Nicolas Caron

20 W passively cooled single-mode all-fiber laser at 2.8 μm

A maximum output power of 20.6 W at 2.825 μm from an erbium-doped all-fiber laser is reported, which we believe is the highest output power for this laser transition in single-mode operation. The slope efficiency of the passively cooled laser was up to 35.4% with respect to the absorbed pump power. Accounting for an estimated round-trip intracavity loss of 1.3 dB, we calculated a theoretical conversion efficiency of 39.5%, which is 15% higher than the Stokes efficiency of 34.3%. We believe this is the first experimental confirmation of the predicted pump energy recycling for this fiber laser. The narrow laser linewidth varied from 0.09 to 0.16 nm from low to maximum output power.

Tumor necrosis factor-alpha (TNF-alpha) stimulates chemotactic response in mouse myogenic cells.

Migration of transplanted myogenic cells occurs during both embryogenesis and regeneration of skeletal muscles and is important for successful myoblast transplantation, but little is known about factors that promote chemotaxis of these cells. Tumor necrosis factor-α (TNF-α) is known to induce chemotactic effect on several cell types. In this study, we investigated its influence on the in vitro and in vivo motility of C2C12 and primary myoblasts. In the in vitro test performed in the blind-well Boyden chambers, we showed that TNF-α (50–400 U/ml) significantly enhanced the ability of myogenic cells to migrate. The dose–response curve for this factor was bell shaped, with maximum activity in the 200 U/ml range. In the in vivo test, intramuscular administration of TNF-α was performed by an Alzet pump connected to a perforated polyethylene microtube inserted in the tibialis anterior (TA) of CD1 mice. In these experiments, myoblasts were injected under the muscle epimysium. The recipient mice were immunosuppressed with FK506. Our results showed that, 5 days after myoblast transplantation, cells migrated further in the muscles infused with TNF-α than in the muscles not exposed to TNF-α. TNF-α not only has a chemotactic activity but may also modify cell migration via its action on matrix metalloproteinase (MMP) expression. The proteolytic activities of the MMPs secreted in the muscles were thus also assessed by gelatin zymography. The results showed an increased of MMP-2 and MMP-9 transcripts in the TNF-α-infused muscles injected with myogenic cells. Myoblast migration during transplantation may be enhanced by overlapping gradients of several effector molecules such as TNF-α, interferon-γ (INF-γ), and interleukins, released at the site of muscle injury. We propose that TNF-α may promote myoblast migration directly through chemotactic activity and indirectly by enhancing MMP activity at the site of muscle injury.

Identification of a putative pathway for the muscle homing of stem cells in a muscular dystrophy model

Attempts to repair muscle damage in Duchenne muscular dystrophy (DMD) by transplanting skeletal myoblasts directly into muscles are faced with the problem of the limited migration of these cells in the muscles. The delivery of myogenic stem cells to the sites of muscle lesions via the systemic circulation is a potential alternative approach to treat this disease. Muscle-derived stem cells (MDSCs) were obtained by a MACS® multisort method. Clones of MDSCs, which were Sca-1+/CD34−/L-selectin+, were found to adhere firmly to the endothelium of mdx dystrophic muscles after i.v. or i.m. injections. The subpopulation of Sca-1+/CD34− MDSCs expressing L-selectin was called homing MDSCs (HMDSCs). Treatment of HMDSCs with antibodies against L-selectin prevented adhesion to the muscle endothelium. Importantly, we found that vascular endothelium from striate muscle of young mdx mice expresses mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a ligand for L-selectin. Our results showed for the first time that the expression of the adhesion molecule L-selectin is important for muscle homing of MDSCs. This discovery will aid in the improvement of a potential therapy for muscular dystrophy based on the systemic delivery of MDSCs.

Mid-infrared chalcogenide glass Raman fiber laser

We report the first demonstration of a Raman fiber laser (RFL) emitting in the mid-infrared, above 3 μm. The operation of a single-mode As2S3 chalcogenide glass based RFL at 3.34 μm is demonstrated by using a low-loss Fabry-Pérot cavity formed by a pair of fiber Bragg gratings. A specially designed quasi-cw erbium-doped fluoride fiber laser emitting at 3.005 μm is used to pump the RFL. A laser output peak power of 0.6 W is obtained with a lasing efficiency of 39% with respect to the launched pump power.

Dynamics of the early immune cellular reactions after myogenic cell transplantation.

The role of immune cells in the early donor cell death/survival following myoblast transplantation is confusing, one of the reasons being the lack of data about the immune reactions following cell transplantation. We used outbred mice as hosts for transplantation of primary cultured muscle cells and T-antigen-immortalized myoblasts. The host muscles were analyzed 1 h to 7 days after cell injection. No net loss of the donor primary cultured cell population was observed in this period. The immune cellular reaction in this case was: 1) a brief (<48 h) neutrophil invasion; 2) macrophage infiltration from days 1 to 7; 3) a specific response involving CTL and few NK cells (days 6 and 7), preceded by a low CD4+ cell infiltration starting at day 3. In contrast, donor-immortalized myoblasts completely disappeared during the 7-day follow-up. In this case, an intense infiltration of CTL and macrophages, with moderate CD4+ infiltration and lower amounts of NK cells, was observed starting at day 2. The nonspecific immune response at days 0 and 1 was similar for both types of donor cells. The present observations set a basis to interpret the role of immune cells on the early death/survival of donor cells following myoblast transplantation.

