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1.
Gene Ther ; 9(21): 1429-37, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12378405

ABSTRACT

A first-line gene therapy for type 1 diabetes should be based on a safe procedure to engineer an accessible tissue for insulin release. We evaluated the ability of the skeletal muscle to release human insulin after electrotransfer (ET)-enhanced plasmid DNA injection in mice. A furin-cleavable proinsulin cDNA under the CMV or the MFG promoter was electrotransferred to immune-incompetent mice with STZ-induced severe diabetes. At 1 week, mature human insulin was detected in the serum of 17/20 mice. After an initial peak of 68.5 +/- 34.9 microU/ml, insulin was consistently detected at significant levels up to 6 weeks after gene transfer. Importantly, untreated diabetic animals died within 3 weeks after STZ, whereas treated mice survived up to 10 weeks. Fed blood glucose (BG) was reduced in correspondence with the insulin peak. Fasting BG was near-normalized when insulin levels were 12.9 +/- 5.3 (CMV group, 2 weeks) and 7.7 +/- 2.6 microU/ml (MFG group, 4 weeks), without frank hypoglycemia. These data indicate that ET-enhanced DNA injection in muscle leads to the release of biologically active insulin, with restoration of basal insulin levels, and lowering of fasting BG with increased survival in severe diabetes. Therefore the skeletal muscle can be considered as a platform for basal insulin secretion.


Subject(s)
DNA/administration & dosage , Diabetes Mellitus, Experimental/therapy , Electroporation , Genetic Therapy/methods , Insulin/genetics , Muscle, Skeletal/metabolism , Animals , Diabetes Mellitus, Experimental/metabolism , Injections, Intramuscular , Male , Mice , Mice, SCID
2.
Science ; 279(5356): 1528-30, 1998 Mar 06.
Article in English | MEDLINE | ID: mdl-9488650

ABSTRACT

Growth and repair of skeletal muscle are normally mediated by the satellite cells that surround muscle fibers. In regenerating muscle, however, the number of myogenic precursors exceeds that of resident satellite cells, implying migration or recruitment of undifferentiated progenitors from other sources. Transplantation of genetically marked bone marrow into immunodeficient mice revealed that marrow-derived cells migrate into areas of induced muscle degeneration, undergo myogenic differentiation, and participate in the regeneration of the damaged fibers. Genetically modified, marrow-derived myogenic progenitors could potentially be used to target therapeutic genes to muscle tissue, providing an alternative strategy for treatment of muscular dystrophies.


Subject(s)
Bone Marrow Cells/physiology , Muscle, Skeletal/cytology , Muscle, Skeletal/physiology , Regeneration , Stem Cells/physiology , Animals , Bone Marrow Cells/cytology , Bone Marrow Transplantation , Cell Differentiation , Cell Movement , Genetic Therapy , Humans , Mice , Mice, SCID , Mice, Transgenic , Muscle Fibers, Skeletal/cytology , Muscular Dystrophies/therapy , Stromal Cells/cytology , Stromal Cells/physiology , beta-Galactosidase/analysis , beta-Galactosidase/genetics
3.
Neuron ; 13(4): 813-21, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7946330

ABSTRACT

Embryonic mouse neural tubes produce a variety of terminally differentiated cells in vitro, mostly neurons and glia. We report here that some of these cells differentiate into skeletal muscle cells. The possibility of mesoderm contamination was ruled out as follows. First, Dil+ muscle cells were present in cultures from a Dil-labeled neuroepithelium. Second, a small fraction of cultured neural tube cells coexpressed muscle myosin and neuronal beta III tubulin within the same cell. Third, embryos generated from embryonic stem cells in which nlacZ was targeted into the myogenic gene myf-5 expressed nlacZ in a localized region of the neural tube. These myf-5+ cells coexpress neuronal and muscle markers in culture. The developmental significance of this phenomenon is discussed in the context of overlapping regulatory networks between myogenesis and neurogenesis.


Subject(s)
Central Nervous System/cytology , Central Nervous System/embryology , DNA-Binding Proteins , Muscle, Skeletal/cytology , Trans-Activators , Animals , Carbocyanines , Cell Differentiation , Cells, Cultured , Fluorescent Antibody Technique , Fluorescent Dyes , Gene Targeting , Immunohistochemistry , Mice , Muscle Proteins/analysis , Muscle Proteins/genetics , Myogenic Regulatory Factor 5 , Sarcomeres/immunology , Tubulin/analysis , beta-Galactosidase/analysis
4.
Atherosclerosis ; 100(2): 133-9, 1993 May.
Article in English | MEDLINE | ID: mdl-8357347

ABSTRACT

To test the affinity of a new F(ab')2 monoclonal antibody (TRF1) against human fragment D dimer of cross-linked fibrin for atherosclerotic plaques free of detectable thrombi, 6 atherosclerotic segments of carotid and femoral artery, and as a control 5 segments of atherosclerosis-free internal mammary artery, were drawn from 11 male patients undergoing bypass surgery. All segments were carefully washed in order to remove possible endoluminal thrombi, and cut to obtain pairs of intimal fragments of similar weight, containing either plaques (n = 16), or fatty streaks (n = 12), or normal endothelium (n = 20). Each fragment underwent a direct binding test to TRF1, or to a non-specific antibody, both labeled with 125I. The activity in each fragment was measured after 3 h of incubation at 37 degrees C, and after washing the fragments every hour for 3 h. TRF1 binding (as percentage of initial activity) was significantly higher (P < 0.001) in atherosclerotic than in normal fragments (26% +/- 11.5%, vs. 9.2% +/- 3.9% in fatty streaks, and 1.9% +/- 0.6% in normal endothelium), and indirect immunofluorescence confirmed TRF1 uptake within the plaque wall. By contrast, the non-specific antibody did not show any significant binding. These preliminary results demonstrate the high specific affinity of TRF1 for atherosclerotic plaques, probably due to the hemorheologic phenomena that activate platelets and provoke the formation of fragment D dimers of cross-linked fibrin on the plaque surface.


Subject(s)
Arteriosclerosis/diagnostic imaging , Iodine Radioisotopes , Arteriosclerosis/pathology , Carotid Arteries/diagnostic imaging , Carotid Arteries/pathology , Femoral Artery/diagnostic imaging , Femoral Artery/pathology , Fibrin/immunology , Fluorescent Antibody Technique , Humans , In Vitro Techniques , Male , Middle Aged , Radionuclide Imaging
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