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Biomedical subjects

T Friedmann

Publications and source records attributed to T Friedmann.

At least 91 records · Page 5Linked to original sources
PubMed1992

Recombination between a herpes simplex virus type 1 vector deleted for immediate early gene 3 and the infected cell genome.

We have used a vector derived from a herpes simplex virus type 1 (HSV-1) mutant deleted for 3.6 kbp of the essential immediate early gene 3 to transduce the Tn5 neomycin phosphotransferase (neor) gene into rodent and primate cells in culture. The transgene was flanked by genomic sequences from the human hprt gene. We demonstrate in this study that sequences introduced by infection with the replication-defective HSV vector can be stably inserted into the cell genome by recombination. Both the efficiency of stable transduction, measured by the number of neor-positive colonies, and the number and length of the transgene sequences inserted into the cell genome were found to be a function of cell type. The transduction efficiency appeared to be influenced, at least in part, by the cytopathic potential of the replication-defective HSV-1 vector, which was more pronounced in primate than in rodent cells.

Animals↗
PubMed1992

Effects of choline and quiescence on Drosophila choline acetyltransferase expression and acetylcholine production by transduced rat fibroblasts.

Rat-1 fibroblasts were transduced to express Drosophila choline acetyltransferase. The presence of an active enzyme in these cells (Rat-1/dChAT) was confirmed using various methods. Rat-1/dChAT fibroblasts released acetylcholine (ACh) into the culture medium. Moreover, intra- and extracellular levels of ACh could be increased by adding exogenous choline chloride. In addition, serum starvation or confluence-induced quiescence caused an 80% decrease in recombinant choline acetyltransferase activity (compared with actively growing cells). ACh release was also repressed in quiescent fibroblast cultures. Exogenous choline could mitigate the decrease in ACh release. These results indicate that Rat-1 fibroblasts can be genetically modified to produce ACh and that ACh release can be controlled by introducing choline into the culture medium. Furthermore, these data demonstrate that the expression of the retroviral promoter used in this study decreases with the onset of quiescence; however, exogenous choline can increase the amount of ACh released by quiescent fibroblasts.

Acetylcarnitine↗
PubMed1992

Cytotoxicity of a replication-defective mutant of herpes simplex virus type 1.

Replication-defective mutants of herpes simplex virus type 1 (HSV-1) may prove useful as vectors for gene transfer, particularly to nondividing cells. Cgal delta 3 is an immediate-early gene 3 (IE 3) deletion mutant of HSV-1 that expresses the lacZ gene of Escherichia coli from the human cytomegalovirus immediate-early control region but does not express viral early or late genes. This vector was able to efficiently infect and express lacZ in cells refractory to traditional methods of gene transfer. However, 1 to 3 days postinfection, Cgal delta 3 induced cytopathic effects (CPE) in many cell types, including neurons. In human primary fibroblasts Cgal delta 3 induced chromosomal aberrations and host cell DNA fragmentation. Other HSV-1 strains that caused CPE, tested under conditions of viral replication-inhibition, included mutants of the early gene UL42, the virion host shutoff function, single mutants of IE 1, IE 2, and IE 3, and double mutants of IE 3 and 4 and IE 3 and 5. Inhibition of viral gene expression by UV irradiation of virus stocks or by preexposure of cells to interferon markedly reduced the CPE. We conclude from these studies that HSV-1 IE gene expression is sufficient for the induction of CPE, although none of the five IE gene products appear to be solely responsible. After infection of human fibroblasts with Cgal delta 3 at a low multiplicity of infection, we were able to recover up to 6% of the input virus 2 weeks later by a superinfection-rescue procedure, even though the virally transduced human cytomegalovirus-lacZ transgene was not expressed at this time. It is therefore likely that inhibition or inactivation of viral IE gene expression, either for establishing latency or for the long-term transduction of foreign genes by HSV-1 vectors, is essential to avoid the death of infected cells.

Animals↗
PubMed1992

Approaches to gene therapy of complex multigenic diseases: cancer as a model and implications for cardiovascular disease and diabetes.

