Biomedical subjects
Publications and source records attributed to G Meola.
The uncommon case is described of a girl severely affected with Duchenne muscular dystrophy. Cytogenetic analysis revealed no numerical or structural abnormalities of the X-chromosome in any of the cells examined (leucocytes and myoblasts). No abnormality in morphology, growth pattern or differentiation was observed in the dystrophic muscle cultures as compared with control cultures.
Human fetal and adult Schwann cells, which had been maintained in culture for 5 weeks according to an explant-reexplantation technique, were labeled by immunoperoxidase using antibodies directed against S-100 protein and laminin in order to find specific antigenic markers. Immunocytochemical analysis of the distribution of both proteins showed that they were expressed in long-term cultures. The localization of S-100 protein and laminin in long-term cultures indicated that the expression of these proteins by human Schwann cells was not axon-dependent and also occurred in absence of myelin synthesis.
Fibronectin expression during human muscle differentiation was investigated by determining its distribution in foetal, normal adult and dystrophic muscle and in foetal, normal adult and dystrophic muscle cultures during myogenesis. Muscle sections and muscle cultures were studied by indirect immunofluorescence staining using polyclonal and monoclonal anti-human antibodies. Mass and clonal muscle cultures were prepared from foetal, adult and dystrophic muscle tissue. Immunofluorescence staining detected fibronectin on the epimysium, perimysium and endomysium of transverse sections of normal adult muscle, while sarcoplasm was devoid of this glycoprotein. In foetal muscle, some fibers showed a prominent ring of fibronectin. In mass and clonal cultures, myoblasts were found to synthesize and accumulate fibronectin while myotubes did not. No difference in fibronectin distribution was observed between Duchenne Muscular Dystrophy (DMD) and control myotubes. An enzyme-linked immunoassay (ELISA), performed on homogenated muscle, sonicated fibroblasts and muscle cells, showed a high fibronectin level in fibroblasts when compared with the other samples tested.
The role of collagen type III and IV (Coll III, IV) in Duchenne muscular dystrophy (DMD) was investigated by the indirect immunofluorescence technique (IIF) both in muscle biopsies and derived cell cultures. Ten dystrophic cases were studied and compared with twelve suitable control cases, extending the IIF analysis to two other representative non-collagenous proteins of the extracellular matrix (ECM), namely fibronectin (FN) and laminin (LM). DMD biopsies generally displayed a thickening of endomysial and/or perimysial connective, as compared to control specimens. All the markers analyzed were found to contribute to this connective proliferation, although from a quantitative point of view the relative involvement decreases progressively from FN through Coll III and IV to LM. However, due to Coll IV selective endomysial localization, Coll IV alteration can be considered a specific indicator of a muscle cell defect. Results from DMD muscle cultures revealed no significant changes in comparison to control cultures, except for Coll IV. A well-organized, Coll IV-containing pericellular matrix was noted in a fraction of DMD cultures as a unique feature seen in no control culture. This alteration was again considered significant since Coll IV is the only marker clearly associated with the myotube component still expressed in vitro. The negative results obtained on the other marker proteins should not however be considered definitive, due to the lack in the simplified in vitro system used of humoral and/or neuronal factors which may be needed to express gene defect(s) in differentiated cells.
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Muscle was cultured from a 7-month-old boy affected by generalized weakness, macroglossia, cardiomegaly, hepatomegaly and increasing dyspnea. Muscle biopsy showed a vacuolar myopathy with glycogen accumulation (Pompe's disease). The muscle was cultured to verify whether the abnormality could be expressed in culture during myogenesis. In the living muscle cultures, phase-contrast microscopy revealed that myotubes as young as two weeks were vacuolated and that the vacuolization was higher in the older cultures compared to parallel control cultures. Fluorescent microscopy by acridine orange staining of the cultures showed a marked increase in acridine orange positive material (presumptive lysosomes) throughout the sarcoplasm. Electron microscopic data revealed myofibrillar destruction in the muscle biopsy and vacuolized cytoplasm in the Schwann cells. Cytochemically, the patient's myotubes stained very intensely for acid phosphatases. The increased acid phosphatase activity was quantitatively confirmed by cytophotometric evaluation performed on patient and control parallel myotubes. This is the first evidence that an increase in acid phosphatases has been quantitatively demonstrated in cultured muscle from a patient with acute infantile onset acid maltase deficiency (Pompe's disease) although the enzymatic activity was assayed at only one time of incubation.
