Raynald Roy

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.

Very efficient myoblast allotransplantation in mice under FK506 immunosuppression

Transgenic CD1 mice expressing β‐galactosidase were used as myoblast donors. The myoblasts were injected in normal or mdx muscles previously irradiated and injected with notexin. Twenty‐eight days after myoblast transplantation, the percentage of muscle fibers β‐galactosidase‐positive was low in mice not immunosuppressed but was high (80%) in those treated with FK506. In mdx mice, muscle fibers expressing β‐galactosidase were also dystrophin positive. Most of the mice not treated with FK506 produced antibodies against the donor myoblasts. These results indicate that FK506 is a very useful immunosuppressive drug for myoblast transplantation in mice. Irradiation and notexin injection used in our experiments are, however, not feasible in humans. Other manipulations capable of increasing the participation of donor myoblasts to regeneration will therefore have to be identified before new clinical trials are attempted.

Dystrophin expression in muscles of duchenne muscular dystrophy patients after high-density injections of normal myogenic cells.

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 patients 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 donors dystrophin in 8 patients. The percentage of myofibers expressing donors 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.

High efficiency of muscle regeneration after human myoblast clone transplantation in SCID mice.

SCID mouse tibialis anterior muscles were first irradiated to prevent regeneration by host myoblasts and injected with notexin to damage the muscle fibers and trigger regeneration. The muscles were then injected with roughly 5 million human myoblasts. 1 mo later, 16-33% of the normal number of muscle fibers were present in the injected muscle, because of incomplete regeneration. However, > 90% of these muscle fibers contained human dystrophin. Some newly formed muscle fibers had an accumulation of human dystrophin and desmin on a part of their membrane. Such accumulations have been demonstrated at neuromuscular junctions before suggesting that the new muscle fibers are innervated and functional. The same pool of clones of human myoblasts produced only < or = 4% of muscle fibers containing human dystrophin when injected in nude mice muscles. Several of the human myoblasts did not fuse and remained in interstitial space or tightly associated with muscle fibers suggesting that some of them have formed satellite cells. Moreover, cultures of 98% pure human myoblasts were obtained from transplanted SCID muscles. In some mice where the muscle regeneration was not complete, the muscle fibers containing human dystrophin also expressed uniformly HLA class 1, confirming that the fibers are of human origin. The presence of hybrid muscle fibers containing human dystrophin and mouse MHC was also demonstrated following transplantation. These results establish that in absence of an immune reaction, transplanted human myoblasts participate to the muscle regeneration with a high degree of efficacy even if the animals were killed only 1 mo after the transplantation.

Risk Factors of Corneal Graft Failure

PURPOSE To measure the association between potential risk factors and corneal graft failure. Two failure outcomes are compared: those with and those without a prior immune allograft reaction. METHODS Based on a single-center observational study design, 539 adult recipients of a corneal graft were followed for a median time of 30 months. Survival analysis was carried out. RESULTS Eighty-two graft failures were recorded. Of 82 failures, 53 (65%) were not preceded by an immune allograft reaction. Presence of blood vessels in the recipient cornea was associated with a twofold increase in risk for both failure outcomes. Three factors increased the risk of failure without an immune reaction: prior glaucoma or uveitis (adjusted relative risk estimate = 3.1), vitreous surgery with the graft (adjusted relative risk estimate = 2.0), and a repeat graft in the study eye (adjusted relative risk estimate = 2.0). Conversely, large graft wound size (adjusted relative risk estimate = 2.0). Conversely, large graft wound size (adjusted relative risk estimate = 2.9) and human leukocyte antigen (HLA)-A, -B incompatibility (adjusted relative risk estimate = 2.2) were associated with failures that followed an immune reaction. CONCLUSION In this study, the authors support the clinical impression that corneal graft failures with and without a prior immune reaction are distinct phenomena. Enhanced surveillance in recipients with glaucoma and early intensive treatment of allograft reactions are recommended to improve the outcome of corneal grafts.

Cytokine and cytotoxic molecule gene expression determined in peripheral blood mononuclear cells in the diagnosis of acute renal rejection.

Background. Prevention of acute rejection is the most prevalent measure used to reduce the long-term risk of chronic allograft rejection.Until now, biopsy was the only useful diagnostic tool for monitoring allograft acute rejection, but invasiveness of this procedure limits its use. The aim of this study was to investigate the implication of peripheral blood immune markers as a predictive diagnostic tool preceding biopsy in acute renal allograft rejection determination. Methods. Of the 61 patients studied, 13 had no rejection episodes, 8 had a proven acute rejection, and 40 were excluded for graft dysfunction causes. Mitogen-induced peripheral blood mononuclear cells were tested for interleukin- (IL) 2, IL-4, IL-5, IL-6, IL-10, IL-15, Interferon-&ggr;, Perforin, Granzyme B, and Fas L using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). An up-regulated mRNA expression value was calculated in which a patient’s sample was deemed positive if its differential expression value was equal or higher than the mean differential expression value calculated from the nonrejecting patients. Results. IL-4, IL-5, IL-6, Interferon-&ggr;, Perforin, and Granzyme B mRNA levels were associated with acute rejection. When at least two of these cytokine markers were up-regulated in a given patient, 75% of the rejecting recipients were identified against 15% of the nonrejecting patients. Conclusions. We have shown that acute rejection episodes in renal transplant recipients were associated with an increase in mRNA expression of cytokines in mitogen-induced peripheral blood mononuclear cells. The evaluation of pro-inflammatory cytokines and cytotoxic molecules prove useful in the clinical identification of acutely rejecting transplant recipients and in the justification of concomitant antirejection therapy before histological diagnosis confirmation.