Erbium-doped all-fiber laser at 2.94 μm

We report what we believe is the first demonstration of laser emission at 2.94 microm in an erbium-doped fluoride fiber laser. The low-loss all-fiber Fabry-Perot laser cavity was formed by two fiber Bragg gratings of 90% and 15% reflectivities in a 6.6 m, 7 mol.% Er-doped double-clad fiber. A maximum cw output power of 5.2 W was measured, which is to our knowledge the highest reported to date for a diode-pumped laser at this wavelength. A coreless endcap was fused at the output fiber end to prevent its deterioration at high output powers. Our results, including the slope efficiency of 26.6% with respect to launched pump power, suggest that erbium could be a better alternative than holmium in the search for a replacement for the flashlamp-pumped Er:YAG at 2.94 microm.

Intramuscular migration of myoblasts transplanted after muscle pretreatment with metalloproteinases.

The effect of pretreatments of host muscles with metalloproteinases (MMPs) or with notexin on the migration of transplanted myoblasts was investigated. Transgenic TnILacZ mice in which the β-galactosidase gene is under the control of a quail fast skeletal troponin I gene promoter were used as donors. A polyethylene microtube with four perforations was inserted in the tibialis anterior (TA) of CD1 mice. Both pretreatment substances and cells were slowly injected through that microtube. Muscles were pretreated 2 days before myoblast injection either with a mixture of collagenase, matrilysin, and notexin or with only collagenase and matrilysin or only notexin. As control for our experiments, TnILacZ and C2C12 myoblasts were also injected in TA muscles not pretreated. Comparison of short and long-term myoblast radial migration was performed using a dye (PKH26) and X-gal staining, respectively. The recipient mice were immunosuppressed with FK506. Two days after myoblast transplantation, the cell movement in muscles pretreated with collagenase, matrilysin, and notexin was slightly greater than in muscles pretreated only with collagenase and matrilysin but was about twice that observed in muscles treated with notexin alone. Almost no radial migration of TnILacZ myoblasts was observed in untreated muscles. The C2C12 myoblasts showed a four-to fivefold higher migration capacity than TnILacZ myoblasts. At 15 days after TnILacZ myoblast transplantation, the farthest positive β-gal muscle fibers show a two- to threefold extension of the initial migration observed at 2 days, demonstrating the ability of myoblasts to continue the migration following all pretreatments and even in the untreated muscles. In addition, more muscle fibers expressed the β-gal reporter gene in muscles pretreated only with MMPs. Our results clearly demonstrate that muscle pretreatments with MMPs increase myoblast migration and fusion with host muscle fibers after transplantation and that the C2C12 cell line producing MMPs has a higher migratory capacity.

Increased myogenic potential and fusion of matrilysin-expressing myoblasts transplanted in mice.

The success of myoblast transplantation in clinical trials has been limited in part by the low dispersion of grafted cells outside the injection site. Our research group previously reported that the culture of myoblasts with concanavalin A, a stimulator of metalloproteinase production, increased their migration. Several lines of evidence also suggested that muscle cell fusion involves metalloproteinase-sensitive mechanisms. To determine whether the increased expression of metalloproteinases had an influence on myoblast fusion and dispersion through the muscle following transplantation, we generated a myoblast cell line expressing human matrilysin (MMP-7). The MMP-7-expressing myoblasts were obtained by the stable transfection of a matrilysin expression vector in a TnILacZ immortomouse myoblast clone. Matrilysin-expressing myoblasts showed a highly increased in vitro fusion index, forming seven times (p < 0.001) more myotubes than the control cell line and three times (p < 0.001) more myotubes than the Immortomyoblast parental clone. Single-site transplantation of matrilysin-expressing myoblasts generated more fibers (p < 0.001), over a greater surface (p < 0.001) than the control cell line. The cotransplantation of matrilysin-expressing myoblasts and of normal human myoblasts in SCID mice increased the number of human dystrophin-positive fibers and myotubes by sixfold. Although no significant increased migration of myoblasts outside the injection sites was observed, our results show that the metalloproteinase activity can improve the myogenic potential of myoblasts in vitro and the fusion of myoblasts with host fibers in vivo. MMP-7 expression may be useful in increasing myoblast transplantation success.

Treatment with anti-CD154 antibody and donor-specific transfusion prevents acute rejection of myoblast transplantation.

BACKGROUND Achieving immunological tolerance to transplanted myoblasts would reduce the adverse effects associated with the sustained immunosuppression required for this experimental therapeutic approach in Duchenne muscular dystrophic patients. METHODS Mdx mice were transplanted with fully allogeneic BALB/c myoblasts in the tibialis anterior muscles. Seven days before transplantation (-7), host mice received 107 total donor spleen cells i.v. (donor-specific transfusion, DST) with 500 microg of anti-CD154 mAb i.p. on days -7, -4, 0, +4. RESULTS Results showed a high level of dystrophin expression in 83, 60, and 20% of the mice 1, 3, and 6 months, respectively, after transplantation of myoblasts. No antibodies against the donor cells were produced up to 3 months after transplantation. However, abundant activated cytotoxic cells were present in muscles still expressing high percentage of dystrophin positive fibers. CONCLUSIONS In conclusion, the DST + anti-CD154 mAb treatments effectively prolonged myoblast survival, but this treatment could not develop tolerance to complete allogeneic myoblast transplantation.

Highly stable and efficient erbium-doped 2.8 μm all fiber laser

We demonstrate the efficient and stable CW laser operation at 2.824 microm of a diode-pumped erbium-doped fluoride fiber laser employing an intracore fiber Bragg grating high reflector. An output power of 5 W and an optical-to-optical conversion efficiency of 32% are reported. The temporal and spectral stability of the laser represent a significant improvement over previous work. This report paves the way to the commercialization of compact and stable fiber lasers for spectroscopic and medical applications.