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1.
Arch Pediatr ; 30(5): 260-265, 2023 Jul.
Article in English | MEDLINE | ID: mdl-37147154

ABSTRACT

OBJECTIVE: Teenagers use the Internet to obtain and exchange information in multiple fields, including about taboo subjects such as sexuality. Our objectives were to determine the prevalence and vulnerability factors related to active cybersexuality among teenagers aged between 15 and 17 years in western Normandy. MATERIAL AND METHODS: This was an observational, cross-sectional, multicenter study integrated into sexual education classes for teenagers between 15 and 17 years old. An anonymous questionnaire, designed for the study, was given at the beginning of each session. RESULTS: The study had a 4-month duration and involved 1,208 teenagers. The results revealed that 66% of them engaged in cybersex, with sexting being the most widespread practice: 21% sent such sexts, 60% received such sexts, and 12% of boys shared such texts with others. Other practices, such as dedipix, dating websites, and skin parties, were more marginal, but 12% of teenagers had met someone in real life after meeting them first online. A history of experiencing violence, a lack of parental control, female gender, poor self-esteem, and consuming toxic drugs were associated with a higher risk of cybersexuality with an odds ratio (OR) of 1.63, 1.95, 2.07, 2.27, and 2.66, respectively. Number of friends on social networks >300 and daily viewing of pornography were also strongly associated with cybersexuality with an OR of 2.83 and 6.18, respectively. CONCLUSIONS: This study shows that cybersex is practiced by two thirds of teens. Vulnerability factors most strongly associated with cybersexuality were female gender, poor self-esteem, consuming toxic drugs, number of friends on social networks >300, and daily viewing of pornography. Cybersexuality involves risks (social exclusion, bullying, dropout, poor self-esteem, breakdown) that are possible to prevent by highlighting this theme during sexual education classes.


Subject(s)
Sexual Behavior , Violence , Male , Humans , Female , Adolescent , Cross-Sectional Studies , Prevalence , Risk Factors
3.
J Gynecol Obstet Biol Reprod (Paris) ; 43(2): 123-45, 2014 Feb.
Article in French | MEDLINE | ID: mdl-24433988

ABSTRACT

OBJECTIVE: State of knowledge about misoprostol's use out of its marketing authorization during the first trimester of pregnancy, in early miscarriage or to induce abortion or medical termination of pregnancy. METHODS: French and English publications were searched using PubMed, Cochrane Library and international learned societies recommendations. RESULTS: Cervical ripening prior to surgical uterine evacuation during the first trimester of pregnancy facilitates cervical dilatation and reduces operative time and uterine retention risk. Misoprostol, mifepristone and osmotic cervical dilators are equally efficient. Concerning first trimester miscarriage, surgical uterine evacuation remains the most effective and the quickest method of treatment (EL 1). Depending on the clinical situation, medical treatment using misoprostol (missed miscarriage) or expectative attitude (incomplete miscarriage) does not increase the risk of complications, neither haemorrhagic nor infectious (EL 1). However, these alternatives generally require longer outpatient follow-up, which leads to more consultations, prolonged bleeding and not planned surgical procedures (EL 1). Concerning missed miscarriage, a vaginal dose of 800 µg of misoprostol, possibly repeated 24 to 48 hours later, seems to offer the best efficiency/tolerance ratio (EL 2). Concerning early abortion, medical method is a safe and efficient alternative to surgery (EL 2). Success rates are inversely proportional to gestational age (EL 2). According to the modalities of its marketing authorization, 400 µg of misoprostol can only be given by oral route, for less than 7 weeks of amenorrhea (WA) pregnancies and after 36 to 48 hours following 600 mg of mifepristone (EL 1). However, 200mg of mifepristone is as efficient as 600 mg (EL 1). Beyond 7WA, misoprostol buccal dissolution (sublingual or prejugal) or vaginal administration are more efficient and better tolerated than oral ingestion (EL 1). Between 7 and 9WA, the best protocol in terms of efficiency and tolerance is the association of 200mg of mifepristone followed 24 to 48 hours later by 800 µg of vaginal, sublingual or buccal misoprostol (EL 1). An additional dose of 400 µg can be given 3 hours later if necessary (EL 3). In case of buccal administration, the dose of 400 µg seems to offer the same efficiency with a better tolerance but further evaluation is needed (EL 2). Between 9 and 12WA, medical treatment is less efficient than surgery and its tolerance is lower (EL 2). However, a protocol of 200mg of mifepristone followed 36 to 48 hours later by 800 µg of vaginal or sublingual misoprostol, plus an additional 400 µg dose every 3-4 hours (until 4-5 doses maximum) seems safe and efficient (EL 5). CONCLUSION: Misoprostol use during the first trimester of pregnancy is a safe and efficient alternative to surgery as long as detailed protocols adjusted to each clinical situation are respected.


