Biomedical subjects

N W Fraser

Publications and source records attributed to N W Fraser.

At least 37 records · Page 2Linked to original sources

PubMed1997

Use of a "replication-restricted" herpes virus to treat experimental human malignant mesothelioma.

Modified, nonneurovirulent herpes simplex viruses (HSVs) have shown promise in the treatment of brain tumors. However, HSV-1 can infect and lyse a wide range of cell types. In this report, we show that HSV-1716, a mutant lacking both copies of the gene coding ICP-34.5, can effectively treat a localized i.p. malignancy. Human malignant mesothelioma cells supported the growth of HSV-1716 and were efficiently lysed in vitro. i.p. injection of HSV-1716 into animals with established tumor nodules reduced tumor burden and significantly prolonged survival in an animal model of non-central nervous system-localized human malignancy without dissemination or persistence after i.p. injection into SCID mice bearing human tumors. These findings suggest that this virus may be efficacious and safe for use in localized human malignancies of nonneuronal origin such as malignant mesothelioma.

Animals↗

PubMed1997

Transfectable and transplantable postmitotic human neurons: a potential "platform" for gene therapy of nervous system diseases.

We have characterized a human embryonal carcinoma cell line (NTera-2 or NT2 cells) that is transfectable and capable of differentiating into postmitotic neuron-like cells (NT2N cells) following treatment with retinoic acid in order to identify a human neuronal cell line that might serve as a "platform" for gene therapy of human neurological diseases. Studies of NT2N cells transplanted into the brain or spinal cord of immunecompetent and immunodeficient rodents show that NT2N cells integrate into the host central nervous system (CNS) and establish the molecular and structural polarity of authentic neurons in vivo. Further, grafted NT2N cells acquire the molecular phenotype of fully mature neurons within 6 months postimplantation and the grafts survive > 1 year in immunodeficient mice without reverting to a neoplastic state. Although grafts of the retinoic acid-naive NT2 cells can form lethal tumors in the CNS, these cells differentiate into postmitotic neuron-like cells and do not form tumors when the grafts are confined to the caudoputamen. Based on the studies reviewed here, we conclude that grafted NT2N cells could serve as a suitable platform for the delivery of exogenous proteins into the CNS for gene therapy of human nervous system diseases.

Animals↗

PubMed1997

beta-Gal enzyme activity driven by the HSV LAT promoter does not correspond to beta-gal RNA levels in mouse trigeminal ganglia.

beta-Galactosidase enzyme expression can be detected in only a small percentage of trigeminal ganglia (TG) neurons acutely and latently infected with herpes simplex virus (HSV), in which the lacZ reporter gene was placed down-stream of the latency associated transcript (LAT) promoter at the LAT locus. However, DNA quantification suggests that a larger percentage of cells is infected than is expressing beta-galactosidase enzyme. To investigate the mechanism involved in regulation of genes expressed from the LAT promoter in trigeminal ganglia, in situ hybridization and histochemical staining assays were employed to determine on a cell-by-cell basis beta-gal gene expression both at the RNA and protein level. Using a LAT promoter-driven beta-gal construct in HSV-1 strain HFEM, it was found that there were 89-fold more cells positive for beta-gal transcript than cells positive for beta-gal enzyme in acutely infected trigeminal ganglia and a 10-fold difference in latently infected trigeminal ganglia. Thus, there is a discordance between beta-gal mRNA and beta-gal enzyme levels in HFEM/LAT-lacZ infected cells during acute and latent infection, and the beta-gal reporter gene activity does not faithfully compare the LAT promoter activity between acute and latently infected tissue. In contrast, in situ hybridization and histochemical staining assays were performed in mice acutely infected with a virus in which 140 bp of the LAT promoter sequences flanking the TATA element were replaced by 1.8 kbp of the neurofilament promoter (HSV-1 HFEM/NF-lacZ). This construct showed a correlation between beta-gal mRNA and enzyme expression in trigeminal ganglia in acute and latent infections. These findings suggest that sequences at the 5' end of the beta-gal transcript influence translation of the beta-gal message.

