Myoblasts were grown from monkey muscle biopsies and infected in vitro with a defective retroviral vector containing a cytoplasmic beta-galactosidase (beta-gal) gene. These myoblasts were then transplanted to 14 different monkeys, 6 of which were immunosuppressed with FK506. Without immunosuppression, only a few myoblasts and myotubes expressing beta-gal were observed 1 week after the transplantation, but no cells expressing beta-gal were observed after 4 weeks. This result was attributed to immune responses since infiltration by CD4+ or CD8+ lymphocytes was abundant 1 week after transplantation but not after 4 weeks. The expression of interleukin 6 (IL-6), interleukin 2 (IL-2), granulocyte/macrophage colony stimulating factor (GM-CSF), transforming growth factor-beta (TGF-beta) and granzyme B mRNAs was increased in the myoblast-injected muscle indicating that the infiltrating lymphocytes were activated. Moreover, antibodies against the donor myoblasts were detected in 3 out of 6 cases. When the monkeys were immunosuppressed with FK506, muscle fibers expressing beta-galactosidase (beta-gal) were present 1, 4 and 12 weeks after the transplantation. There was neither significant infiltration by CD4 or CD8 lymphocytes, nor antibodies detected. The mRNA expression of most cytokines was significantly reduced as compared to the nonimmunosuppressed monkeys. These results indicate that FK506 is effective in controlling short-term immune reactions following myoblast transplantation in monkeys and suggest that it may prove useful for myoblast transplantation in Duchenne Muscular Dystrophy patients.
Seleno-glutathione peroxidase (GSHPx) is considered to be the major enzymatic activity in charge of removing excess cytosolic and mitochondrial H2O2 in most tissues including brain. Intracellular GSHPx activity is therefore hypothesized to be one important factor that contributes to minimize hydroxyl radical formation via Fenton-type reactions. An animal model was developed to challenge this hypothesis in vivo and evaluate the role of GSHPx in hydroperoxide metabolism and oxidative stress homeostasis. Three lines of transgenic mice, homozygous for the integration of 1 to 3 GSHPx transgene copies, have been generated. The transgene was placed under transcriptional control of a metallothionein promoter (hMT-IIA). This promoter was chosen because metallothionein expression, normally low in most tissues, can be induced by several inflammatory cytokines, protein kinase C activators, and stress agents including heavy metals. The data reported here provide information on the constitutive expression of GSHPx mRNA and enzyme in various brain regions of healthy untreated adult tg-MT-GPx mice. Northern and/or Western analysis indicated that transgenic GSHPx was expressed constitutively in all brain regions investigated in tg-MT-GPx-6 mice, including the cerebral cortex, brainstem, hippothalamus, cerebellum, substantia nigra, and striatum. Similar results were obtained with the two other transgenic lines, tg-MT-GPx-11 and -13. Depending on the brain region, the GSHPx immunoreactivity detected in tissue extracts with an immunoaffinity-purified polyclonal antibody was about 2- to 5-fold stronger in transgenic extracts than in their non-tg counterparts (western blots). In contrast, the corresponding increase in GSHPx activity measured in these extracts was smaller, for example, about 1.5-fold in transgenic mesencephalon. Immunocytochemical data indicated that GSHPx-like staining was distinctly more intense in transgenic midbrain brain sections than in corresponding non-tg sections. Interestingly, only a subset of the cells displayed higher density staining that most likely reflects increased amounts of GSHPx protein. This observation suggests that the stained cells, not yet identified, may have larger GSHPx activity increments than the cell-average increments measured in tissue extracts. Current work is in progress to determine whether transgenic GSHPx expression may be induced by inflammatory processes or perturbations of heavy metal metabolism.