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A case of M-type PFK deficiency due to the synthesis of a structurally abnormal and catalytically inactive M-subunit was reported. PFK activity was reduced (39% of normal) in red cells, normal in leukocytes and platelets, and absent in muscle. The red cell enzyme was not inhibited by antiserum to human muscle PFK and displayed normal biochemical properties (Km for ATP and fructose-6-phosphate, storage stability at +4 degrees C and -80 degrees C, optimum pH, electrophoretic pattern and molecular weight). The complete lack of PFK activity in muscle was confirmed on both histological preparations and muscle extracts. Double immunodiffusion analysis using an antinormal M-PFK serum revealed that the enzyme molecule was present and immunologically identical with normal, although it was catalytically inactive. The muscle abnormality was also confirmed by electromyography, ischemic exercise testing, histochemistry and electron microscopy. Moreover, PFK activity was investigated in myoblast cultures maintained up to 25 days, and it was found to be absent.
Two sisters with congenital nemaline myopathy are described. In both cases almost 70% of muscle fibers contained rods which were selectively localized in the larger ones. The variability coefficient was abnormally increased. Histochemical reactions showed that almost all the muscle fibers were type 1. In one case many fibers contained one or more core-like lesions. The parents and two siblings of the patients were clinically normal; EMG examination also showed normal motor unit potentials. Muscle biopsy was normal in the father; in the mother a slight type 1 predominance was detected without rods or other signs of myopathy. The disease seems to be transmitted by an autosomal recessive trait, although incomplete penetrance of a dominant trait cannot be excluded.
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The authors report a patient with systemic carnitine deficiency in which, despite restoration of normal serum carnitine level by therapy, the fatal course of the disease progressed. Postmortem studies of cardiac muscle with Oil red O failed to show lipid accumulation which was still present in muscle and liver. Carnitine level in liver was less than 10% of the controls values; total carnitine in muscle was 50% of the controls with differences in its esterification pattern; whereas the free carnitine was 1/10 of normal, the long chain esterified one was 3 times higher than the control. Heart carnitine content was normal. The authors suggest that although no therapeutic effects were detected in skeletal muscle, the normal carnitine content and the absence of lipidosis in the heart suggest that carnitine therapy may have been beneficial in our patient enabling the heart to utilize fatty acids.
Two cases of pentazocine-induced neuromuscular syndrome in addicted patients are reported. Histochemical and quantitative histographic analyses of muscle biopsies performed in areas distant from site of injection, disclosed type 2 atrophy. A generalized myotoxic effect of pentazocine is suggested.
A critical review of the published cases reports suggests that the muscle changes could be induced by Pentazocine by mechanisms other than local myotoxicity. The mechanism of myotoxicity in Pentazocine-myopathy remains uncertain and for this reason a tissue culture study was performed in order to investigate the close relationship between myopathy and Pentazocine. The effects of Pentazocine on the extent of myogenesis in human foetal muscle cultures were investigated. The phase contrast study, the light microscopy the quantitative assessment of myogenesis (the number of nuclei incorporated into myotubes were counted and expressed as a percentage of the total number of nuclei) showed a complete block of myogenesis in Pentazocine treated culture and a delayed fusion process was present in D-arabinofuranosylcytosine (Ara-C)-Pentazocine-treated cultures. The present preliminary data, however, suggest that muscle stem cells could participate in the replacement of skeletal muscle by fibrous tissue in neuromuscular syndrome induced by Pentazocine.
The authors have investigated the uncommon occurrence of a boy affected with Duchenne muscular dystrophy (DMD) whose mother showed myopathic features in the clinical history, EMG, biochemical tests and muscle biopsy. This study suggests that the patient's mother is a manifesting carrier of X-linked DMD with clinical, neurophysiological, biochemical and histological findings of X-linked DMD with an almost complete inactivation of the paternal X-chromosome (lyonization).
The following aspects have been investigated in 10 patients affected by Huntington's disease )HD): --extensive haematological investigations; --red cell enzyme activities and level of the most important glycolytic intermediate compounds; --protein, lipid and carbohydrate composition of the erythrocyte membrane and membrane polarity; --effects of in vitro aging on red cell membranes. Lack of 4.5 protein band in SDS-PAGE and 14-fold decrease in membrane-bound catalase were found in the in vitro aged red cells from the 10 HD patients examined. Na+ + K+ATPase was slightly higher than normal in all the patients. Red cells from 5 out of 8 patients showed a decrease in reduced glutathione and phosphoenolpyruvate levels and/or an increase in hexokinase, glucose-6-phosphate dehydrogenase, pyruvate kinase and glutathione reductase activities. The haematological investigations, the protein lipid and carbohydrate composition of the fresh red cells, the membrane polypeptide aggregates and the membrane polarity evaluated by microspectrofluorometric analysis were normal.
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