Subject(s)
Abortion, Induced/methods , Misoprostol/administration & dosage , Off-Label Use , Abortifacient Agents, Nonsteroidal , Administration, Buccal , Administration, Intravaginal , Administration, Sublingual , Cervical Ripening , Female , France , Humans , Misoprostol/adverse effects , Pregnancy , Pregnancy Trimester, First
4.
Transplantation ; 72(1): 38-44, 2001 Jul 15.
Article in English | MEDLINE | ID: mdl-11468532

ABSTRACT

BACKGROUND: Overcoming adverse effects of immunosuppressors can be achieved by combining different drugs, thus allowing a dosage reduction. Myoblast transplantation is a potential therapy for Duchenne muscular dystrophy. Our research group previously established that FK506 (tacrolimus) is an effective immunosuppressive drug for myoblast transplantation in mice and monkeys. METHODS: In the present study, a reduced dose of FK506 at 1.0 mg/kg/day was used in combination with mycophenolate mofetil (MMF; 80 mg/kg/day) as an immunosuppressive protocol for myoblast transplantation. Graft success was evaluated by quantifying the number of dystrophin-positive fibers per muscle section that were injected with normal cells. RESULTS: MMF used alone could not prevent immune rejection of the transplanted myoblasts. MMF given in combination with FK506 immediately after transplantation reduced the success of myoblast transplantation by about 50%. A low dose of FK506 combined with MMF after the establishment of the graft (3 weeks) maintained graft success and controlled immune infiltration compared with a low dose of FK506 alone. However, lymphocyte infiltration was observed at longer term using a low dose of FK506 combined with MMF. CONCLUSIONS: The diminution of graft success when combining FK506 and MMF by the time of myoblast transplantation could be attributed to the inhibition of myoblast fusion by MMF. The use of MMF and FK506 after the establishment of the graft did not reduce graft success, however, this combination was not effective at controlling long-term immune rejection in comparison with the optimal dose of FK506 alone.


Subject(s)
Cell Transplantation , Immunosuppressive Agents/therapeutic use , Muscle, Skeletal/cytology , Mycophenolic Acid/therapeutic use , Tacrolimus/therapeutic use , Animals , Cell Division/drug effects , Cell Fusion , Cells, Cultured , Dose-Response Relationship, Drug , Drug Synergism , Drug Therapy, Combination , Graft Rejection/prevention & control , Graft Survival/drug effects , Immune System/pathology , Immunosuppressive Agents/administration & dosage , Mice , Mice, Inbred Strains , Mice, Inbred mdx , Mice, Transgenic , Muscle, Skeletal/pathology , Mycophenolic Acid/analogs & derivatives , Tacrolimus/administration & dosage
5.
Protein Eng ; 13(9): 611-5, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11054455

ABSTRACT

Transfection and transduction studies involving the use of the full-length dystrophin (11 kb) or the truncated mini-gene (6 kb) cDNAs are hampered by the large size of the resulting viral or non-viral expression vectors. This usually results in very low yields of transgene-expressing cells. Moreover, the detection of the few transgene-expressing cells is often tedious and costly. For these reasons, expression vectors containing the enhanced green fluorescent protein (EGFP) fused with the N-termini of mini- and full-length human dystrophin were constructed. These constructs were tested by transfection of Phoenix cells with Effectene, resulting after 48 h in a green fluorescent signal in 20% of cells. Analysis of the cell extracts by immunoblotting with the use of a monoclonal antibody specific to the dystrophin C-terminus confirmed the expression of EGFP-mini- (240 kDa) and EGFP-full-length human dystrophin (450 kDa) fusion proteins. Moreover, following the in vivo electroporation of the plasmids containing the EGFP-mini- and full-length dystrophin in mouse muscles, both fluorescent proteins were observed in cryostat sections in their normal location under the plasma membrane. This indicates that the fusion of EGFP to dystrophin or mini-dystrophin did not interfere with the normal localization of the protein. In conclusion, the fusion of EGFP provides a good tool for the search of the best methods to introduce mini- or full-length dystrophin cDNA in the cells (in vitro) or muscle fibers (in vivo) for the establishment of a treatment by gene therapy of Duchenne muscular dystrophy patients.