The general concept of gene therapy is now well established and accepted by the medical, scientific and public policy communities, and is rapidly being implemented in human experimental studies. In addition to the initial models of single gene defects, target diseases have now come to include multigenic and multifactorial diseases such as human cancer, neurodegenerative diseases such as Parkinson's disease and firms of cardiovascular disease. While many conceptual and technical obstacles must still be overcome before therapy for disorders such as coronary artery disease and diabetes mellitus will easily be approached at the genetic level, the early results with several multigenic disease models gives some cause for optimism that gene therapies for even those complicated disorders will eventually become available.

Animals↗
PubMed1992

Direct gene transfer to the liver with herpes simplex virus type 1 vectors: transient production of physiologically relevant levels of circulating factor IX.

We have used gene transfer vectors derived from a replication-defective mutant of herpes simplex virus type 1 (HSV-1) expressing the hepatitis B virus surface antigen (HBsAg), Escherichia coli beta-galactosidase (beta-gal), or canine factor IX (cFIX) from the immediate early promoter of human cytomegalovirus (hCMV) to infect mouse liver by direct injection or through the portal vein. By either route, high levels of transgene expression were demonstrated by the detection of immunoreactive HBsAg or cFIX in the circulation and by histochemical detection of beta-gal activity in situ. The results were striking in that the serum level of cFIX reached 10% of the normal murine levels. Although the level of transgene expression from the hCMV promoter was transient, a significant number of persistent vectors could be rescued from the livers of recipient mice up to 2 months after inoculation. Replacement of the hCMV promoter with the HSV-1 latency-associated transcript (LAT) promoter resulted in reduced but prolonged expression of both HBsAg and cFIX. The very high level of factor IX expression suggests that clinically useful gene transfer may eventually be feasible through direct vector delivery to the liver.

Animals↗
PubMed1992

Functional analysis of brain dopamine systems in a genetic mouse model of Lesch-Nyhan syndrome.

The Lesch-Nyhan syndrome is a neurogenetic disorder caused by congenital deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). The disorder is characterized by prominent neurobehavioral abnormalities which appear to result in part from dysfunction of striatal dopamine systems. HPRT-deficient (HPRT-) mutant strains of mice have been produced as animal models for this syndrome, but these animals exhibit none of the neurobehavioral abnormalities seen in Lesch-Nyhan patients. The present studies describe the behavioral responses of three strains of mice carrying one of two mutations in the HPRT gene to agents which interact with brain dopamine systems. HPRT- mice are more sensitive than age- and sex-matched littermates to the motor-activating properties of dopamine-releasing agents (amphetamine, amfonelic acid and methylphenidate), but not dopamine uptake inhibitors (GBR 12909 and nomifensine). The enhanced sensitivity of the HPRT- mice to the dopamine-releasing agents is not caused by dopamine receptor supersensitivity, because the HPRT- mice do not show enhanced motor responses to the direct D1/D2 dopamine receptor agonist apomorphine or to the selective D1 dopamine receptor agonist SKF 38393. The function of regulatory dopamine autoreceptors, as assessed by suppression of spontaneous motor activity by low doses of R(-)-propylnorapomorphine, also appears normal in the HPRT- mice. Biochemical analysis shows that the HPRT- mice have significantly lower levels of dopamine (-45%), but normal levels of tyrosine, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 3-methoxytyramine in the caudoputamen. In contrast to the deficit in caudoputamen dopamine, no deficits were noted in the accumbens of the HPRT- mice. These results indicate the existence of an inherent abnormality in the dopamine systems in the brains of HPRT- mice, despite their apparently normal spontaneous behavior.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1992

Long-term transgene expression from genetically modified hepatocytes grafted to the rat liver.

Primary cultures of rat hepatocytes embedded and grown to high density in a wafer of reticulated polyurethane have been infected in vitro with a retroviral vector expressing the hepatitis B virus surface antigen (HBsAg). Wafers containing infected cells and implanted directly between the liver lobes of recipient rats became vascularized and firmly attached to the liver parenchyma. Stable levels of HBsAg were detected in the blood circulation for at least 4 months after grafting. Hepatocytes recovered from the recipient rats up to 4 months after implantation were recultured under selective conditions and found to produce high levels of HBsAg.