In vitro biocompatibility study of electrically conductive polypyrrole-coated polyester fabrics.

This study investigated the basic biocompatibility aspects of two types of polypyrrole (PPy)-coated polyester fabrics for possible use as vascular prostheses. These PPy-coated fabrics, PPy-Phos and PPy-Plas, were sterilized with ethylene oxide (EO) and the following characterizations were performed: surface morphology by scanning electron microscope, EO residuals analysis by the headspace method, acute systemic toxicity in the mouse model, hemolysis, blood coagulation time, viability and proliferation of endothelial cells measured with the WST-1 method, and activation of polymorphonuclear (PMN) cells indicated by the specific expression of interleukin 8 mRNA measured by reverse transcription polymerase chain reaction. Virgin polyester fabrics, expanded poly(tetrafluoroethylene) (ePTFE), and medical-grade Bionate 80A poly(carbonate urethane) were used as references in the cell culture experiments. The PPy-coated fabrics revealed different surface morphologies by showing more PPy lamina and clusters on the PPy-Plas. Neither of the PPy-coated fabrics had an adverse effect on hemolysis and coagulation time, and they did not cause any acute systemic toxicity. The EO residual level was as low as 5 ppm or less, which is considered quite acceptable. Although exhibiting a relatively low initial cell adhesion at 24 h, the two PPy-coated samples showed no cytotoxicity at 72 and 168 h. Bionate 80A and ePTFE recorded cytotoxicity at 72 and 168 h, respectively. The virgin fabrics also demonstrated a decrease of viable cells at 72 h that was not significant. The activation of PMN cells induced by both PPy-coated fabrics, the ePTFE, and the negative control was significantly lower than that induced by their respective tumor necrosis factor-alpha controls. These results therefore highlighted the potential of PPy-coated fabrics for use as cardiovascular prostheses. It was suggested that cell adhesion moieties should be incorporated into the PPy/fabric composite to increase cell adhesion and subsequent cell proliferation.

Myoblast transplantation in monkeys: control of immune response by FK506.

Myoblasts were grown from monkey muscle biopsies and infected in vitro with a defective retroviral vector containing a cytoplasmic β-galactosidase (β-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 β-gal were observed 1 week after the transplantation, but no cells expressing β-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-β) 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 (β-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.

An albumin-coated polyester arterial graft: in vivo assessment of biocompatibility and healing characteristics

The albumin-coated vascular graft (ACG) and its uncoated polyester substrate, the Vascular II (V-II), were evaluated in terms of biocompatibility and biofunctionality using two in vivo animal studies. Biocompatibility and immunoreactivity were assessed by implanting intraperitoneally in the rat small segments of the ACG and the V-II graft and harvesting them with their surrounding tissue 3d, 1, 2 and 4 weeks later. Cytofluorometric determination of total T cells (CD3), the ratio of CD4/CD8 subsets and the percentage of IL-2 receptor-positive T cells in the peripheral blood has revealed that no significant difference in any of the T cell populations was found between the ACG and the V-II graft. The cellular reactivity of the ACG in terms of acid phosphatase activity at the implant side was significantly greater at 3 d but not at longer periods. Biofunctionality was evaluated by implanting both grafts as a thoracoabdominal vascular bypass in dogs for 11 different periods ranging from 4 h to 6 months. The rate of albumin resorption was such that traces were still present at 1 month, but no longer observable at 2 months. Tissue incorporation into the graft wall was earlier for the V-II (2 weeks) than for the ACG (4 weeks), which showed complete encapsulation, tissue incorporation and endothelialization after 2 months in vivo. Only small differences were observed between both grafts in terms of platelet and fibrin uptake on the luminal surface. The prostacyclin/thromboxane A2 ratio increased to a level higher that 1.0 aorta within 1 month for the V-II and 4 months for the ACG. In conclusion, the Bard ACG has demonstrated excellent biocompatibility in terms of blood T cell behaviour and acid phosphatase activity at the implant site. Finally, its healing response is equivalent to that of the uncoated Dacron prosthesis once the albumin coating has been resorbed.

In vitro cellular response to polypyrrole-coated woven polyester fabrics : Potential benefits of electrical conductivity

Electrically conducting polypyrrole-treated films have recently been shown to influence the morphology and function of mammalian cells in vitro. This type of polymer represents a possible alternative biomaterial for use in vascular implantation. The present study compared the in vitro biocompatibility of the five different polyester woven fabrics having increasing levels of electrical conductivity ranging from 4.5 x 10(4) to 123 omega/square with that of low density polyethylene and polydimethylsiloxane primary reference materials. Biocompatibility was measured in terms of four different types of in vitro cellular response, including (a) an indirect and (b) a direct control organotypic culture assay using endothelial cells, (c) a polymorphonuclear (PMN) cell activation study using flow-cytometric measurements of CD11/CD18 integrin molecule expression, and (d) a semiquantification of interleukin (IL)-6 mRNA expression on monocytes/macrophages using reverse-transcriptase polymerase chain reaction. The organotypic culture study revealed that the fabrics with high levels of conductivity exhibited lower cell migration, proliferation, and viability. The PMN activation study of blood from 10 healthy adult donors demonstrated that the two most conductive fabrics were able to identify the more reactive donors. The levels of IL-6 mRNA expression by monocytes/macrophages decreased as the conductivity level of the fabrics increased. The results of the present study therefore indicate that high levels of conductivity (< 200 omega/square) on polyester fabrics are detrimental to the growth, migration, and viability of endothelial cells; induce elevated PMN activation; and affect the intracellular metabolism of monocytes. They also point to a specific range of conductivity (10(3) < 10(4) omega/square) which is associated with an optimum in vitro cellular response.