Animals↗

PubMed1997

Herpes simplex virus 1716, an ICP 34.5 null mutant, is unable to replicate in CV-1 cells due to a translational block that can be overcome by coinfection with SV40.

Herpes simplex virus (HSV) mutants lacking the gene encoding infected cell protein (ICP) 34.5 exhibit an attenuated phenotype in models of pathogenesis and have been used for experimental cancer therapy. Recently it was shown that the HSV ICP 34.5 protein functions to prevent the host cell-induced double-stranded RNA-activated protein kinase (PKR)-dependent translational block that normally occurs during virus infection. We now report that an HSV ICP 34.5 mutant called HSV-1716 is unable to replicate in the simian kidney cell-derived line CV-1, due to a translational block. Moreover, we find that this block can be overcome by simian virus 40 (SV40). This has been shown directly by infecting CV-1 cells with SV40 and HSV-1716 simultaneously, and indirectly via HSV-1716 infection of COS-1 cells (CV-1 cells transformed by an origin-defective mutant of SV40 that codes for wild-type T antigen). The translational block is restored when infections are done in the presence of the phosphatase inhibitor okadaic acid. These results support, but do not directly prove, contentions that HSV ICP 34.5 interacts with the PKR pathway to restore translation in non-permissive cells, and that SV40 large T antigen has a similar functional role, but acts downstream of the site of ICP 34.5 interaction (eIF2alpha) in the pathway. Study of this CV-1/COS-1 system should allow further clarification of the virus-host interactions that underlie the restricted replication of HSV-1 ICP 34.5 gene null mutants.

Animals↗

PubMed1997

Treatment of experimental subcutaneous human melanoma with a replication-restricted herpes simplex virus mutant.

Modified, non-neurovirulent herpes simplex viruses (HSV) have shown promise for the treatment of brain tumors, including intracranial melanoma. In this report, we show that HSV-1716, an HSV-1 mutant lacking both copies of the gene coding-infected cell protein 34.5 (ICP 34.5), can effectively treat experimental subcutaneous human melanoma in mice. In vitro, HSV-1716 replicated in all 26 human melanoma cell lines tested, efficiently lysing the cells. Therapeutic infection of subcutaneous human melanoma nodules with HSV-1716 led to viral replication that was restricted to tumor cells by immunohistochemistry. Moreover, HSV-1716 treatment significantly inhibited progression of preformed subcutaneous human melanoma nodules in SCID mice and caused complete regression of some tumors. This work expands the potential scope of HSV-1-based cancer therapy.

Analysis of Variance↗

PubMed1997

The herpes simplex virus type 1 2.0-kilobase latency-associated transcript is a stable intron which branches at a guanosine.

We have used a minigene construct of the herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene to analyze its transcripts in transient transfection assays. A 2.8-kb fragment of the approximately 8.5-kb LAT gene encompassing the 2.0-kb LAT was cloned into a eukaryotic expression vector downstream of the cytomegalovirus immediate-early gene promoter. Northern hybridization of RNA isolated from transfected COS-1 cells identified three LAT-specific transcripts, 3.4, 2.0, and 1.4 kb in size. Mapping of these transcripts by Northern hybridization indicated that the 1.4- and 2.0-kb RNAs are nonoverlapping, while the 3.4-kb RNA overlaps both smaller RNAs. Reverse transcription-PCR (RT-PCR) and partial sequencing of the 1.4-kb RNA revealed that this RNA is the spliced exons of the 3.4-kb primary transcript. The 2.0-kb LAT appears to be an intron accumulating after splicing of the minor LAT (mLAT) pre-mRNA. The splice donor and acceptor sites for the 2.0-kb LAT identified in transfected and HSV-1-infected cells are identical. Mapping of the branch point of this intron by RT-PCR in transfected and HSV-1-infected cells, as well as in latently infected murine trigemial ganglia, shows that it is a guanosine. This branch site does not bear homology to consensus mammalian branch site sequences. These data provide evidence that the 2.0-kb LAT is an intron of the mLAT pre-mRNA with a unique branch point.

Animals↗

PubMed1997

Gene expression during reactivation of herpes simplex virus type 1 from latency in the peripheral nervous system is different from that during lytic infection of tissue cultures.