Subject(s)
Dystrophin/metabolism , Genetic Vectors/genetics , Luminescent Proteins/metabolism , Animals , Cell Line , Cytomegalovirus/genetics , Dystrophin/genetics , Genetic Therapy/methods , Green Fluorescent Proteins , Humans , Indicators and Reagents , Luminescent Proteins/genetics , Mice , Mice, Inbred C57BL , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/metabolism , Promoter Regions, Genetic , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transfection
6.
Hum Gene Ther ; 11(9): 1277-88, 2000 Jun 10.
Article in English | MEDLINE | ID: mdl-10890738

ABSTRACT

Clinical use of human granulocyte-colony stimulating factor (hG-CSF) to treat various diseases involving neutropenia has been previously shown to (1) successfully increase circulating neutrophils, (2) reduce condition-related infections, and (3) cause few side effects in patients. To alleviate the symptoms of neutropenia, the patient must receive frequent injections of recombinant hG-CSF. Permanent ways to deliver stable levels of the molecule to the patient are being investigated. Among them, the transplantation of hG-CSF-secreting cells has been proposed and performed successfully in rodents, using fibroblast cell lines and primary muscle cells. We thus investigated whether similar results could be obtained by intramuscular myoblast transplantation in a large animal model. When 1-3 x 10(8) myoblasts were injected into three Macaca mulatta, hG-CSF was detected at high levels (300-900 pg/ml), which in turn led to a four- to fivefold increase in circulating neutrophils. However, both the concentrations of hG-CSF and neutrophil levels were found to decrease over time. Nonetheless, neutrophils were found at higher levels from the fourth week until the end the experiment (up to 29 weeks) in G-CSF monkeys compared with control animals. These results show that transplantation of hG-CSF-secreting myoblasts may indeed be a therapeutic option for the treatment of neutropenic patients.


Subject(s)
Cell Transplantation , Gene Transfer Techniques , Granulocyte Colony-Stimulating Factor/genetics , Muscle, Skeletal/cytology , Animals , Cell Division , Dystrophin/analysis , Gene Expression , Granulocyte Colony-Stimulating Factor/metabolism , Humans , Injections, Intramuscular , Macaca mulatta , Mice , Mice, Inbred BALB C , Mice, SCID , Muscle, Skeletal/metabolism , Neutrophils/cytology , Recombinant Proteins/metabolism , Time Factors , beta-Galactosidase/genetics
7.
Cell Transplant ; 8(5): 465-76, 1999.
Article in English | MEDLINE | ID: mdl-10580341

ABSTRACT

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.


Subject(s)
Cell Transplantation/methods , Matrix Metalloproteinase 7/genetics , Muscle, Skeletal/cytology , Animals , Cell Line , Cells, Cultured , Dystrophin/biosynthesis , Dystrophin/genetics , Female , Humans , Matrix Metalloproteinase 7/biosynthesis , Mice , Mice, Inbred BALB C , Mice, SCID , Muscle, Skeletal/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , beta-Galactosidase/analysis , beta-Galactosidase/genetics
8.
Biochem Biophys Res Commun ; 248(3): 648-54, 1998 Jul 30.
Article in English | MEDLINE | ID: mdl-9703980

