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Biomedical subjects

G Karpati

Publications and source records attributed to G Karpati.

At least 73 records · Page 4Linked to original sources
PubMed1997

Abnormal oxidative metabolism in exercise intolerance of undetermined origin.

Twenty-four patients with exercise intolerance of undetermined origin were examined by muscle phosphorus magnetic resonance spectroscopy (31P-MRS) to test for a possible underlying defect in oxidative metabolism. Results were compared to those of 37 normal controls and 22 patients with well-defined mitochondrial disorders. In 17 (71%) patients with exercise intolerance, ADP recovery after exercise, an index of mitochondrial function, was abnormally slow. The energy state of phosphate-containing metabolites at rest was abnormal in 33% of patients. In 17/22 patients with well-defined mitochondrial disorders, ADP recovery was similarly slow. Abnormalities at rest were slightly more prevalent (50%) in this group of patients. Other 31P-MRS measurements did not add to the overall sensitivity in detecting abnormalities in either of these groups. We suggest that many patients with exercise intolerance of undetermined cause may have impaired muscle oxidative metabolism, that is an important causative factor in the pathophysiology of their symptoms.

Adenosine Diphosphate↗
PubMed1997

Efficient muscle-specific transgene expression after adenovirus-mediated gene transfer in mice using a 1.35 kb muscle creatine kinase promoter/enhancer.

Replication-defective (E1-E3-deleted) human adenovirus vectors are a promising means of therapeutic gene delivery to skeletal muscle cells. Since the tropism of adenovirus is nonselective, muscle-specific expression of systemically administered vectors can only be achieved by the use of a tissue-specific promoter/enhancer that is small enough to fit the insert capacity of the vector. We have generated two replication-defective adenovirus recombinants (AV) in which the reporter gene (either firefly luciferase or E. coli beta-galactosidase) was driven by a truncated (1.35 kb) muscle creatine kinase (MCK) promoter/enhancer or by the fast troponin I (TnI) promoter/enhancer. Highly efficient and muscle-specific transgene expression was demonstrated in immunodeficient mice after local injection of AV into muscles at an early age. In nonmuscle tissues (brain, liver, kidney, lung), the transgene expression was extremely low even though in these tissues in situ polymerase chain reaction showed as high an infectivity of the cells by the AV as in muscle. The relatively small size, the good efficiency and the muscle specificity of the MCK promoter would make it ideal to drive the 6.3 kb (truncated) dystrophin cDNA in first generation AV (with a limited (8 kb) insert capacity) designed for gene therapy of Duchenne muscular dystrophy.

Adenoviridae↗
PubMed1997

Complete restoration of a wild-type mtDNA genotype in regenerating muscle fibres in a patient with a tRNA point mutation and mitochondrial encephalomyopathy.

Replicative segregation of mitochondrial DNA (mtDNA) can produce large differences in the proportions of wild-type and mutant mtDNAs in different cell types of patients with mitochondrial encephalomyopathy. This is particularly striking in the skeletal muscle of patients with Kearns-Sayre syndrome (KSS), a sporadic disease associated with large-scale mtDNA deletions, and in sporadic patients with tRNA point mutations. Although the skeletal muscle fibres of these patients invariably contain a large proportion of mutant mtDNAs, mutant mtDNAs are rare or undetectable in satellite cells cultured from the same muscle biopsy specimens. Since satellite cells are responsible for muscle fibre regeneration, restoration of the wild-type mtDNA genotype might be achieved in these patients by encouraging muscle regeneration. To test this concept, we re-biopsied a patient with a KSS phenotype and a mtDNA point mutation in the tRNAleu(CUN)gene and analysed muscle fibres regenerating at the site of the original muscle biopsy. Regenerating fibres were identified by morphological criteria and by expression of neural cell adhesion molecule (NCAM). All such fibers were positive for cytochrome c oxidase (COX) activity by cytochemistry and essentially homoplasmic for wild-type mtDNA, while the majority of non-regenerating fibres were COX-negative and contained predominantly mutant mtDNAs. These results demonstrate that it may be possible to improve muscle function in similar patients by methods that promote satellite cell incorporation into existing myofibres.

