Biomedical subjects

H Lochmüller

Publications and source records attributed to H Lochmüller.

At least 73 records · Page 4Linked to original sources

PubMed1999

Technology evaluation: CRIB (CNTF delivery) CytoTherapeutics Inc.

To achieve continuous, site-specific delivery of therapeutic molecules to the central nervous system (CNS), a new therapeutic approach was developed combining in vitro gene transfer with a new delivery device. Xenogenic cells genetically modified to secrete specific bioactive substances were encapsulated into polymer-based fibers. A semipermeable membrane allows for passage of nutrients and cell-released therapeutic agents, but restricts inward diffusion of larger molecules and cells of the host's immune system, thus facilitating xenograft survival. This novel technique was successfully tested for the in vitro and in vivo delivery of various therapeutic agents, including neurotrophic factors, neurotransmitters and hormones. Phase I clinical trials were reported for the treatment of amyotrophic lateral sclerosis (ALS) and chronic cancer pain.

Amyotrophic Lateral Sclerosis↗

PubMed1998

Tenascin is a useful marker in the diagnosis of inflammatory myopathies.

The expression of tenascin, NCAM, vimentin and HSP-70 was examined in 15 patients with dermatomyositis and 12 patients with polymyositis. All biopsies with dermatomyositis showed a confluent network of tenascin expression in the extracellular space. This network surrounded numerous fibers (39 up to 320). Tenascin expression preceded other histological and immunohistochemical signs of dermatomyositis such as the expression of N-CAM and vimentin and the appearance of perifascicular atrophy and inflammatory response. Tenascin expression was found in 3 biopsies where microtubular inclusions in the arteriolar walls were absent. In contrast, tenascin expression was confined to the surroundings of necrotic fibers also expressing vimentin or N-CAM in polymyositis. In general, tenascin was found in the vicinity of inflammatory cells, only. These findings suggest that tenascin is induced by ischemic stress in dermatomyositis. Widespread tenascin expression in a fascicular distribution is a specific marker, if an inflammatory myopathy has been proven by standard techniques. In some cases, tenascin staining might confirm the clinical diagnosis of dermatomyositis, where other histological findings are inconclusive.

Adolescent↗

PubMed1998

High-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscle of dystrophic dogs: prolongation of expression with immunosuppression.

Replication-deficient adenovirus vectors (AdV) have been successfully used to transfer a truncated human dystrophin cDNA to skeletal muscle of dystrophin-deficient mdx mice. A dystrophin-deficient golden retriever dog model (GRMD) has been identified, which, unlike the mouse model, leads to a clinicopathological phenotype similar to that of Duchenne muscular dystrophy (DMD). We show for the first time that high-level dystrophin expression in skeletal muscle of GRMD dogs can be achieved by AdV-mediated gene transfer. However, a humoral and cellular immune response of the host against antigens of viral and transgene origin (similar to that occurring in mdx mice after AdV-mediated dystrophin gene transfer) leads to a decline of dystrophin expression over a 2-month period. Immunosuppression by cyclosporin significantly prolonged transgene expression. The GRMD model may help to solve the open questions pertaining to dystrophin gene transfer such as systemic delivery and improvement of muscle function before human trials for gene replacement therapy in DMD may be considered.

Adenoviridae↗

PubMed1998

Interferons impair early transgene expression by adenovirus-mediated gene transfer in muscle cells.

Recombinant adenovirus (AVR) promises to be an efficient vector in gene therapy for neuromuscular diseases, but in preclinical experiments the expression of therapeutic genes is shorter lived in immunocompetent animals than in immunocompromised hosts. Interferons (IFN), which are known to have a role both in early antiviral activity and in late cytotoxic immunoreaction against the virus or transduced cells, may influence the efficiency of gene transfer. In this study we investigated the role of IFNs in determining the efficiency of gene transfer by AVR. AVRs expressing beta-galactosidase (beta-gal) from either a cytomegalovirus (CMV) or a troponin-I promoter were used. Muscle cells were infected by AVR after exposure to various IFNs. The alphaIFN treatment significantly reduced (up to fivefold) the CMV promoter-driven gene expression in muscle cells in vitro and in immature muscles in vivo, while the least effective inhibitor was betaIFN. The decrease in gene expression by IFNs was more pronounced with the CMV-driven transgene than troponin-I promoter-driven one and was due to a decrease in transcript level. Intrinsic IFNs that are triggered by AVR administration can decrease the efficiency of gene transfer in muscle cells. Therefore the use of muscle specific promoters in AVR and/or IFN inhibitory agents will likely improve the prospects of effective gene therapy by AVR.