ABSTRACT

Transplantation of normal myoblasts into dystrophic muscles is a potential treatment for Duchenne muscular dystrophy (DMD). However, the success of such grafts is limited by the immune system responses. To avoid rejection problems, autologous transplantation of the patient's corrected myoblasts has been proposed. Regretfully, the low proliferative capacity of DMD myoblasts in culture (due to their premature senescence) limits such procedure. On the other hand, modification of dermal fibroblasts leading to the myogenic pathway using a master regulatory gene for myogenesis is an interesting alternative approach. In this study, the retrovirally encoded MyoD1 cDNA was introduced in dermal fibroblasts of TnI LacZ mice to provoke their conversion into myoblast-like cells. In vitro and in vivo assays were done and the results were compared to those obtained with uninfected fibroblasts and myoblasts. Some MyoD1-expressing fibroblasts were able to fuse and to express beta-galactosidase (under the transcriptional control of the Troponin I promoter), dystrophin and desmin in vitro. Thirty days following implantation of these modified fibroblasts in muscles of mdx mice, an average of 7 beta-Gal+/Dys-muscle fibers were observed. No beta-Gal+ fibers were observed after the transplantation of uninfected fibroblasts. Our results indicate that the successful implantation of myoblasts obtained from genetically modified fibroblasts is indeed feasible. However, the in vitro conversion rate and the in vivo fusion of genetically modified fibroblasts must be largely increased to consider this approach as a potential therapy for DMD and other myopathies.


Subject(s)
MyoD Protein/biosynthesis , Skin Transplantation/physiology , Skin/metabolism , Animals , Animals, Newborn , Desmin/biosynthesis , Dystrophin/biosynthesis , Fibroblasts/cytology , Fibroblasts/metabolism , Fibroblasts/transplantation , Mice , Mice, Transgenic , Promoter Regions, Genetic , Quail , Recombinant Fusion Proteins/biosynthesis , Skin/cytology , Trans-Activators/biosynthesis , Troponin I/genetics , beta-Galactosidase/biosynthesis
9.
Neuromuscul Disord ; 8(2): 95-110, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9608563

ABSTRACT

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.


Subject(s)
Cell Transplantation , Immunosuppressive Agents/therapeutic use , Muscles/transplantation , Animals , Biopsy , Cells, Cultured , Cyclosporine/therapeutic use , Dogs , Drug Therapy, Combination , Mice , Mice, SCID , Muscles/cytology , Muscles/immunology , Muscular Dystrophy, Animal/surgery , Mycophenolic Acid/analogs & derivatives , Mycophenolic Acid/therapeutic use , Tacrolimus/therapeutic use , Transplantation, Heterologous , Transplantation, Homologous , beta-Galactosidase/biosynthesis
10.
Biochem Biophys Res Commun ; 247(1): 94-9, 1998 Jun 09.
Article in English | MEDLINE | ID: mdl-9636661

ABSTRACT

Myoblast transplantation and gene therapy are two promising therapeutical approaches for the treatment of Duchenne Muscular Dystrophy (DMD). So far, both strategies have met many hurdles, mainly because of immune reactions. In this study, we investigated a third and novel strategy based on the combination of these two basic ones, i.e., transplantation of genetically modified myoblasts. We first derived a primary culture from a muscle biopsy of a young DMD patient (3 years old). Adenoviral-mediated dystrophin gene transfer into these DMD cultures and expression of the dystrophin transgene were achieved in vitro. The transduced cultures were then transplanted the same day in immunodeficient SCID mouse muscles. Three weeks following the graft, many human dystrophin-positive fibers were observed throughout sections of the injected muscles. However, many fibers expressed human MHC antigens without expressing human dystrophin due to the low percentage of infected primary muscle cells in vitro (even when a high MOI [400] was used) and to a reduction and even to a complete loss of transgene copy number during myoblast replication. From our results, we conclude that, although not at a high proportion, (1) DMD primary myoblast cultures are infectable by adenoviruses; (2) they can be efficiently transplanted back in a muscle, leading to normal fusion of infected myoblasts with the host fibers; and (3) they can correct the dystrophin deficiency in the host fibers by the expression of a mini-dystrophin transgene.


