tacrolimus and Muscular-Dystrophy--Animal

The clinical trials of myoblast transplantation in Duchenne Muscular Dystrophy (DMD) patients produced disappointing results. The main problems responsible for these poor results have since then been identified and partially resolved. One of them was related to the use of an inadequate immunosuppression and, since then, immunosuppression with FK506 has permitted successful myoblast transplantation not only in mice but also in monkeys. The requirement for a sustained immunosuppression may be eventually avoided by developing a state of tolerance to the allogeneic cells or by autologous transplantation of genetically corrected myoblasts or stem cells. The rapid death of 75-80% of the injected myoblasts during the first five days has also contributed to the limited success of the early trials. This death was due to an inflammatory reaction and has been compensated in animal experiments by the injection of a larger number of cells (30 millions per cc). Finally, the myoblasts migrated only 0.5 mm away from their site of injection. This problem is currently compensated in animal experiments by injecting the myoblasts at every mm. The number of injections required may eventually be reduced by transfecting myoblasts with one or several metalloproteinase genes. The very good results obtained during the last two years in primates permit us to undertake a new phase I clinical trial to verify that myoblast transplantation can lead to the formation of muscle fibers expressing normal dystrophin in muscles of DMD patients.

Topics: Animals; Cell Transplantation; Cellular Senescence; Clinical Trials as Topic; Graft Enhancement, Immunologic; Graft Survival; Haplorhini; Hematopoietic Stem Cell Transplantation; Humans; Immune Tolerance; Immunosuppression Therapy; Immunosuppressive Agents; Metalloendopeptidases; Mice; Mice, Inbred mdx; Minor Histocompatibility Antigens; Muscle, Skeletal; Muscular Diseases; Muscular Dystrophies; Muscular Dystrophy, Animal; Tacrolimus; Transfection; Treatment Failure

Sliced male C57Bl/10Sn (H2-b) donor muscles were grafted into the female histocompatible muscles of untreated, FK506-treated, and T-cell depleted (with or without thymic tolerization) dystrophic (mdx; H2-b) and normal (C57Bl/10Sn; H2-b) hosts, and also into histoincompatible normal (Balb/c; H2-d) hosts. The fate of male donor nuclei was monitored on tissue sections by in situ hybridization with a Y-chromosome specific probe. The results demonstrate that the dystrophic environment is more conducive than normal muscle to donor myoblast migration, with the distance moved being threefold greater at 12 weeks in dystrophic hosts. T-cell depletion was significantly more effective than FK506 treatment at enhancing donor myoblast emigration in both histocompatible and histoincompatible hosts at 3 weeks. Furthermore, the effects of T-cell depletion were sustained in histoincompatible hosts at 12 weeks. These data endorse the use of host T-cell depletion as a promising long-term strategy to improve myoblast transfer therapy (MTT) in the clinical situation.

Topics: Animals; Cell Movement; Female; Immunosuppression Therapy; Lymphocyte Depletion; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred mdx; Muscle, Skeletal; Muscular Dystrophy, Animal; T-Lymphocytes; Tacrolimus; Transplantation Chimera; Transplantation, Homologous; Y Chromosome