Herpes simplex virus (HSV) replicates in peripheral tissues and forms latent infections in neurons of the peripheral nervous system. It can be reactivated from latency by various stimuli to cause recurrent disease. During lytic infection in tissue culture cells, there is a well-described temporal pattern of (i) immediate-early, (ii) early, and (iii) late gene expression. However, latency is characterized by little if any expression of genes of the lytic cycle of infection. During reactivation, the pattern of gene expression is presumed to be similar to that during the lytic cycle in tissue culture, though recent work of W. P. Halford et al. (J. Virol. 70:5051-5060, 1996) and P. F. Nichol et al. (J. Virol. 70:5476-5486, 1996) suggests that it is modified in neuronal cell cultures. We have used the mouse trigeminal ganglion explant model and reverse transcription-PCR to determine the pattern of viral and cellular gene expression during reactivation. Surprisingly, the pattern of viral gene expression during lytic infection of cell cultures is not seen during reactivation. During reactivation, early viral transcripts were detected before immediate-early transcripts. The possibility that a cellular factor upregulates early genes during the initial reactivation stimulus is discussed.

Animals↗

PubMed1997

Selection of a nonconsensus branch point is influenced by an RNA stem-loop structure and is important to confer stability to the herpes simplex virus 2-kilobase latency-associated transcript.

Herpes simplex virus type 1 latent infection in sensory neurons is characterized by the highly restricted transcription of viral genes. The latency-associated transcripts (LAT) family members are the only transcripts that can be identified in large amounts in latently infected cells. The most abundant LAT species is a 2-kb RNA that results from splicing of a rare primary transcript. Analysis of a LAT mutant virus (TB1) in cell culture revealed an aberrant splicing pattern and production of a stable small (0.95-kb) LAT intron. A panel of deletion constructs expressing truncated LAT in transiently transfected cells mapped the region influencing stability to the 3' end of the LAT intron. This region encompasses the branch point and a putative stable stem-loop hairpin structure immediately upstream of the splice acceptor consensus polypyrimidine tract. Mutagenic analysis of the sequence in this region confirmed our hypothesis that the stem-loop structure is important for efficient splicing by influencing the selection of a nonconsensus branch point. Changes in this structure correlate with changes in branch point selection and production of an unstable 2-kb LAT.

Alternative Splicing↗

PubMed1997

Repression of the HSV-1 latency-associated transcript (LAT) promoter by the early growth response (EGR) proteins: involvement of a binding site immediately downstream of the TATA box.

During herpes simplex virus (HSV) latency, in neurons of the nervous system, a single family of viral transcripts (the Latency-Associated Transcripts or LATs) are synthesized. Within the LAT promoter region, we have identified a consensus sequence for the EGR proteins in an unusual position immediately downstream of the TATA box. The early growth response (EGR) proteins are rapidly induced in cells by stimuli which also induce HSV to reactivate from latency. In order to determine if EGR proteins play any role in control of LAT transcription, we have analyzed the interactions between EGR proteins and the LAT promoter. Gel retardation and DNase I protection assays demonstrated that EGR1 zinc finger protein bound specifically to the LAT promoter region EGR consensus sequence. To determine if EGR proteins could modulate transcription through the LAT promoter, cotransfection assays were performed using chloramphenicol acetyltransferase (CAT) reporter constructs driven by either the wild-type LAT promoter or a LAT promoter with a mutated EGR binding site. Contransfection of the wild-type LAT promoter construct with EGR expression plasmids resulted in inhibition of the basal level of CAT activity with EGR-2 but not EGR-1 or 3. However, normal levels of CAT activity were observed in cotransfections using the mutant LAT promoter CAT construct suggesting that repression was mediated by the binding of EGR-2 proteins to the LAT promoter. Furthermore, data from combination binding assays using EGR1 and TATA binding protein (TBP) in vitro support the hypothesis that binding of EGR proteins to the LAT promoter prevents binding of TBP and thus suppresses transcription. These results may provide a link between stress responses in neurons of the CNS which activate the EGR family of proteins and HSV reactivation from latency due to the same stress response.