Biopsy↗
PubMed1997

Adenovirus-mediated wild-type p53 gene transfer and overexpression induces apoptosis of human glioma cells independent of endogenous p53 status.

Mutation or inactivation of the p53 tumor suppression gene is an early alteration in the transformation of glial cells to gliomas. To study the effect of exogenous wild-type p53 on glioma cell growth, human glioma lines U251 MG, U87 MG and A172 were infected with an adenovirus vector expressing either wild-type p53 or bacterial lacZ. Rapid cell death occurred only in the p53-transduced cell lines and was characterized by nuclear condensation, formation of nucleosomal DNA ladders, and positive in situ end-labeling of DNA, suggesting that apoptosis had been induced. The U87 MG cell line that contains wild-type p53 as evidenced by wild-type p53-dependent transcription activity also underwent apoptosis within 2 to 3 days after infection. These results suggest that the presence of endogenous wild-type p53 does not preclude apoptosis by overexpression of exogenous p53.

Adenoviridae↗
PubMed1997

Gene therapy research for Duchenne and Becker muscular dystrophies.

Gene therapy is a promising option for the definitive treatment of Duchenne and Becker muscular dystrophies. Presently, gene therapy for Duchenne and Becker muscular dystrophies is still in the preclinical stage with dystrophin-deficient animals (the mdx mouse and a golden retriever dog strain) serving as convenient models. The thrust of research during the past 18 months has focused on two approaches: adenovirus-mediated dystrophin gene transfer and upregulation of a natural dystrophin analogue, utrophin. In the area of adenovirus-mediated gene transfer, substantial progress has been made in characterizing and mitigating the deleterious immune responses to the vector and transgene proteins. Furthermore, new adenovirus vectors have been created with reduced immunogenicity and increased insert gene capacity, which enhance the longevity of the transgene expression. Additional efforts are underway to develop safe and efficient routes of administration of the adenovirus vector carrying the dystrophin expression cassette. The prospects of utrophin upregulation as an attractive strategy for treatment of Duchenne and Becker muscular dystrophies was greatly enhanced by the demonstration of a substantial mitigation of the dystrophic phenotype of the transgenic mdx mouse overexpressing utrophin.

Animals↗
PubMed1996

Impairment of force generation after adenovirus-mediated gene transfer to muscle is alleviated by adenoviral gene inactivation and host CD8+ T cell deficiency.

Recombinant adenovirus vectors (AdV) hold promise as a means of delivering therapeutic genes to muscle in diseases such as Duchenne muscular dystrophy (DMD). However, we have previously shown that the use of AdV is hampered by the development of reduced force-generating capacity, which occurs within 1 week and is progressive up to at least 1 month after AdV delivery in immune-competent animals. Determinations of muscle force production provide a sensitive and clinically important measure of potential adverse effects of AdV-mediated gene transfer on muscle cell function. In the present study, we investigated the role of AdV-related gene expression and host T lymphocyte responses in the genesis of muscle dysfunction following AdV injection of muscle. We report that UV-irradiation of AdV particles, which reduced AdV transcriptional activity without impairing infectivity (as confirmed by in situ polymerase chain reaction), significantly reversed early (4 days post-injection) AdV-induced contractile impairment in immune-competent mice as well as in mice lacking effective CD8+ T cell activity. The superimposed additional reduction in force-generating capacity normally found between 4 and 30 days post-AdV delivery in immune-competent mice, along with the associated loss of transgene (beta-galactosidase) expression, was largely abrogated by the absence of an intact CD8+ T lymphocyte response. Furthermore, short-term administration of a neutralizing antibody against CD4+ T cells significantly prolonged transgene expression and showed a trend toward mitigation of AdV-induced reductions in force-generating capacity. Cellular infiltration and humoral immune responses against the vector and transgene product were also blunted to varying degrees in the setting of CD8+ or CD4+ T cell deficiency. We conclude that AdV-related gene expression has an early negative (probably toxic) effect on muscle cell function that is independent of CD8+ T cell-mediated immunity. In contrast, further progression of contractile impairment and the accompanying loss of transgene expression from AdV-injected muscle are largely dependent upon the activity of CD8+ T cells. These results have implications for the design of future generation vectors and the potential need for immunosuppressive therapy after AdV-mediated gene transfer to muscle.

