myoblast replication was slightly delayed in the desmin (-/-) mice until day 8. These results indicate a slightly extended phase of myoblast proliferation with delayed fusion in vivo in mature regenerating desmin (-/-) skeletal muscle.

Complement and myoblast transfer therapy: donor myoblast survival is enhanced following depletion of host complement C3 using cobra venom factor, but not in the absence of C5.

Myoblast transfer therapy (MTT) is a potential cell therapy for myopathies such as Duchenne Muscular Dystrophy and involves the injection of cultured muscle precursor cells ('myoblasts') isolated from normal donor skeletal muscles into dystrophic host muscle. The failure of donor myoblast survival following MTT is widely accepted as being due to the immune response of the host. The role of complement as one possible mechanism for the initial, very rapid death of myoblasts following MTT was investigated. Donor male myoblasts were injected into the tibialis anterior (TA) muscles of female host mice that were: (i) untreated; (ii) depleted of C3 complement (24 h prior to MTT) using cobra venom factor (CVF); and/or (iii) deficient in C5 complement. Quantification of surviving male donor myoblast DNA was performed using the Y-chromosome specific (Y1) probe on slot blots for samples taken at 0 h, 1 h, 24 h, 1 week and 3 weeks after MTT. Peripheral depletion of C3 was confirmed using double immunodiffusion, and local depletion of C3 in host TA muscles was confirmed by immunostaining of muscle samples. Cobra venom factor treatment significantly increased the initial survival of donor myoblasts, but there was a marked decline in myoblast numbers after 1 h and little long-term benefit by 3 weeks. Strain specific variation in the immediate survival of donor male myoblasts following MTT in untreated C57BL/10Sn, DBA-1 and DBA-2 (C5-deficient) female hosts was observed. Cobra venom factor depletion of C3 increased initial donor male myoblast survival (approximately twofold at 0 h) in C57BL/10Sn and DBA-1 host mice and approximately threefold in DBA-2 hosts at 0 h and 1 h after MTT. The rapid and extensive number (approximately 90%) of donor male myoblasts in untreated DBA-2 mice (that lack C5) indicates that activation of the membrane attack complex (MAC) plays no role in this massive initial cell death. The observation that myoblast survival was increased in all mice treated with CVF suggests that CVF may indirectly enhance donor myoblast survival by a mechanism possibly involving activated C3 fragments.

Problems and solutions in myoblast transfer therapy.

Duchenne muscular dystrophy is a severe X-linked neuromuscular disease that affects approximately 1/3500 live male births in every human population, and is caused by a mutation in the gene that encodes the muscle protein dystrophin. The characterization and cloning of the dystrophin gene in 1987 was a major breakthrough and it was considered that simple replacement of the dystrophin gene would ameliorate the severe and progressive skeletal muscle wasting characteristic of Duchenne muscular dystrophy. After 20 years, attempts at replacing the dystrophin gene either experimentally or clinically have met with little success, but there have been many significant advances in understanding the factors that limit the delivery of a normal dystrophin gene into dystrophic host muscle. This review addresses the host immune response and donor myoblast changes underlying some of the major problems associated with myoblast-mediated dystrophin replacement, presents potential solutions, and outlines other novel therapeutic approaches.