Adenylate cyclase activity and its hormonal stimulation were measured in endometrial tissue, and sex steroid levels were quantified in uterine tissue collected from pregnant and estrous rabbits. The tissues from pregnant animals were separated into implantation (ES) and interimplantation (IES) sites. Adenylate cyclase activity was measured in broken cell preparations by enzymatic conversion of α-32P-adenosine triphosphate (ATP) into 32P-cyclic adenosine 3′, 5′-monophosphate using Mg2+-ATP as a substrate. The activity was measured with no addition (basal) and after stimulation with guanosine triphosphate (GTP), NaF, or increasing doses (1 nM to 100 μM) of isoproterenol (ISO) and prostaglandin E2 (PGE2). The presence of GTP was necessary to observe a stimulation by ISO and PGE2. During pregnancy, adenylate cyclase activity was reduced compared to activity at estrus on Day 6.5 (IES and ES) and on Day 9 (IES); however, it reached its highest level at ES (Day 9). The regulation of isoproterenol response followed a similar pattern. Dose responses to PGE2 were markedly affected by physiological status. The response was higher during pregnancy than at estrus, and response (percent of GTP), as well as sensitivity, was higher in IES than in ES on Day 6.5 and even greater on Day 9. The levels of estradiol (E2) were reduced during pregnancy, but comparable in ES and IES; however, progesterone (P) levels were reduced in ES, and the E2/P ratio was significantly higher (p<0.01) in ES (15 ± 1, 17 ± 2) than in IES (8 ± 1, 6 ± 0.8) on Days 6.5 and 9, respectively. Our results demonstrate that the presence of the embryo is associated with a local alteration in adenylate cyclase activity and sex steroid level at implantation sites.
Three Duchenne muscular dystrophy (DMD) patients received injections of myogenic cells obtained from skeletal muscle biopsies of normal donors. The cells (30 x 10 (6)) were injected in 1 cm3 of the tibialis anterior by 25 parallel injections. We performed similar patterns of saline injections in the contralateral muscles as controls. The patients received tacrolimus for immunosuppression. Muscle biopsies were performed at the injected sites 4 weeks later. We observed dystrophin-positive myofibers in the cell-grafted sites amounting to 9 (patient 1), 6.8 (patient 2), and 11% (patient 3). Since patients 1 and 2 had identified dystrophin-gene deletions these results were obtained using monoclonal antibodies specific to epitopes coded by the deleted exons. Donor dystrophin was absent in the control sites. Patient 3 had exon duplication and thus specific donor-dystrophin detection was not possible. However, there were fourfold more dystrophin-positive myofibers in the cell-grafted than in the control site. Donor-dystrophin transcripts were detected by RT-PCR (using primers reacting with a sequence int eh deleted exons) only in the cell-grafted sites in patients 1 and 2. Dystrophin transcripts were more abundant in the cell-grafted than in the control site in patient 3. Therefore, significant dystrophin expression can be obtained in teh skeletal muscles of DMD patients following specific conditions of cell delivery and immunosuppression.
A clinical trial was conducted to test a new protocol of normal muscle precursor cell (MPC) allotransplantation in skeletal muscles of patients with Duchenne muscular dystrophy (DMD). Cultured MPCs obtained from one of the patient's parents were implanted in 0.25 or 1 cm3 of a Tibialis anterior in 9 patients with DMD. MPC injections were placed 1 to 2 mm from each other, and a similar pattern of saline injections was done in the contralateral muscle. The patients were immunosuppressed with tacrolimus. Muscle biopsies were performed at the injected sites 4 weeks later. In the biopsies of the cell-grafted sites, there were myofibers expressing donor's dystrophin in 8 patients. The percentage of myofibers expressing donor's dystrophin varied from 3.5% to 26%. Evidence of small myofiber neoformation was observed in some patients. Donor-derived dystrophin transcripts were detected by reverse transcriptase-polymerase chain reaction in the cell-grafted sites in all patients. The protocol of immunosuppression was sufficient to obtain these results, although it is not certain whether acute rejection was efficiently controlled in all the cases. In conclusion, intramuscular allotransplantation of normal MPCs can induce the expression of donor-derived dystrophin in skeletal muscles of patients with DMD, although this expression is restricted to the sites of MPC injection.