Subject(s)
Cell Transplantation/methods , Dystrophin/genetics , Gene Transfer Techniques , Muscle Fibers, Skeletal/transplantation , Muscular Dystrophies/genetics , Transduction, Genetic , Adenoviridae/genetics , Animals , Cells, Cultured , Child, Preschool , Desmin/biosynthesis , Dystrophin/biosynthesis , Genetic Vectors/biosynthesis , Genetic Vectors/chemical synthesis , HLA Antigens/biosynthesis , Histocompatibility Antigens Class I/biosynthesis , Humans , Mice , Mice, Inbred BALB C , Mice, SCID , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Muscular Dystrophies/metabolism , Muscular Dystrophies/pathology
11.
Muscle Nerve ; 21(5): 567-76, 1998 May.
Article in English | MEDLINE | ID: mdl-9572235

ABSTRACT

Duchenne muscular dystrophy is an X-linked devastating disease due to the lack of expression of a functional dystrophin. Unfortunately, the dystrophin-deficient mdx mouse model does not present clinical signs of dystrophy before the age of 18 months, and the role of dystrophin in fiber integrity is not fully understood. The fragility of the skeletal muscle fibers was investigated in transgenic mice expressing beta-galactosidase under the control of a muscle specific promoter. Adult mdx/beta-galactosidase (dystrophin-negative) and normal/beta-galactosidase (dystrophin-positive) mice were submitted to one short session of eccentric, downhill running exercise. The leakage of muscle enzymes creatine kinase and beta-galactosidase was investigated before, 1 h after, and 3 days after the running session. A significant and transient rise in the level of these enzymes was noted in the serum of mdx mice following the exercise session. Thus, the lack of dystrophin in the mdx model led to local microdamages to the exercised muscle allowing leakage of proteins from the fibers. The peak leakage was transient, suggesting that muscle fiber lesions were rapidly repaired following this short, noninvasive eccentric running session.


Subject(s)
Mice, Inbred mdx/physiology , Muscle, Skeletal/physiopathology , Running , Animals , Behavior, Animal/physiology , Creatine Kinase/metabolism , Female , Male , Mice , Muscle, Skeletal/enzymology , Reference Values , beta-Galactosidase/metabolism
12.
Transplantation ; 65(6): 793-9, 1998 Mar 27.
Article in English | MEDLINE | ID: mdl-9539090

ABSTRACT

BACKGROUND: The inflammatory reaction that occurs during the 5 days after transplantation led at 3 days to the death of 70% of injected myoblasts. Use of anti-inflammatory agents appeared to be a possible way to increase myoblast survival. The application of gene transfer techniques to cell transplantation offers the potential for the prevention of inflammatory reaction. METHODS: In this study, transforming growth factor-beta1 (TGF-beta1) gene was introduced in myoblasts with a retroviral vector to permit the secretion of this anti-inflammatory cytokine. Survival of (1) infected myoblasts expressing TGF-beta1 or (2) normal myoblasts transplanted with genetically modified cloned myoblasts was compared with survival of normal myoblasts. RESULTS: Expression of TGF-beta1 by myoblasts or by cotransplanted cells decreased myoblast mortality after 3 days by roughly 20% (66.0+/-3.0% in control vs. 46.3+/-4.2% and 46.2+/-5.9%). The increase of myoblast survival by TGF-beta1 expression was correlated with a lower polymorphonuclear cell and macrophage infiltration in muscles compared with control. In addition, cytotoxicity of neutrophils against myoblasts was assayed in vitro. The oxidation of myoblasts by activated neutrophils was decreased after infection of the myoblasts with the TGF-beta1 retroviral vector. CONCLUSIONS: These data demonstrate that the insertion of TGF-beta1 decreases inflammatory reaction observed after myoblast transplantation and thus prolongs their survival.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Muscle, Skeletal/transplantation , Transforming Growth Factor beta/pharmacology , Animals , Cell Line , Cells, Cultured , Gene Expression , Humans , Inflammation/pathology , Leukocyte Count , Mice , Muscle, Skeletal/cytology , Muscles/pathology , Neutrophils , Peroxidase/metabolism , Recombinant Proteins , Transforming Growth Factor beta/genetics
13.
Muscle Nerve ; 21(1): 91-103, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9427228