3T3 Cells↗

PubMed1998

Drug-induced systemic lupus erythematosus after 8 years of treatment with carbamazepine.

This is the report of a late-onset carbamazepine-induced systemic lupus erythematosus in a 34-year-old patient who had been treated with daily carbamazepine for 8 years because of complex partial seizures. When carbamazepine was discontinued, symptoms rapidly improved and antinuclear antibodies disappeared. So far, few cases have been reported of carbamazepine-induced lupus erythematosus within months after the start of treatment. This is the first case report about carbamazepine-induced systemic lupus erythematosus with serological confirmation after years of treatment without side effects.

Adult↗

PubMed1998

Myotonic ADR-MDX mutant mice show less severe muscular dystrophy than MDX mice.

In Duchenne muscular dystrophy (DMD) and its murine model, the dystrophic mouse (MDX), the skeletal musculature lacks dystrophin. The presumed function of this cytoskeletal protein is to protect the sarcolemma against mechanical stress during muscle activity. To test this hypothesis in vivo, we bred a double mutant mouse that combines two genetic defects: the dystrophin-deficiency of the MDX mouse and the Cl- channel myotonia of the arrested development of righting response (ADR) mouse. We hypothesized that high mechanical muscle activity would aggravate muscular dystrophy in double mutant ADR-MDX mice. On the contrary, ADR-MDX mice showed fewer signs of muscle fiber necrosis and fibrosis than MDX mice at all ages. Plasma creatine kinase levels were slightly increased in ADR-MDX, but significantly lower when compared to MDX mice. Sections of ADR-MDX muscle showed a uniform pattern of oxidative muscle fibers. Similar findings have been obtained in dystrophin-positive ADR mice, they result from a complete fiber-type IIB to IIA transformation in myotonic muscle. Our results suggest that small, oxidative fibers of myotonic mice are less sensitive to dystrophin deficiency. Therefore, ADR-MDX mice develop less severe muscular dystrophy than MDX mice do, although their muscles are continually stressed. The new ADR-MDX double mutant mouse is the first animal model combining both a dystrophinopathy and a channelopathy. The results presented here give new insights into the pathomechanism of muscular dystrophy and may be helpful for the therapeutic management of DMD.

Age Factors↗

PubMed1997

Congenital myasthenic syndromes: clinical and genetic analysis of 18 patients.

Congenital myasthenic syndromes (CMS) are a group of rare gentic disorders in which neuromuscular transmission is compromised by a variety of mechanisms, other than autoimmunity. Recently, substantial progress has been made by the identification of mutations in acetylcholine receptor (AChR) genes which cause CMS. We report on the clinical and genetic analysis of 18 independent CMS patients. All patients were clinically classified as sporadic cases of CMS (group III according to ENMC consensus). In order to investigate the prevalence of AChR mutations in this group we analyzed structural domains of the AChR genes at strategically important sites - the channel pore-lining regions (M2 domains) of the alpha, beta and epsilon subunits, and the extracellular domain close the acetylcholine (ACh) binding site. All patients showed wild-type sequence in these regions, mutations were not detected. Therefore, we conclude, that point mutations in domains which are known to cause slow channel congenital myasthenic syndromes (SCCMS) are rare in group III-patients in Germany. Determining the genetic defects causing CMS may have implications for diagnosis and genetic counseling of CMS patients. Moreover, this may be important for the therapeutic management of CMS as some patients may profit form quinidine sulfate. Therefore, further efforts will be undertaken to elucidate the underlying defects of CMS.

Adolescent↗

PubMed1997

Delayed colonic transit times in amyotrophic lateral sclerosis assessed with radio-opaque markers.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder, characterized by progressive loss of motor neurons. However, ALS has been recognized to involve several non-motor systems, subclinically. Cardiac and sudomotor autonomic involvement in ALS has been described. Recently, delayed gastric emptying was reported. The aim of this study was to assess colonic transit time in patients with ALS. Therefore, measurement of total and segmental colonic transit times using radio-opaque markers was performed in 14 patients with ALS and 14 healthy age-matched volunteers. Multiple-ingestion, single-radiograph technique was used. Segmental and colonic transit times were calculated from the number of retained markers. Nine of 14 patients with ALS showed markedly delayed colonic transit times if compared to healthy controls. Colonic transit in ALS patients was significantly delayed in the right and left colon; the rectosigmoid transit did not show major delay. The colonic transit times did not correlate with bulbar involvement, Norris score, walking disability or duration of the disease. In summary, colonic dysfunction in ALS may be a result of inactivity or inadequate fiber intake. However, it also may represent a gastrointestinal autonomic involvement due to nerval degeneration. Considering ALS as a multisystem disorder including the autonomic nervous system may have implications for research into pathogenesis and therapy of neurodegenerative disease.