Adenoviridae↗
PubMed1996

Dystrophin expression in muscles of mdx mice after adenovirus-mediated in vivo gene transfer.

We have generated high-titer adenoviral recombinants (AVR) expressing a 6.3-kb partial dystrophin cDNA insert under the control of either the Rous sarcoma virus (RSV) or cytomegalovirus (CMV) promoter. These AVR preparations were free of both E1-containing AVR and AVR with a nonfunctional dystrophin expression cassette. With these optimal AVR preparations, we have obtained a high degree of short-term (10 days) expression of a truncated (approximately 200 kD) dystrophin in dystrophin-deficient mdx muscles injected in the neonatal period; a lesser degree of expression of dystrophin was found in muscles injected in the young adult age and in old animals. Microscopic indices of muscle damage revealed that the truncated dystrophin provided a significant protection of the transduced muscle fibers. However, by 60 days post-injection, a substantial reduction of the number of dystrophin-positive fibers was noted, even in the neonatally injected muscles, and near-total elimination of dystrophin-positive fibers occurred in muscles injected in the adult age. These effects appeared to be brought about by the activity of CD8+ cytotoxic lymphocytes directed against the transduced cells, leading to their eventual elimination. In severe combined immunodeficiency (SCID) mice, lacking both humoral and cellular immune competence, muscles transduced (either in the neonatal or adult age) by AVR containing a CMV-LacZ expression cassette maintained the early (10 day) transduction level up to 30 days post-injection. Systemic administration of AVR (i.e., into the left ventricle of the heart) led in 5 days to a high number of dystrophin-positive fibers in heart, diaphragm, and intercostal muscles but not in limb muscles.

Adenoviruses, Human↗
PubMed1996

Impairment of muscle mitochondrial oxidative metabolism in McArdles's disease.

Impairment of muscle glycogenolysis in McArdle's disease (myophosphorylase deficiency) leads to exercise intolerance and exercise-induced myalgia. The pathophysiology of these symptoms is not entirely clear. We used phosphorus magnetic resonance spectroscopy to measure muscle phosphate metabolite concentrations and intracellular pH during brief ischemic exercise and in the period of aerobic metabolic recovery after exercise, with special attention to cytoplasmic adenosine 5'-diphosphate (ADP). In 5 patients with McArdle's disease, calculated muscle intracellular ADP concentrations at the beginning of recovery were higher than in normal control subjects (70-425 mmol/L, control mean: 73 +/- 40 mmol/L, P < 0.05). The half-time for intracellular ADP recovery after exercise, an index of maximal mitochondrial oxidative phosphorylation, was 0.16 +/- 0.07 in normal controls and was independent of metabolic state or intracellular pH. ADP recoveries were abnormally slow in all patients with McArdle's disease (range: 0.32-0.83 min, mean = 0.2 min, P < 0.0001). These results are indicative of a limitation in the rate of oxidative phosphorylation in muscle of patients with McArdle's disease, most likely due to impaired substrate delivery to mitochondria. This impairment of mitochondrial function may contribute to the exercise-related symptoms in McArdle's disease.

Adenosine Diphosphate↗
PubMed1996

Toxicity of replication-defective adenoviral recombinants in dissociated cultures of nervous tissue.

Replication-defective human type 5 adenoviral recombinants (AVR) are very efficient means of introducing foreign genes into neurons in vitro and in vivo; however, a significant reduction in the number of cells expressing reporter genes has been reported to occur over time. In vitro, this may be due to direct toxicity of the protein product of the transgene or adenoviral molecules. In vivo, in addition, an immune attack by the host could eliminate the transduced cells. To assess the direct toxicity of AVR or reporter gene products, a quantitative study of survival of transduced neurons over a period of 4 weeks was conducted in primary neural cultures. Cultures of dissociated murine spinal cord-dorsal root ganglia were exposed to AVR containing the Escherichia coli lacZ (E. coli lacZ) gene under control of either the very efficient cytomegalovirus enhancer/promoter or the fast muscle troponin I promoter, which is not active in these cells. Two factors contributed to loss of neuronal and nonneuronal cells: (i) direct toxicity of (E1 + E3)-deleted replication-incompetent AVR at high titers [> or = 5 x 10(8) viral particles/ml or multiplicity of infection (m.o.i.) 1000] and (ii) high levels of expression of the reporter gene product, beta-galactosidase, at titers that result in 55-75% transduction efficiency (5 x 10(7)-5 x 10(8) viral particles/ml or m.o.i. 100-1000). Despite the efficacy of adenoviral vectors in introducing foreign genes into primary, postmitotic cells, specific precautions must be taken in their use because of the narrow margin between concentrations of recombinants that transduce a sufficient percentage of cells and those that are cytotoxic.