Animals↗

PubMed1996

Correlation between herpes simplex virus type 1 rate of reactivation from latent infection and the number of infected neurons in trigeminal ganglia.

The presence of wild-type herpes simplex virus type 1 (HSV-1) and several latency associated transcript (LAT) region mutants within the trigeminal ganglia (TG) of latently infected mice was examined. A combination of methods including conventional in situ hybridization to detect viral LAT and an in situ DNA polymerase chain reaction (PCR) to detect viral DNA was used. These data show that, for all virus strains in which a comparison was possible, the population of neurons expressing detectable levels of LAT was approximately one-third the total number of viral DNA-containing cells. In addition, in situ PCR analysis revealed that mutants such as 17 delta Sty. 17 delta BstE, and 17 delta S/N, which contain deletions within the LAT locus which do not affect the kinetics of viral reactivation from explanted murine TG, are present in as many neurons as wild-type virus. This was true regardless of the ability to induce accumulation of intact 2.0-kb LAT. On the other hand, mutant 17 delta N/H, which contains a deletion removing the LAT promoter and surrounding genomic region and reactivates slowly from explanted TG, was present in only one-sixth as many neurons as wild-type virus. These data show that detection of mutants unable to synthesize or accumulate 2.0-kb LAT (such as 17 delta N/H) is possible with in situ DNA PCR and that the slow reactivation phenotype of 17 delta N/H correlates with a reduced number of HSV DNA-containing neurons.

Animals↗

PubMed1996

Herpes simplex 1716--an ICP 34.5 mutant--is severely replication restricted in human skin xenografts in vivo.

HSV-1716 is a replication-restricted, neuroattenuated ICP 34.5 gene mutant of herpes simplex virus type 1 (HSV-1). Because of the attenuated phenotype of ICP 34.5 mutants in rodent models of HSV disease, they have been promoted as potential vaccine strains and gene therapy vectors and have been used by us and others as therapeutic agents for the treatment of experimental malignant tumors. However, all data on the phenotype of HSV-1716 and other ICP 34.5 mutants are from animal model systems, while humans are the natural hosts of HSV-1. To achieve an initial characterization of the phenotype of 1716 in human tissue, we have studied its replication in mature human skin xenografts on SCID mice. We find that replication of 1716 is severely restricted in such human skin grafts relative to both parental wild-type HSV-1 strain 17+ and the HSV-1716 revertant virus 1716R, in which the 759-bp ICP 34.5 gene deletions have been repaired. Moreover, the replication of both 1716 and 17+ is significantly better in the human skin grafts than it is in mouse skin. The implications of these findings are discussed.

Animals↗

PubMed1996

Selective vulnerability of mouse CNS neurons to latent infection with a neuroattenuated herpes simplex virus-1.

Herpes simplex viruses that lack ICP34.5 are neuroattenuated and are presently being considered for cancer and gene therapy in the nervous system. Previously, we documented the focal presence of the latency-associated transcripts (LATs) in the hippocampi of immunocompromised mice after intracranial (IC) inoculation of an ICP34.5-deficient virus called strain 1716. To characterize further the biological properties of strain 1716 in the CNS of immunocompetent mice, we determined the extent of viral gene expression in different cell types and regions of the CNS after stereotactic IC inoculation of this virus. At survival times of > 30 d after inoculation, we found that (1) infectious virus was not detectable by titration and immunohistochemical studies; (2) neurons harbored virus as demonstrated by the detection of the LATs by in situ hybridization (ISH); (3) transcripts expressed during the lytic cycle of infection were not detected by ISH; and (4) subsets of neurons were selectively vulnerable to latent infection, depending on the site of inoculation. These results suggest that the absence of ICP34.5 does not abrogate latent infection of the CNS by strain 1716. Additional studies of strain 1716 in the model system described here will facilitate the elucidation of the mechanisms that regulate the selective vulnerability of CNS cells to latent viral infection and lead to the development of ICP34.5 mutant viruses as therapeutic vectors for CNS diseases.

Animals↗

PubMed1996

Therapy of a murine model of pediatric brain tumors using a herpes simplex virus type-1 ICP34.5 mutant and demonstration of viral replication within the CNS.