Animals↗
PubMed1991

Efficient gene expression in mammalian cells from a dicistronic transcriptional unit in an improved retroviral vector.

We have studied the properties of dicistronic transcriptional units in retroviral vectors. In these vectors, the promoter in the 5' retroviral long terminal repeat (LTR) controls expression of both an upstream cistron (luc) encoding firefly luciferase and a downstream cistron (neo), a selectable marker encoding neomycin phosphotransferase (NPTII). By assaying for simultaneous expression of luc and neo after transfection or infection of hamster BHK, rat 208F, and mouse retroviral packaging cell lines, we have identified important factors that affect expression from the downstream cistron, including the presence of intercistronic ATG sequences, the length of the intercistronic sequence and conformity of the sequence surrounding the downstream start codon to the eukaryotic consensus sequence. Optimized dicistronic vectors produced amounts of NPTII comparable to a vector in which neo was driven by a strong internal promoter consisting of a modified Rous sarcoma virus LTR. Additionally, they produced higher virus titers and demonstrated improved stability of gene expression in the absence of selection. By virtue of their physical compactness and elimination of the need for a separate promoter for every gene, dicistronic transcriptional units allow the introduction of larger genes into retroviral vectors and may allow for more than two genes to be placed in a single vector.

Avian Sarcoma Viruses↗
PubMed1991

Gene transfer into rat airway epithelial cells using retroviral vectors.

Primary cultures of epithelial cells from adult rat tracheas were maintained in vitro on collagen matrices and were exposed to a murine retrovirus vector expressing the E. coli beta-galactosidase gene. Infection was carried out on cells grown as monolayers under medium and on cells grown on raised platforms. Cells maintained at an air-medium interface were highly susceptible to infection with the vector, showing an efficiency of infection of 20-25%, compared with an efficiency of less than 1% for cells grown under medium. Infected beta-galactosidase-expressing cells were seeded into denuded tracheas and were capable of partially repopulating the denuded tracheas grafted subcutaneously into host rats. The susceptibility of these cells to retroviral infection suggests an approach to the treatment of some pulmonary genetic disorders such as cystic fibrosis.

Aerosols↗
PubMed1991

Gene therapy techniques.

The most dramatic event of the past year in the field of gene therapy has been the initiation of clinical trials involving the introduction of genetically altered cells into human beings. Four studies, three involving new approaches to cancer therapy and one involving the treatment of adenosine deaminase deficiency, are presently under way. There has also been significant recent progress in the technology of gene transfer relevant to gene therapy. This progress, along with the recent clinical therapy trials, is the subject of this review.

Animals↗
PubMed1991

Amphetamine-induced behavioral phenotype in a hypoxanthine-guanine phosphoribosyltransferase-deficient mouse model of Lesch-Nyhan syndrome.

In humans, congenital deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a disorder known as the Lesch-Nyhan syndrome. Patients with this disorder exhibit a prominent neurobehavioral phenotype that results in part from dysfunction of catecholaminergic systems in the striatum. HPRT-deficient mice produced as animal models for this syndrome curiously exhibit no spontaneous neurobehavioral abnormalities. However, the present study demonstrates that HPRT-deficient mice are more sensitive than their HPRT-normal littermates to the ability of amphetamine to stimulate locomotor or stereotypic behaviors. This behavioral supersensitivity to amphetamine indicates the existence of an underlying subclinical abnormality of catecholaminergic systems in the brains of HPRT-deficient mice, analogous to findings in human Lesch-Nyhan patients.

Animals↗
PubMed1991

Dose-related analgesic effects of flupirtine.

1. Flupirtine is a novel and, in all probability, centrally acting, analgesic. The present investigation was conducted in order to investigate dose-related effects of perorally administered flupirtine in man, with special regard to specifically analgesic actions, employing a model based on pain-related chemosomatosensory evoked potentials and subjective intensity estimates of painful stimuli. 2. Plasma concentrations of flupirtine measured 2 h after dosing linearly increased as a function of the administered dose. 3. It was possible to reproduce our own previously obtained results, which established the analgesic action of 200 mg flupirtine administered perorally. 4. Intensity estimates linearly decreased as a function of the administered dose, whereas chemosomatosensory evoked potential amplitudes non-linearly changed in relation to the administered dose. 5. In the spontaneous EEG, a dose-dependent increment in the power-spectra was observed, and this mainly in the alpha- and beta-range.