Adenoviridae↗
PubMed1996

A distinctive type of infantile inflammatory myopathy with abnormal myonuclei.

Four infants developed progressive muscle weakness after a normal initial postnatal development. All patients had a moderate elevation of serum creatine kinase (CK) activity. Muscle biopsies revealed, in addition to myopathic features, endomysial and perivascular inflammation. Electron microscopy disclosed prominent myonuclear abnormalities. Corticosteroids in 3 patients were moderately beneficial. This appears to be a clinicopathologically distinct form of inflammatory myopathy of infants.

Adrenal Cortex Hormones↗
PubMed1996

Chronic inflammatory demyelinating polyneuropathy (CIDP) in diabetics.

Seven diabetic patients developed a progressive, moderately severe, motor rather than sensory neuropathy predominantly affecting the legs. This met clinical and electrophysiological criteria for chronic inflammatory demyelinating polyneuropathy (CIDP). Nerve biopsies showed a variety of abnormalities, none of which clearly distinguished between diabetic polyneuropathy and CIDP. The patients were treated with combinations of corticosteroids, azathioprine, plasmapheresis and intravenous immune globulin; all improved substantially. We believe that CIDP may masquerade as unusually severe and progressive diabetic distal symmetric polyneuropathy. It is important to recognize CIDP in diabetics because, unlike diabetic polyneuropathy, CIDP is treatable.

Adult↗
PubMed1996

The principles of gene therapy for the nervous system.

Research pertaining to gene transfer into cells of the nervous system is one of the fastest growing fields in neuroscience. An important application of gene transfer is gene therapy, which is based on introducing therapeutic genes into cells of the nervous system by ex vivo or in vivo techniques. With the eventual development of efficient and safe vectors, therapeutic genes, under the control of a suitable promoter, can be targeted to the appropriate neurons or glial cells. Gene therapy is not only applicable to the treatment of genetic diseases of the nervous system and the control of malignant neoplasia, but it also has therapeutic potential for acquired degenerative encephalopathies (Alzheimer's disease, Parkinson's disease), as well as for promoting neuronal survival and regeneration in various pathological states.

Animals↗
PubMed1996

Clinical phenotypes of different MPZ (P0) mutations may include Charcot-Marie-Tooth type 1B, Dejerine-Sottas, and congenital hypomyelination.

Hereditary demyelinating peripheral neuropathies consist of a heterogeneous group of genetic disorders that includes hereditary neuropathy with liability to pressure palsies (HNPP), Charcot-Marie-Tooth disease (CMT), Dejerine-Sottas syndrome (DSS), and congenital hypomyelination (CH). The clinical classification of these neuropathies into discrete categories can sometimes be difficult because there can be both clinical and pathologic variation and overlap between these disorders. We have identified five novel mutations in the myelin protein zero (MPZ) gene, encoding the major structural protein (P0) of peripheral nerve myelin, in patients with either CMT1B, DSS, or CH. This finding suggests that these disorders may not be distinct pathophysiologic entities, but rather represent a spectrum of related "myelinopathies" due to an underlying defect in myelination. Furthermore, we hypothesize the differences in clinical severity seen with mutations in MPZ are related to the type of mutation and its subsequent effect on protein function (i.e., loss of function versus dominant negative).

Adult↗
PubMed1996

Mutations in the gene-encoding SERCA1, the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+ ATPase, are associated with Brody disease.

