Johnny Huard

Results of a Triple Blind Clinical Study of Myoblast Transplantations without Immunosuppressive Treatment in Young Boys with Duchenne Muscular Dystrophy

The effects of myoblast transplantations without an immunosuppressive treatment on muscle strength, and the formation of dystrophin-positive fibers was studied in five young boys with Duchenne muscular dystrophy (DMD) using a triple blind design. Injections of myoblasts were made into one biceps brachii (BB), and the opposite BB, used as a control, was sham-injected; the experimenters and the patient were blind to the myoblast-injected side. At the same time, myoblasts were also injected in the left tibialis anterior (TA) of these patients. The strength developed during maximal static contractions of the elbow flexor and extensor muscles was measured with a Kin-Com dynamometer. No increase in static elbow flexion torque was measured at any time from 2 mo up to 18 mo after the transplantation. One month after the transplantation, the percentage of dystrophin-positive fibers in the myoblast-injected TA ranged from 0 to 36%, while it ranged from 0 to 4% on the control side. The expression of dystrophin in these fibers, however, was generally low, and most likely less than 10% of the normal level. In the biceps brachii of both sides 6 mo after the transplantation, less than 1.5% of dystrophin-positive fibers were detected. The injections also triggered a humoral immune response of the host. Antibodies were capable of fixing the complement, and of lysing the newly formed myotubes. One of the antigens recognized by this immune response is possibly dystrophin. These results strongly suggest that myoblast transplantations, as well as gene therapy for DMD, cannot be done without immunosuppression.

Use of fluorescent latex microspheres (FLMs) to follow the fate of transplanted myoblasts

A potential treatment for Duchenne muscular dystrophy (DMD) is injection of normal myoblasts into dystrophic muscles to induce formation of muscle fibers. To develop this therapy it is important to identify the injected myoblasts and the muscle fibers that they form in the host muscles. Fluorescent latex microspheres (FLMs) were used for this purpose in this study. Normal myoblasts were labeled with FLMs and injected into dystrophin-deficient (mdx) mice. The FLMs clearly indicated the location of injected myoblasts in the host muscle. Muscle fibers containing dystrophin were localized by immunofluorescence and immunoperoxidase. They were observed in clusters near the myoblasts labeled with FLMs. FLMs were also observed in some of these dystrophin-positive fibers in each cryostat section. These results indicate that: labeling myoblasts with FLMs can be used to trace the injected myoblasts in the muscle and to identify the muscle fibers that they formed; injected myoblasts remain near the injected site and do not migrate very far; most of the dystrophin-positive muscle fibers around the injected myoblasts result from fusion of the injected myoblasts; and the low percentage of dystrophin-positive muscle fibers is likely related to limited diffusion and lack of fusion of many injected myoblasts.

Localization of dystrophin in the Purkinje cells of normal mice

A monoclonal antibody that reacts with a mid rod fragment of dystrophin was used to localize this protein in the central nervous system (CNS). Due to a low abundance of dystrophin in the CNS, an immunoperoxidase reaction amplified with a biotin-avidin system was used. All Purkinje cells in normal mice were dystrophin positive while the mdx mouse cerebellum was completely devoid of reaction. Dystrophin staining was present in the soma and dendrites of Purkinje cells but not in their axons. This uniform dystrophin labelling in the normal mouse Purkinje cells indicates that this protein is not only localized in synaptic contact regions of the CNS.

Utilization of an antibody specific for human dystrophin to follow myoblast transplantation in nude mice

Human myoblasts were transplanted in nude mice. The efficacy of these transplantations was analyzed using a monoclonal antibody (NCLDys3) specific for human dystrophin. This antibody did not reveal any dystrophin in nude mice that did not receive a human myoblast transplantation. However, about 30 days after a human myoblast transplantation, dystrophin-positive muscle fibers were observed. They were not abundant, and were present either in small clusters or isolated. This technique follows the fate of myoblast transplantation in animals that already have dystrophin, and distinguishes between new dystrophin-positive fibers due to the transplantation and the revertant fibers in mdx mice. Moreover, this technique does not require any labelling of the myoblasts before transplantation. It can also be used to detect dystrophin produced following the fusion of myoblasts transfected with the human dystrophin gene.