Administration, Oral↗
PubMed1991

Activity of the simian virus 40 early promoter-enhancer in herpes simplex virus type 1 vectors is dependent on its position, the infected cell type, and the presence of Vmw175.

We have studied some of the parameters governing the expression of a foreign promoter-reporter gene construct incorporated into herpes simplex virus (HSV) type 1. These include the genetic background of the parental virus, the site of transgene insertion within the HSV genome, and the infected cell type. The genetic background of the vector constructs denoted delta 3 was an HSV type 1 mutant deleted for nearly the entire coding portion of Vmw175 (ICP4), the product of the essential immediate-early gene IE3. For vectors denoted +, the IE3 deletion had been repaired by marker rescue. We used as a reporter gene the bacterial chloramphenicol acetyltransferase (CAT) gene, driven by the simian virus 40 (SV40) early promoter and enhancer region. The SV40-cat hybrid gene was inserted either into the HSV thymidine kinase (TK) locus to create the vectors TKScat delta 3 and TKScat+ or into an intergenic site within the BamHI z fragment of the short unique portion of the viral genome to create the vectors GScat delta 3 and GScat+. In Vero and BHK cells infected with TKScat delta 3, CAT activity was first detected at 10 h postinfection and continued to accumulate until 36 h postinfection. In cells of primate origin infected with the replication-competent vector TKScat+, or in primate cells which complement the IE3 deficiency and which were infected with TKScat delta 3, CAT activity was significantly lower than in cells of rodent origin. However, levels of CAT were increased in the presence of cycloheximide, suggesting that the low production of CAT in primate cells was due to repression of SV40-cat hybrid gene expression. In contrast with results with TKScat delta 3 and TKScat+, CAT activity was not detectable in any of the tested cell types infected with GScat delta 3 or GScat+ except under conditions of cycloheximide reversal. These results show that while HSV gene products expressed in the presence of Vmw175 inhibited SV40-cat expression in the tk locus in a cell-type-specific manner, HSV gene products expressed in the presence or absence of Vmw175 inhibited SV40-cat expression in the BamHI z locus independently of cell type.

Animals↗
PubMed1991

Isolation of a herpes simplex virus type 1 mutant deleted for the essential UL42 gene and characterization of its null phenotype.

We isolated a cell line, designated V9, stably transformed with the herpes simplex virus type 1 (HSV-1) UL42 gene, which is one of seven genes required in trans for the replication of plasmids containing an HSV origin of replication (C. A. Wu, N. J. Nelson, D. J. McGeoch, and M. D. Challberg, J. Virol. 62:435-443, 1988). V9 cells inducibly expressed the product of the UL42 gene, the 65-kDa DNA-binding protein (65KDBP), and were used as a permissive host to construct a mutant virus deleted for this essential gene. The UL42 deletion mutant, designated Cgal delta 42, displayed a tight early phenotype in nonpermissive Vero cells producing no infectious progeny, viral DNA, or late gene products but accumulated selected immediate-early and early transcripts with kinetics similar to those of wild-type virus. Wild-type levels of viral DNA and infectious progeny were produced in permissive V9 cells, despite the fact that V9 cells infected with Cgal delta 42 accumulated less than 1% of the UL42 RNA and protein found in Cgal+ virus-infected V9 or Vero cells. These results indicate that only small quantities of the 65KDBP are required for the synthesis of HSV DNA and the production of infectious virus. Although we could find no evidence that the superinduction of the 65KDBP in V9 cells infected with Cgal+ repressed expression of HSV-1 genes as observed in cells expressing another DNA-binding protein, ICP8 (P. K. Orberg and P. A. Schaffer, J. Virol. 61:1136-1146, 1987), the induction of the 65KDBP in V9 cells correlated with an approximately 2-h-earlier shift in the expression of genes from all three kinetic classes. The availability of the UL42 mutant should facilitate the construction of more subtle UL42 mutants which will be useful in the elucidation of the interrelationship between the 65KDBP and other DNA replication proteins as well as in the characterization of additional important functional domains.

