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

Department of Anatomy and Human Biology, University of Western Australia, Crawley, Western Australia, Australia.
Immunology and Cell Biology (Impact Factor: 4.15). 07/2001; 79(3):231-9. DOI: 10.1046/j.1440-1711.2001.01006.x
Source: PubMed


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.

Download full-text


Available from: Stuart Hodgetts,
  • Source
    • "ICAM-1/CD54 and DAF/CD55 are members of the immunoglobulin superfamily, and are involved in immune cell adhesion during the inflammatory process and in protection against complement-mediated cell lysis. Their expression pattern in skeletal muscle is rather poorly documented, although there is growing interest in their use as therapeutic targets to reduce the inflammatory response resulting from ischemia/reperfusion (Huda et al., 2004; Mollnes et al., 2002) and to improve the rate of myoblast transfer for cell therapy of muscular dystrophies (Gasque et al., 1996; Hodgetts and Grounds, 2001; Merly et al., 1998). DAF is normally absent from mature mammalian muscle (Navenot et al., 1997), but is expressed at low levels on cultured human myoblasts (Gasque et al., 1996). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Coxsackieviruses A (CVA) are associated with several clinical manifestations such as aseptic meningitis and paralytic syndromes in humans. Most CVA are difficult-to-cultivate, which impedes their propagation and isolation from clinical material. Here, we tested the ability of cultivable (CVA-13, CVA-14), and difficult-to-cultivate (CVA-6, CVA-22) strains to infect primary cultures of skeletal muscle cells established from newborn mice. We found that such cultures sustained the multiplication of these CVA, as evidenced by the development of a cytopathic effect, already in the initial preparation or after passaging once. Cultures established for no more than 24h were sensitive to infection whereas older preparations were resistant. Using confocal microscopy after double-immunolabeling of the VP1 capsid protein and the muscle cell marker myosin, we demonstrated that only the myoblasts were infected, resulting in VP1 expression throughout their cytoplasm. Inoculation of infected cultures to suckling mice resulted in paralysis indicating that infection was productive. The nature of candidate receptors for virus entry in such cultures and the influence of cell culture conditions on the expression of these putative receptors are discussed. This work suggests that primary cultures of skeletal muscle cells could be used to propagate and isolate any CVA strain.
    Virus Research 02/2007; 123(1):30-9. DOI:10.1016/j.virusres.2006.07.016 · 2.32 Impact Factor
  • Source
    • "Our hypothesis to explain these observations was that inflammatory cells (neutrophils and macrophages) that rapidly infiltrate the muscle and interact with the donor cells (Skuk et al., 2002a), kill many donor cells by releasing toxic oxygen radicals and proteases (Guerette et al., 1997b). We have not observed morphologic evidence indicating that complement depletion in mice reduces the early cell death (Skuk and Tremblay, 1998), although some researchers have suggested a role of complement in this phenomenon (Hodgetts and Grounds, 2001). In vitro, human myoblasts spontaneously activated complement, but the abundant expression of complement-regulatory proteins in the membrane made them resistant to complement killing (Gasque et al., 1996). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The implantation of cultured myogenic cells into the body (myoblast transplantation) is an experimental strategy that is being explored for the potential treatment of myopathies. Its potential benefits should be: (1) to slow down or to stop muscle degeneration, and/or (2) to increase force in wasted muscles. For these objectives, myoblast transplantation may act by two actions: (1) genetic complementation (as a vehicle of normal genes in the case of genetic myopathies), and (2) increasing the myogenic pool of the muscle. During the last decade, myoblast transplantation seemed stagnant in a contradiction of experiments producing good results in mice, against the poor results of human trials. This contradiction was apparent, since the conditions used in mouse models were largely different from those used in dystrophic patients. Our monkey experiments demonstrated that promising results can be observed in large muscles of primates, but under conditions that differ from those previously used in patients. These conditions are: (1) an appropriate immunosuppression, and (2) a careful distribution of sufficient quantities of myoblasts into the recipient muscles. Most of the work on myoblast transplantation is addressed to improve this method by: (1) reducing or avoiding the toxicity of sustained immunosuppression, (2) favoring donor-myoblast migration into the recipient muscle, and (3) defining the factors implicated in the early donor-cell survival following intramuscular implantation. Other research subjects in this field are the potential use of pluripotent stem cells instead of satellite cells, and the potential delivery of the exogenous myogenic cells by the blood stream.
    Journal of Muscle Research and Cell Motility 02/2003; 24(4-6):285-300. DOI:10.1023/A:1025425823322 · 2.09 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Journal of Cellular and Molecular Medicine 11/2000; 5(1):33-47. · 4.01 Impact Factor
Show more