Dystrophin-like immunoreactivity in monkey and human brain areas involved in learning and motor functions.

Two antidystrophin antibodies against different fragments of dystrophin were used to detect this polypeptide in monkey and human brains. Dystrophin was revealed by immunoperoxidase amplified with the biotin/avidin system and by immunoblotting. A dystrophin-like immunoreactivity was uniformly expressed in several brain regions implicated in learning and motor functions. Dystrophin function is not clear but our results raise the possibility that this protein may be involved in the cognitive impairment observed in several Duchenne muscular dystrophy (DMD) patients.

In Vitro Bromodeoxyuridine Labeling of Nuclei: Application to Myotube Hybridization'

Rat myoblast nuclei were labeled with various concentrations of bromodeoxyuridine (BrdU), an analogue of thymidine, for 24 or 48 hr. Almost every myoblast was labeled with BrdU at concentrations between 10(-7) M and 10(-5) M. When the cells were labeled with 0.5 microM or more, the percentage of labeled cells remained over 90% and 80% at 2 and 5 days, respectively. However, when the cells were labeled with BrdU concentration lower than 10(-7) M the percentage of labeled nuclei decreased more rapidly with time. The BrdU-labeled cells were mixed with an unlabeled population to determine whether their capacity to fuse was reduced. At a BrdU concentration of 0.5 x 10(-6) M, labeled myoblasts fused to a similar extent as unlabeled myoblasts, and a high percentage of marked cells were still perceptively labeled after 5 days. In contrast, the fusion capacity of myoblasts incubated with more than 10(-6) M BrdU was inhibited after only few rounds of DNA synthesis. These myoblasts were eventually able to fuse, however, when the BrdU diminished in the DNA due to cell division. These results indicate that labeling with BrdU at a concentration of 0.5 x 10(-6) M and an incorporation time of 48 hr is optimal to obtain perceptible immunocytochemical staining without affecting myoblast fusion. Such BrdU immunolabeling could be used as a nuclear marker for hybridization studies.

A new technique to identify hybrid myotubes in vitro without culture fixation.

Fluorescent latex microspheres (FLMs) were used to label myoblasts and to permit the observation of hybrid myotubes before culture fixation. This type of labeling did not affect survival, development, or fusion of these cells. The FLMs were retained for several weeks. Labeled mouse myoblasts were co-cultured with unlabeled rat myoblasts to verify whether the marker was released and spread from labeled to unlabeled cells. The nuclear stain Hoechst 33258 was used to distinguish the myoblasts from both species and permitted the demonstration that there was virtually no re-uptake. Hybrid myotubes were also obtained by co-culturing mouse myoblasts containing rhodamine FLMs and rat myoblasts containing green FLMs. These mixed cultures were observed repeatedly with a fluorescent microscope without any cytotoxic effect. Several myotubes were observed before fixation of the cultures to contain both types of fluorescent labels. Subsequent fixation and staining with Hoechst dye confirmed that these myotubes were hybrids.

Mosaic expression of dystrophin in the cerebellum of heterozygote dystrophic (mdx) mice

The monoclonal NCLDys1 revealed the presence of dystrophin in the Purkinje cells of normal mice but not of mdx mice and a mosaic staining in Purkinje cells of heterozygote mdx mice. Dystrophin was present in the soma and the dendrites of the dystrophin positive Purkinje cells and was absent in both regions of the dystrophin negative Purkinje cells. However, the polyclonal antibody d10 produced a uniform labeling of all Purkinje cells not only in the normal mice but also in mdx and heterozygote mdx mice. This staining was attributed to a reaction of this antibody not only with dystrophin but also with a different isoform of dystrophin or with a dystrophin related protein present even in mdx mice.