Dog myoblasts obtained from muscle biopsies were infected in vitro with a defective retroviral vector containing a cytoplasmic beta-galactosidase (beta-Gal) gene. These myoblasts were initially transplanted in the irradiated muscles of SCID mice and beta-Gal positive muscle fibers were observed. beta-Gal myoblasts were also transplanted back either in the donor dogs (autotransplantation model) or in unrelated recipient dogs (allotransplantation model). Following these myoblast injections, a rapid inflammatory reaction developed within the muscle as indicated by an expression of P-selectin and of pro-inflammatory cytokine mRNAs (interleukin 6 (IL-6) and transforming growth factor beta (TGF-beta), and by a neutrophil infiltration. Following either auto- or allotransplantation in inadequately or non-immunosuppressed dogs, a specific immune reaction also developed within 2 weeks as indicated by the infiltration of CD4+ and of CD8+ lymphocytes, the increased expression of IL-10 and granzyme B mRNAs and the presence of antibodies reacting with the injected cells. Some dogs were immunosuppressed with several combinations of FK506, cyclosporine (CsA) and RS-61443. In dogs immunosuppressed with CsA combined with RS-61443, only a few myoblasts and myotubes expressing beta-Gal were observed 1-2 weeks after the transplantation, but no muscle fibers expressing beta-Gal were observed after 4 weeks, and antibodies against the injected cells were formed. In dogs immunosuppressed with FK506 alone, although no antibodies against the injected cells were produced, there were no small cells and no muscle fibers expressing beta-Gal 1 month after the transplantation. However, FK506 triggered diarrhea and vomiting in dogs. When the dogs were immunosuppressed with FK506 combined with CsA and RS-61443, muscle fibers expressing beta-Gal were present 4 weeks after the transplantation and no antibodies reacting with donor myoblasts were detected. These results indicate that the combination of three immunosuppressive agents (i.e., FK506, CsA and RS-61443) is effective in controlling the specific immune reactions following myoblast transplantation in dogs and they underline that the outcome of myoblast transplantation is dependent in part on an adequate immunosuppression. These results obtained here in normal dogs may justify myoblast transplantation in dystrophic dogs despite the side effects of FK506.

Topics: Animals; beta-Galactosidase; Biopsy; Cell Transplantation; Cells, Cultured; Cyclosporine; Dogs; Drug Therapy, Combination; Immunosuppressive Agents; Mice; Mice, SCID; Muscles; Muscular Dystrophy, Animal; Mycophenolic Acid; Tacrolimus; Transplantation, Heterologous; Transplantation, Homologous

Myoblast transplantation is a potential treatment for Duchenne muscular dystrophy. One of the problems possibly responsible for the limited success of clinical trials is the rapid death of the myoblasts after transplantation. To investigate this problem, myoblasts expressing beta-galactosidase were injected in the tibialis anterior muscles of mice. Beta-galactosidase activity was reduced by 74.7% after 3 days. Myoblast death observed at 3 days was reduced to 57.2% when the hosts were irradiated. This result suggested that host cells were contributing to this phenomenon. Transplantation in SCID and FK506-treated mice did not reduce cell death, indicating that mortality was not due to an acute specific reaction. In contrast, administration of the anti-LFA-1 (TIB-213) mAb markedly reduced myoblast death at 3 days without altering leukocyte tissue infiltration. We postulated that neutrophils were mediating myoblast mortality by an LFA-1-dependent mechanism. To test this hypothesis, IL-1beta-activated myoblasts were loaded with 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethylester) (DCFH), a marker for oxidative stress. Addition of neutrophils and zymosan-activated serum resulted in a time-dependent DCFH fluorescence; this neutrophil-induced oxidation was considerably inhibited by TIB-213. These results indicate that an effective control of the inflammatory reaction will be necessary for any new clinical trials of myoblast transplantation and suggest that neutrophil-mediated myoblast injury occurs by an LFA-1-dependent pathway.

Topics: Animals; Anti-Inflammatory Agents; Anti-Inflammatory Agents, Non-Steroidal; Antibodies, Monoclonal; Apoptosis; beta-Galactosidase; Biomarkers; Cell Adhesion; Cell Death; Cell Line, Transformed; Fluoresceins; Genes, Reporter; Graft Rejection; Immunosuppressive Agents; Inflammation; Interleukin-1; Lymphocyte Function-Associated Antigen-1; Macrophage-1 Antigen; Methylprednisolone; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred mdx; Mice, SCID; Muscle, Skeletal; Muscular Dystrophy, Animal; Naproxen; Necrosis; Neutrophils; Oxidative Stress; Peroxidase; Piroxicam; Radiation Chimera; Stem Cell Transplantation; Tacrolimus; Whole-Body Irradiation; Zymosan