ABSTRACT

Myoblast cultures from normal and Tg-MDA (transgenic mouse overexpressing dystrophin 50-fold) mice were transplanted into dystrophin-deficient mdx mouse muscles. Four weeks after transplantation, dystrophin-positive fibers were observed four times more frequently in cross sections of muscles injected with Tg-MDA. Myoblasts from Tg-MDA mice also expressing the beta-gal transgene (Tg-MDA/beta-gal) and myoblasts from beta-gal transgenic mice containing one normal dystrophin gene (normal/beta-gal) were also transplanted into mdx mouse muscles. Four weeks after transplantation, the fiber length positive for dystrophin (nuclear domain) was shorter (439 +/- 326 microm) than the beta-gal nuclear domain (1466 +/- 713 microm) of the same fiber when normal/beta-gal myoblasts were transplanted, but increased (1302 +/- 487 microm) when Tg-MDA/beta-gal myoblasts were used. These experiments show that despite the presence in Tg-MDA myoblasts of constructions which lead in vivo in transgenic mice to an overexpression of dystrophin 50-fold, the membrane area over which dystrophin was expressed was increased only threefold. This observation is also expected for vector-mediated gene therapy.


Subject(s)
Dystrophin/biosynthesis , Muscle, Skeletal/transplantation , Animals , Cell Transplantation/methods , Cells, Cultured , Computer Simulation , DNA Primers , DNA, Complementary , Dystrophin/genetics , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Mice, Transgenic , Models, Biological , Muscle, Skeletal/cytology , Muscle, Skeletal/physiology , Nerve Fibers/physiology , Nerve Fibers/ultrastructure , Polymerase Chain Reaction , Recombinant Fusion Proteins/biosynthesis , Regression Analysis , beta-Galactosidase/biosynthesis
14.
J Immunol ; 159(5): 2522-31, 1997 Sep 01.
Article in English | MEDLINE | ID: mdl-9278346

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal/pharmacology , Graft Rejection/pathology , Lymphocyte Function-Associated Antigen-1/physiology , Muscle, Skeletal/cytology , Muscular Dystrophy, Animal/therapy , Stem Cell Transplantation , Animals , Anti-Inflammatory Agents/therapeutic use , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Antibodies, Monoclonal/therapeutic use , Apoptosis , Biomarkers , Cell Adhesion , Cell Death , Cell Line, Transformed , Fluoresceins , Genes, Reporter , Graft Rejection/prevention & control , Immunosuppressive Agents/therapeutic use , Inflammation , Interleukin-1/pharmacology , Lymphocyte Function-Associated Antigen-1/immunology , Macrophage-1 Antigen/physiology , Methylprednisolone/therapeutic use , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred mdx , Mice, SCID , Muscular Dystrophy, Animal/pathology , Naproxen/therapeutic use , Necrosis , Neutrophils/immunology , Neutrophils/pathology , Oxidative Stress , Peroxidase/analysis , Piroxicam/therapeutic use , Radiation Chimera , Tacrolimus/therapeutic use , Whole-Body Irradiation , Zymosan/pharmacology , beta-Galactosidase/analysis
15.
Transplantation ; 63(6): 893-9, 1997 Mar 27.
Article in English | MEDLINE | ID: mdl-9089231

ABSTRACT

Myoblasts obtained from donors histoincompatible for several non-major histocompatibility complex antigens (i.e., including minor histocompatibility antigens) and from syngeneic donors were transplanted without any immunosuppression into the muscles of male dystrophic C57BL/10J mdx/mdx mice. Myoblasts from syngeneic mice resulted in the formation of a high percentage of dystrophin-positive fibers 16 weeks after the transplantation. There was no evidence of a cellular immune reaction against the donor myoblasts, i.e., no infiltration by CD4 or CD8 lymphocytes and no increased expression of granzyme B and interferon-gamma mRNAs. Transplantation of myoblasts obtained from donors histoincompatible only for non- major histocompatibility complex antigens produced a transient increase of dystrophin-positive fibers at 4 weeks after transplantation for some donor strains but not for others. For donor strains that did produce an increase at 4 weeks, the number of dystrophin-positive fibers was reduced 16 weeks after the transplantation. There was evidence of a cellular immune reaction-infiltration by CD4 and by CD8 lymphocytes and increased expression of granzyme B and interferon-gamma mRNAs. Transplantation of myoblasts obtained from male C57BL/10J +/+ mice into female C57BL/10J mdx/mdx mice also led to the presence of only a few dystrophin-positive fibers with the same signs of cellular immune reaction. In this later case, the cellular immune response was attributed to the H-Y minor antigens. Finally, antibodies against fetal calf serum were detected after both syngeneic and nonsyngeneic transplantations, indicating that the culture medium may also be a source of antigens. In mice, the presence of these antibodies against culture medium did not reduce the success of a first syngeneic transplantation.