Animals↗
PubMed1991

Pseudotype formation of murine leukemia virus with the G protein of vesicular stomatitis virus.

Mixed infection of a cell by vesicular stomatitis virus (VSV) and retroviruses results in the production of progeny virions bearing the genome of one virus encapsidated by the envelope proteins of the other. The mechanism for the phenomenon of pseudotype formation is not clear, although specific recognition of a viral envelope protein by the nucleocapsid of an unrelated virus is presumably involved. In this study, we used Moloney murine leukemia virus (MoMLV)-based retroviral vectors encoding the gene for neomycin phosphotransferase to investigate the interaction between the VSV G protein and the retroviral nucleocapsid during the formation of MoMLV(VSV) pseudotypes. Our results show that VSV G protein can be incorporated into the virions of retrovirus in the absence of other VSV-encoded proteins or of retroviral envelope protein. Infection of hamster cells by MoMLV(VSV) pseudotypes gave rise to neomycin phosphotransferase-resistant colonies, and addition of anti-VSV serum to the virus preparations completely abolished the infectivity of MoMLV(VSV) pseudotypes. It should be possible to use existing mutants of VSV G protein in the system described here to identify the signals that are important for the formation of MoMLV(VSV) pseudotypes.

Animals↗
PubMed1991

Knock-in and knock-out. Transgenes, Development and Disease: A Keystone Symposium sponsored by Genentech and Immunex, Tamarron, CO, USA, January 12-18, 1991.

The meeting clearly reflected the recent rapid and staggering progress in the use of transgenic mice. Being able to manipulate single units of genetic information in their natural position in a cell genome and to examine the results of that manipulation in the background of a terminally differentiated organism has been an outstanding achievement on the road toward a deeper and generalizable understanding of complex life forms. However, there are many examples of genetic diseases in humans (for instance, Lesh-Nyhan syndrome, Duchenne muscular dystrophy, phenylketonuria) whose genotypic "equivalent" in transgenic mice results in very different phenotypes or no phenotypic changes at all. Mice are clearly not human beings. Why not? No doubt, we will find out!

Animals↗
PubMed1990

Limited bidirectional activity of two housekeeping gene promoters: human HPRT and PGK.

Typical of other housekeeping genes, the promoter for the human hypoxanthine phosphoribosyl transferase-encoding gene (HPRT) is G + C-rich, lacks a TATA box and has multiple transcription start points. To test the hypothesis that these features may result in relaxed control over the direction of transcription, we examined the effect of orientation on the ability of the HPRT promoter to control expression of the following reporter genes in transfected cells: luc (firefly luciferase), cat (bacterial chloramphenicol acetyltransferase) and neo (neomycin resistance). A 376-bp fragment containing the HPRT promoter efficiently expressed the luc gene irrespective of orientation, and the 5' ends of luciferase RNA produced in cells transfected with inverted promoter constructs mapped to within the HPRT promoter, indicating that the HPRT promoter has bidirectional activity. However, in the presence of two divergently-flanking reporter genes expression from the inverted HPRT promoter was only 10-20% compared to the noninverted orientation. Furthermore, the inverted HPRT promoter expressed cat less well than luc, and was unable to express neo sufficiently well to produce any colonies under appropriate selection conditions. Attempts to detect endogenous divergent HPRT transcripts were unsuccessful. The promoter of another housekeeping gene, encoding 3-phosphoglycerate kinase (PGK), expressed moderate levels of cat (40%) but not luc (less than 5%) in the inverted orientation. By comparison, two TATA-box containing promoters functioned extremely poorly when inverted. This study indicates that two plasmid-borne housekeeping promoters have at least a limited potential for bidirectional activity, but the functional significance of this is unclear if the corresponding endogenous housekeeping promoters express divergent transcripts at similarly low levels. The poor activity of the HPRT and PGK promoters in the inverted orientation suggests that there is a mechanism which influences the direction of transcription from these promoters.

Chloramphenicol O-Acetyltransferase↗