Laminin-2 is a component of skeletal and cardiac basal lamina expressed in normal mouse and human. Laminin alpha2 chain (LAMA2), however, is absent from muscles of some congenital muscular dystrophy patients and the dystrophia muscularis (dy/dy) mouse model. LAMA2 restoration was investigated following cell transplantation in vivo in dy/dy mouse. Allogeneic primary muscle cell cultures expressing the beta-galactosidase transgene under control of a muscular promoter, or histocompatible primary muscle cell cultures, were transplanted into dy/dy mouse muscles. FK506 immunosuppression was used in noncompatible models. All transplanted animals expressed LAMA2 in these immunologically-controlled models, and the degrees of LAMA2 restoration were shown to depend on the age of the animal at transplantation, on muscle pretreatment, and on duration time after transplantation in some cases. LAMA2 did not always colocalize with new or hybrid muscle fibers formed by the fusion of donor myoblasts. LAMA2 deposition around muscle fibers was often segmental and seemed to radiate from the center to the periphery of the injection site. Allogeneic conditionally immortalized pure myogenic cells expressing the beta-galactosidase transgene were characterized in vitro and in vivo. When injected into FK506-immunosuppressed dy/dy mice, these cells formed new or hybrid muscle fibers but essentially did not express LAMA2 in vivo. These data show that partial LAMA2 restoration is achieved in LAMA2-deficient dy/dy mouse by primary muscle cell culture transplantation. However, not all myoblasts, or myoblasts alone, or the muscle fibers they form are capable of LAMA2 secretion and deposition in vivo.

Topics: Age Factors; Animals; beta-Galactosidase; Cell Transplantation; Cells, Cultured; Elapid Venoms; Female; Gamma Rays; Humans; Immunosuppressive Agents; Laminin; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophy, Animal; Tacrolimus

Adenovirus (AV)-mediated gene transfer into skeletal muscles of adult immune-competent animals has been limited by the fact that a cell-mediated immune attack of the host against transduced muscle fibers prevented efficient long-term transgene expression. More recently, various immunomodulating strategies have been shown to improve the longevity of transgene expression after AV-mediated gene transfer. In this study we treated adult dystrophic (mdx) mice with daily subcutaneous injections of the immunosuppressive drug FK506 (tacrolimus) over 5, 10, 30 and 60 days after AV-mediated dystrophin gene transfer and compared the transduction level with saline-injected mdx controls. We show that daily FK506 treatment after AV-mediated dystrophin gene transfer into adult mdx muscle results in the maintenance of the initial transgene expression for at least 2 months, even when FK506 treatment was discontinued after 1 month. This is in keeping with the marked reduction of inflammatory infiltrates and the reduced activation level (inducible nitric oxide synthase) of macrophages in adenoviral recombinant (AVR)-injected muscles of FK506-treated animals. Moreover, we find that FK506 efficiently suppresses the humoral immune response against both the vector proteins and the transgene protein product (dystrophin). Furthermore, we demonstrate that continuous FK506 treatment over 30 days significantly improves the efficiency of gene transfer when the same vector is readministered to an animal which had been transduced 20 days earlier. In conclusion, the data suggest that sensitization by the initial antigenic load of the AVR application plays a pivotal role in triggering the humoral and cellular immune response of the host, which can be significantly counteracted by relatively short-term immunosuppressive treatment. These findings have important implications for the design of future human trials for gene replacement therapy in Duchenne muscular dystrophy.

Topics: Adenoviridae; Animals; Antibodies; Dystrophin; Gene Expression; Gene Transfer Techniques; Humans; Immunocompetence; Immunosuppression Therapy; Mice; Mice, Inbred mdx; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophy, Animal; Tacrolimus; Time Factors; Transgenes

Topics: Animals; Biopsy; Cell Transplantation; Gene Transfer Techniques; Immunosuppression Therapy; Macaca mulatta; Muscle Fibers, Skeletal; Muscles; Muscular Dystrophy, Animal; Stem Cell Transplantation; Tacrolimus; Transplantation, Homologous

Topics: Animals; Animals, Newborn; Biopsy; Cell Transplantation; Cells, Cultured; DNA Primers; Dystrophin; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred mdx; Muscles; Muscular Dystrophy, Animal; Polymerase Chain Reaction; RNA; RNA, Messenger; Tacrolimus; Transplantation, Heterologous; Transplantation, Homologous