Subject(s)
Graft Rejection/immunology , Histocompatibility Antigens/immunology , Muscle, Skeletal/cytology , Muscle, Skeletal/transplantation , Muscular Dystrophy, Animal/therapy , Transplantation, Homologous/immunology , Transplantation, Isogeneic/immunology , Actins/biosynthesis , Animals , Animals, Newborn , Antibody Formation , Cells, Cultured , Female , Immunity, Cellular , Interferon-gamma/biosynthesis , Isoantibodies/biosynthesis , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Inbred mdx , Polymerase Chain Reaction , Transplantation, Homologous/methods , Transplantation, Homologous/pathology , Transplantation, Isogeneic/methods , Transplantation, Isogeneic/pathology
16.
Cell Transplant ; 6(2): 101-7, 1997.
Article in English | MEDLINE | ID: mdl-9142441

ABSTRACT

Myoblast transplantation is a potential treatment for Duchenne Muscular Dystrophy. This article confirms by experiments in mice that one problem that has limited the success of clinical trials of this procedure is a rapid (within 3 days) inflammatory reaction which kills most of the injected myoblasts. The death of the transplanted myoblasts can be prevented by treating the host with a mAb against LFA-1. This led to a 27-fold increase in the number of muscle fibers expressing a reporter gene present in the donor myoblasts when the host is also adequately immunosuppressed with FK506. Therefore, both the nonspecific inflammatory reaction and the specific immune response should be adequately controlled following myoblast transplantation.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Cell Transplantation , Lymphocyte Function-Associated Antigen-1/physiology , Muscle, Skeletal/cytology , Transplantation, Homologous/immunology , Animals , Ascites , Crosses, Genetic , Enzyme-Linked Immunosorbent Assay , Female , Genes, Reporter , Immunoglobulin G , Immunosuppressive Agents/therapeutic use , Inflammation/prevention & control , Lymphocyte Function-Associated Antigen-1/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Transgenic , Rats , Tacrolimus/therapeutic use , Transplantation, Homologous/pathology , beta-Galactosidase/biosynthesis , beta-Galactosidase/genetics
17.
Muscle Nerve ; 19(7): 829-35, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8965835

ABSTRACT

Myoblasts from C57BL/10SnJ+/+ were transplanted in major histocompatibility complex (MHC)-compatible mice (i.e., C57BL10SnJ+/+ and C57BL/10SnSc mdx/mdx) and in MHC-noncompatible (BALB/c+/+) mice. The recipients were killed 1-21 days after transplantation. C57BL10SnJ+/+ myoblasts were also transplanted in a few BALB/c+/+ mice treated with FK506 and killed 7 days thereafter. Our results showed that after MHC-noncompatible transplantation, interferon-gamma (IFN-gamma) mRNA expression is increased from day 5 to day 21, indicating the presence of a cellular immune reaction. Short-term immunosuppressive treatment with FK506 inhibited the transcription of IFN-gamma mRNA compared with that in untreated mice. Myoblast-specific antibodies were also detected 2 and 3 weeks after MHC-incompatible transplantation, indicating that the cellular immune reaction, revealed by the increase in IFN-gamma, was followed by a humoral reaction.


Subject(s)
Cell Transplantation , Histocompatibility , Immunosuppressive Agents/pharmacology , Interferon-gamma/genetics , Muscles/cytology , RNA, Messenger/antagonists & inhibitors , Tacrolimus/pharmacology , Animals , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Polymerase Chain Reaction , Postoperative Period , RNA, Messenger/metabolism
18.
J Neuropathol Exp Neurol ; 55(6): 687-97, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8642394

ABSTRACT

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.