Abdominal Pain↗

PubMed1997

The immunosuppressant FK506 prolongs transgene expression in brain following adenovirus-mediated gene transfer.

First generation, replication-defective adenoviral vectors are highly effective for gene transfer into the central nervous system, but the host's immune response limits the utility of this vector for possible therapy of neurological disease or long-term gene transfer studies in experimental animals. We have demonstrated the effectiveness of FK506 (tacrolimus), a powerful immunosuppressant that readily crosses the blood-brain barrier, in maintaining adenovirus-mediated reporter gene transfer following stereotaxic injection of the recombinant (AdCMVlacZ) into mouse striatum. After 28 days, beta-galactosidase expression was reduced by 75% relative to day 10 in immunocompetent animals, accompanied by an inflammatory reaction in the region of transduced cells; however, in mice receiving daily s.c. injections of FK506, beta-galactosidase activity was maintained at the 10 days post-injection level.

Adenoviridae↗

PubMed1997

Generation, validation, and large scale production of adenoviral recombinants with large size inserts such as a 6.3 kb human dystrophin cDNA.

Human, serotype 5 (Ad 5), replication-defective recombinant adenoviruses (AdVs) expressing a 6.3 kb partial dystrophin cDNA (Becker) under the control of either the CMV early or the RSV LTR promoter/enhancer in combination with various polyadenylation sequences (polyA), were developed for gene transfer studies aimed at Duchenne muscular dystrophy. Based on previous experience, a strategy for generation, screening and validation of AdVs with relatively large size gene expression cassette inserts was established. Here we focus on some aspects of stability and safety of such AdVs as gene therapeutic tools based on relevant molecular biological methods. Furthermore, the quality of our best AdV-minidystrophin construct was validated following its large scale production and purification as well as its delivery in mdx mice. These results are of interest for establishing other AdVs, where the combined length of a tissue specific promoter, the gene of interest and the polyA sequences reach the upper limit of the packaging capacity of first generation AdVs.

Adenoviruses, Human↗

PubMed1997

The scope of gene therapy in humans: scientific, safety and ethical considerations.

Gene therapy entails the introduction of specific, functional genes into cells for therapeutic or preventive purposes. Therapeutic genes may be introduced into cells in vitro (and the treated cells then introduced into the patient) or directly in vivo. Gene therapy may be used for several purposes including: (a) Gene replacement in genetic diseases; (b) Destroying malignant cells in neoplastic diseases; (c) Modifying immune responses; (d) Providing trophic molecules; (e) Immunisation against infectious diseases. In designing gene therapy strategies the following major items need to be investigated and optimised: (1) Relevant biological characteristics of the treatable cells and tissues; (2) Gene vectors (viral or non-viral); (3) The gene expression cassette (promoter+cDNA+poly A signal); (4) The safest and most effective route of administration; (5) Longevity of the transgene expression. Before human trials for gene therapy can commence for a given disease, extensive preclinical studies are to be carried out in an appropriate experimental models to assure the feasibility, safety and efficiency of the enterprise. For each disease or application, the risk/benefit ratio must be determined. Permission by regulatory agencies, institutional review boards (IRB) and thorough informed consent are essential. Although gene therapy holds great promise for many human diseases, it is still too early to predict the extent and timetable of its successful implementation in individual diseases.

Ethics, Medical↗

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

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↗

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

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

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

Adenovirus-mediated gene transfer of the tumor suppressor, p53, induces apoptosis in postmitotic neurons.

Programmed cell death is an ongoing process in both the developing and the mature nervous system. The tumor suppressor gene, p53, can induce apoptosis in a number of different cell types. Recently, the enhanced expression of p53 has been observed during acute neurological disease. To determine whether p53 overexpression could influence neuronal survival, we used a recombinant adenovirus vector carrying wild type p53 to transduce postmitotic neurons. A control consisting of the same adenovirus vector background but carrying the lacZ reporter expression cassette was used to establish working parameters for the effective genetic manipulation of sympathetic neurons. We have found that recombinant adenovirus can be used at titers sufficiently high (10 to 50 multiplicity of infection) to transduce the majority of the neuronal population without perturbing survival, electrophysiological function, or cytoarchitecture. Moreover, we demonstrate that overexpression of wild type p53 is sufficient to induce programmed cell death in neurons. The observation that p53 is capable of inducing apoptosis in postmitotic neurons has major implications for the mechanisms of cell death in the traumatized mature nervous system.

Adenoviridae↗