To develop improved therapies for medulloblastoma, we studied the ability of a neuroattenuated HSV-1 ICP34.5 mutant (variant-1716) to replicate within and destory an authentic medulloblastoma cell line known as Med 283 (D283) using immunohistochemistry, in situ hybridization, and viral titrations. In vitro studies showed that variant-1716 replicates in and destroys monolayers of D283 cells with kinetics similar to wild-type strain 17+. When D283 tumor-bearing animals were treated with variant-1716 injected directly into the tumor, there was a statistically significant increase in survival (p < .02) compared to mock-treated tumor-bearing mice. Additionally, several novel observations emerged from this study. Most importantly, we demonstrated focal acute viral replication within murine brain cells, a finding not previously reported with HSV-1 ICP-34.5 variants. Further, the brains of tumor-bearing animals treated with variant-1716 demonstrated persistent viral replication within tumors for several weeks. Thus, we conclude that variant-1716 causes a statistically significant increase in survival of experimental medulloblastomas, but further analysis of the replication of HSV-1 ICP34.5 mutants within the mammalian CNS is necessary to assess its potential long-term toxicity.

Animals↗

PubMed1996

Reactivation of herpes simplex virus from latently infected mice after administration of cadmium is mouse-strain-dependent.

It was previously reported that administration of cadmium (Cd) to CBA mice latently infected with herpes simplex virus (HSV) results in a high incidence of virus reactivation in vivo. In the present study, Cd-inducible reactivation was used to compare CBA with four other laboratory mouse strains. HSV reactivation, as measured by the recovery of infectious particles from latently infected trigeminal ganglia following Cd treatment, occurred predominantly in the CBA strain and was almost entirely absent from other strains tested. There was no correlation of strain-dependent Cd toxicity with the recovery of infectious virus. In situ examination of Cd-treated ganglia from latently infected CBA and BALB/c mice revealed that viral antigens were expressed exclusively in CBA specimens, but that viral replicative transcripts were expressed in both strains, although more strongly in CBA than in BALB/c specimens. We conclude that Cd treatment had induced reactivation of HSV from both mouse strains, and that the reactivation process was completed in CBA but not in BALB/c mice.

Animals↗

PubMed1996

In vivo epinephrine reactivation of ocular herpes simplex virus type 1 in the rabbit is correlated to a 370-base-pair region located between the promoter and the 5' end of the 2.0 kilobase latency-associated transcript.

A rabbit ocular model of epinephrine-induced herpes simplex virus type 1 reactivation was employed to study the effect of a deletion in the latency-associated transcript domain. A viral construct derived from 17Syn+, designated 17deltaSty, has a deletion of 370 nucleotides between genomic positions 118880 and 119250. 17deltaSty has been shown to reactivate with wild-type virus kinetics from explants of trigeminal ganglia from latently infected mice. To determine the behavior of this mutant in an in vivo, inducible reactivation system, rabbit corneas were infected with 17Syn+, 17deltaSty, or its rescuant, 17detlaSty-Res. After viral latency was established, transcorneal epinephrine iontophoresis was performed. The rabbits latently infected with 17deltaSty exhibited a significantly reduced ability to undergo adrenergically induced reactivation, i.e., viral shedding in the tears, compared with rabbits infected with either 17Syn+ or 17deltaSty-Res. However, quantitative PCR demonstrated similar numbers of viral genomes in the trigeminal ganglia from rabbits latently infected with all three viruses, and all three viruses reactivated in vitro with wild-type kinetics in an explant cocultivation assay. These studies indicate that the 370-bp region deleted in the 17deltaSty construct plays a role in epinephrine-induced reactivation.

Animals↗

PubMed1996

A herpes simplex virus type 1 mutant with a deletion immediately upstream of the LAT locus establishes latency and reactivates from latently infected mice with normal kinetics.