Brody disease is a rare inherited disorder of skeletal muscle function. Symptoms include exercise-induced impairment of skeletal muscle relaxation, stiffness and cramps. Ca2+ uptake and Ca2+ ATPase activities are reduced in the sarcoplasmic reticulum, leading to the prediction that Brody disease results from defects in the ATP2A1 gene on chromosome 16p12.1-12.2, encoding SERCA1, the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+ ATPase. A recent search, however, did not reveal any mutations in the ATP2A1 gene in three Brody patients. We have now associated Brody disease with the autosomal recessive inheritance of three ATP2A1 mutations in two families, suggesting that the disease is genetically heterogeneous. One mutation occurs at the splice donor site of intron 3, while the other two mutations lead to premature stop codons, truncating SERCA1, deleting essential functional domains and raising the intriguing question: how have these Brody patients partially compensated for the functional knockout of a gene product believed to be essential for fast-twitch skeletal muscle relaxation?

Calcium-Transporting ATPases↗
PubMed1996

A novel heteroplasmic tRNAleu(CUN) mtDNA point mutation in a sporadic patient with mitochondrial encephalomyopathy segregates rapidly in skeletal muscle and suggests an approach to therapy.

A novel mtDNA point mutation was detected in the tRNAleu(CUN) gene (G to A at position 12315) in a sporadic patient with chronic progressive external ophthalmoplegia, ptosis, limb weakness, sensorineural hearing loss and a pigmentary retinopathy. The mutation disrupts base pairing in the T psi C stem at a site which has been conserved throughout evolution. Although the other mtDNA tRNAleu gene (UUR) is a hotspot for mutation, this is the first pathogenic mutation to be reported in the gene coding for tRNAleu(CUN). MtDNAs carrying the mutation constituted 94% of total mtDNAs in two separate muscle biopsies. Single fibre analysis showed that skeletal muscle fibres without detectable cytochrome c oxidase activity (COX-ve fibres) contained predominantly mutant mtDNAs (93-98%) while fibres with apparently normal COX activity had up to 90% mutant mtDNAs, demonstrating that the G12315A mutation is functionally recessive. Immunofluorescence studies with specific antibodies to mtDNA- or nuclear-encoded subunits of COX were consistent with a defect in mitochondrial protein translation. The mutation was not present in blood cells or cultured fibroblasts and surprisingly, it could not be detected in satellite cells cultured from the patient's muscle. This pattern, which may by typical of patients who have inherited new germline pathogenic mtDNA mutations, possibly reflects loss of the mutation by random genetic drift in mitotic tissues and proliferation of mitochondria containing the mutant mtDNA in post-mitotic cells. The absence of mtDNA carrying the mutation in satellite cells suggests that regeneration of skeletal muscle fibres from satellite cells could restore a wild-type mtDNA genotype and normal muscle function.

Base Sequence↗
PubMed1996

Combined aerobic training and dichloroacetate improve exercise capacity and indices of aerobic metabolism in muscle cytochrome oxidase deficiency.

There is no generally effective therapy for mitochondrial myopathies. In this study, we measured responses to combined aerobic training and oral dichloroacetate (DCA) therapy in a 25-year-old woman with a mitochondrial myopathy caused by cytochrome oxidase deficiency. The patient trained for 14 weeks, and DCA therapy was begun after 8 weeks. Independent indices of aerobic capacity and oxidative metabolism showed substantial improvement. Venous lactate concentrations at rest, and after a constant amount of work, decreased by approximately 50% after 8 weeks of aerobic training, and by more than 70% with the combination of training and DCA treatment. Heart rate at rest and after a constant amount of submaximal work decreased progressively. Aerobic capacity on a graded submaximal exercise test improved by 71% from baseline by the end of the treatment period. 31P magnetic resonance spectroscopy measurements of rate constants for recovery of muscle phosphocreatine increased 1.7-fold and metabolically active adenine diphosphate increased 2.8-fold after 8 weeks of training alone, and 4.5-fold and 23.0-fold after 14 weeks of training plus DCA treatment. Responses to the SF-36 Health Survey suggested a marked reduction in handicap. Thus, in this open study of a patient with cytochrome oxidase deficiency, a combination of aerobic training and DCA treatment resulted in substantial improvements in biochemical indices, exercise performance, and handicap. We conclude that exercise limitation in patients with mitochondrial myopathy may arise from effects of chronic deconditioning in addition to the effects of primary mitochondrial dysfunction and may be partially reversed by training and administration of DCA.

Adult↗