Subject(s)
Immunosuppressive Agents/pharmacology , Muscle Fibers, Skeletal/transplantation , Tacrolimus/pharmacology , Animals , Base Sequence , Chlorocebus aethiops , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Interleukin-2/genetics , Interleukin-2/immunology , Interleukin-6/genetics , Interleukin-6/immunology , Macaca mulatta , Molecular Sequence Data , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/immunology , Muscle, Skeletal/cytology , Muscle, Skeletal/embryology , Muscle, Skeletal/immunology , Polymerase Chain Reaction , RNA, Messenger/analysis , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/immunology
19.
Transplantation ; 60(9): 1011-6, 1995 Nov 15.
Article in English | MEDLINE | ID: mdl-7491674

ABSTRACT

Normal C57BL/10SnJ myoblasts were transplanted into the tibialis anterior of C57BL/10SnJ, C57BL/ScSn mdx, or BALB/c mice. These transplantations allowed us to investigate the immune response not only against MHC but also against dystrophin introduced in the dystrophic muscles by such transplantations. Recently, our group reported following myoblast transplantations cellular infiltration of the host muscle by class II MHC cells, macrophages, and lymphocytes expressing CD4 or CD8 and IL-2 receptors. In the present study, activation of these infiltrating lymphocytes was investigated by measuring the expression of granzyme B mRNA. We used reverse-transcriptase polymerase chain reaction to detect granzyme B mRNA at various intervals after myoblast transplantations. To standardize the results, the mRNA were reverse transcribed using an oligo (dt) so that beta-actin mRNA could also be amplified from the same cDNA preparation. Granzyme B mRNA was increased for at least 3 weeks after MHC alloincompatible grafts. The absence of increased granzyme B expression after allocompatible transplantation in mdx mice suggests that dystrophin is not sufficiently immunogenic to induce short term acute rejection. These results indicate that lymphocytes infiltrated in muscles injected with histoincompatible myoblasts are activated and sustain the requirement for an adequate immunosuppression after such transplantations.


Subject(s)
Gene Expression , Muscles/transplantation , Serine Endopeptidases/biosynthesis , Transplantation, Homologous/physiology , Animals , Base Sequence , CD4 Antigens/analysis , CD4 Antigens/biosynthesis , CD8 Antigens/analysis , CD8 Antigens/biosynthesis , DNA Primers , Granzymes , Histocompatibility Antigens Class II/analysis , Histocompatibility Testing , Humans , Infant , Lymphocytes/immunology , Macrophages/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred mdx , Molecular Sequence Data , Muscles/enzymology , Muscles/immunology , Polymerase Chain Reaction , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Receptors, Interleukin-2/analysis , Receptors, Interleukin-2/biosynthesis , Transplantation, Homologous/immunology
20.
Muscle Nerve ; 18(9): 980-6, 1995 Sep.
Article in English | MEDLINE | ID: mdl-7643878

ABSTRACT

A mutagenesis RT-PCR method was used to detect normal dystrophin mRNA following the injection of normal myoblasts in mdx mice using two immunosuppressors. A specific sequence of the dystrophin mRNA (257 bp) was amplified by RT-PCR from the muscle total RNA. MaeIII digestion of the amplified products allowed us to distinguish the normal messenger of dystrophin from the dystrophic one and to establish the percentage of normal and of dystrophic (mdx) dystrophin mRNA. Normal dystrophin mRNA was detected using this technique in mdx muscles transplanted with histocompatible normal myoblasts. For this type of transplantation, no significant difference in the percentage of normal dystrophin mRNA was observed between immunosuppressed mice and those not immunosuppressed. No normal dystrophin mRNA was, however, observed in mdx mice following histoincompatible normal myoblast transplantation without immunosuppression. When such transplantations were done in mice immunosuppressed with cyclosporine or FK-506, normal dystrophin mRNA accounted for 31% and 36% of the total dystrophin mRNA, respectively. In fact, one animal immunosuppressed with FK-506 expressed as much as 57% of normal dystrophin mRNA. These results thus show that FK-506 makes it possible to restore dystrophin expression to a level comparable to that observed in DMD carriers that are usually asymptomatic.


Subject(s)
Dystrophin/biosynthesis , Muscles/embryology , Muscular Dystrophy, Animal/metabolism , Stem Cell Transplantation , Animals , Antibodies/immunology , Base Sequence , Cell Transplantation , Cells, Cultured , DNA Probes , Flow Cytometry , Immunosuppression Therapy , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Molecular Sequence Data , Muscles/cytology , Muscles/metabolism , Mutagenesis , Polymerase Chain Reaction , RNA, Messenger/metabolism , Sarcolemma/metabolism
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