The latency associated transcripts (LATs) are the only abundant viral gene products detected during latent herpes simplex virus (HSV) infection of peripheral nerves in animals and people. A LAT promoter has been identified and mutant viruses with lesions removing the promoter and surrounding region have been observed to reactivate slowly from trigeminal ganglia (TG) explanted from latently infected mice. Previous work has shown that most mutants with lesions limited to regions downstream of the LAT promoter reactivate normally. Therefore, to help map the boundaries of the slow reactivation phenotype, a mutant virus with lesions located immediately upstream of the LAT promoter was constructed and called 17 delta S/N. 17 delta S/N contains a 437 nucleotide (nt) deletion 332 nts upstream of the TATAA box of the LAT promoter. In productively infected cells, 17 delta S/N failed to synthesize detectable amounts of the 1.1 and 1.8 kb transcripts which are produced during wild-type infections and are specified by a region just upstream of the LAT promoter. However, 17 delta S/N did produce normal amounts of LAT in tissue culture as well as in neurons derived from latently infected cells, as ascertained by Northern blot and in situ hybridization analysis. Moreover, in latently infected mice, 17 delta S/N established and maintained infection in as many neurons as did wild type virus, as determined by in situ polymerase chain reaction (PCR) to detect viral DNA. Finally, the virus reactivated from TG derived from latently infected mice with kinetics indistinguishable from those of wild-type virus. Therefore, reactivation from latency, in this model system, does not appear to require function from the viral genomic region located immediately upstream of the LAT promoter.

Animals↗

PubMed1996

Increased susceptibility to the pathogenic effects of wild-type and recombinant herpesviruses in MPS VII mice compared to normal siblings.

In previous studies, we have shown that a herpesvirus vector can transfer a therapeutic cellular gene (beta-glucuronidase) from peripheral sites of inoculation into the central nervous system in mice with a model neurodegenerative disease caused by a deficiency of this enzyme (mucopolysaccharidosis type VII, Sly disease). The vector corrects the enzymatic deficiency in transduced cells but the number of cells corrected is too low to alter the pathology of the disease. The recombinant vector virus, which has the foreign gene substituted into the viral LAT locus, had reduced pathogenicity after corneal inoculation compared to the wild-type virus from which it was derived (HSV-1 strain 17+). We therefore attempted to increase the number of corrected cells in the MPS VII brain by increasing the inoculating dose of the vector. However, the vector was acutely pathogenic in the diseased mice at doses that were non-pathogenic in normal littermates. The pathogenic effect of the vector virus in the mutants could be blocked by passive immunization with human gamma-globulin containing anti-HSV-1 antibodies on the day of infection but not when given at the peak of viral replication (day 4). However, effective protection also blocked transduction by the vector, thereby abrogating the effects of increased vector dosage. The effect was virus specific because inoculation of a high dose of a non-pathogenic variant of strain 17+ virus (1716) directly into the brains of MPS VII mice was not lethal. We found no apparent differences in the acute inflammatory response in mutant versus normal animals. These data suggest that the increased susceptibility to vector virulence was related to the overall compromised state of health of the diseased animals, which is further supported by the observations that the mutant mice are more sensitive to stress and to anesthetics than normal littermates. These findings indicate that adverse effects of gene transfer vectors for genetic diseases may not be fully apparent when tested in normal animals.

Anesthesia↗

PubMed1995

Treatment of experimental intracranial murine melanoma with a neuroattenuated herpes simplex virus 1 mutant.

Brain metastases occur commonly in the setting of a variety of human cancers. At present, such cases are invariably fatal and highlight a need for research on new therapies. We have developed a mouse brain tumor model utilizing the Harding-Passey melanoma cell line injected intracranially into C57Bl/6 mice. Tumors develop in 100% of the mice and can be detected by magnetic resonance imaging as early as 5 days post cell injection. Death from tumor progression occurs between 12 and 16 days post cell injection. Stereotactic injection of the neuroattenuated HSV-1 strain 1716 into brain tumors 5 or 10 days postinjection of the melanoma cells results in a statistically significant increase in the time to development of neurological symptoms and in complete tumor regression and the long-term survival of some treated animals. Moreover, viral titration studies and immunohistochemistry suggest that replication of this virus is restricted to tumor cells and does not occur in the surrounding brain tissue. These results suggest that HSV-1 mutant 1716 shows particular promise for use as a therapeutic agent for the treatment of brain tumors.

Animals↗