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Biomedical subjects

S M Brown

Publications and source records attributed to S M Brown.

At least 91 records · Page 5Linked to original sources
PubMed1996

Selective in vitro replication of herpes simplex virus type 1 (HSV-1) ICP34.5 null mutants in primary human CNS tumours--evaluation of a potentially effective clinical therapy.

Primary tumours of the central nervous system (CNS) are an important cause of cancer-related deaths in adults and children. CNS tumours are mostly glial cell in origin and are predominantly astrocytomas. Conventional therapy of high-grade gliomas includes maximal resection followed by radiation treatment. The addition of adjuvant chemotherapy provides little improvement in survival time and hence assessment of novel therapies is imperative. We have evaluated the potential therapeutic use of the herpes simplex virus (HSV) mutant 1716 in the treatment of primary brain tumours. The mutant is deleted in the RL1 gene and fails to produce the virulence factor ICP34.5. 1716 replication was analysed in both established human glioma cell lines and in primary cell cultures derived from human tumour biopsy material. In the majority of cultures, virus replication occurred and consequential cell death resulted. In the minority of tumour cell lines which are non-permissive for mutant replication, premature shut-off of host cell protein synthesis was induced in response to lack of expression of ICP34.5. Hence RL1-negative mutants have the distinct advantage of providing a double hit phenomenon whereby cell death could occur by either pathway. Moreover, 1716, by virtue of its ability to replicate selectively within a tumour cell, has the potential to deliver a 'suicide' gene product to the required site immediately. It is our opinion that HSV which fails to express ICP34.5 could provide an effective tumour therapy.

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

The herpes simplex virus type 2 strain HG52 RL1 gene contains a 154 bp intron as predicted from sequence analysis.

The published sequence of the herpes simplex virus (HSV) type 2 strain HG52 neurovirulence gene RL1 indicated that, unlike the HSV-1 homologue, the gene contains a 154 bp intron. This intron contains six complete and one partial copy of a 19 bp repeat which encodes a stop codon; thus all three forward frames are blocked. By RT-PCR of poly(A)+ RNA, using primers on either side of the proposed intron, we have isolated a partial cDNA clone corresponding to the predicted spliced mRNA.

Base Sequence↗
PubMed1996

The role of the gene 71 product in the life cycle of equine herpesvirus 1.

Equine herpesvirus type 1 (EHV-1) gene 71 encodes a heavily O-glycosylated 192 kDa protein with no identified herpesvirus homologue. Isolation of a deletion mutant in gene 71 (ED71) demonstrated that its protein product is not essential in vitro. To investigate the role of the gene 71 protein in the virus life cycle, ED71 has been characterized in vitro in terms of cellular adsorption, penetration, egress and transmission compared to wild-type and revertant virus. ED71 virions adsorbed to cells less efficiently than wild-type and revertant virus with a consequential effect on virus penetration; virus egress was significantly impaired and the timing of release was also delayed. The percentage of both full and empty capsids accumulating in the nuclei of ED71-infected cells was significantly higher than in wild-type virus-infected cells but the most notable differences were the low number of particles and the low ratio of enveloped to unenveloped capsids in the cytoplasm. The primary mode of transmission of the mutant virus is by direct cell-to-cell spread and the fact that a neutralizing antiserum did not reduce ED71 plaque size, supported the conclusion that deletion of gene 71 impairs the ability of virus to spread via release and readsorption to uninfected cells. Thus, deletion of EHV-1 gene 71 results in a defect in virus maturation and capsid envelopment. Progeny virus is consequently impaired in adsorption/penetration presumably due to the particles lacking the glycoprotein spikes predicted to be encoded by this gene and hence spreads by direct cell-to-cell contact.

Animals↗
PubMed1996

Gene delivery to the heart in vivo and to cardiac myocytes and vascular smooth muscle cells in vitro using herpes virus vectors.

Herpes simplex virus 1 (HSV1), while usually thought of as neurotrophic, can also efficiently infect a wide variety of non-neuronal cell types and so might be developed as a vector for gene delivery to non-neuronal as well as neuronal cells. Here we have tested three different disabled HSV vectors for their ability to deliver a lacZ gene to primary cardiac myocytes and vascular smooth muscle cells in vitro, and used the most efficient virus to transfect the rat heart in vivo. We also assessed the degree of cytopathic effect of the various viruses on the cardiac myocytes in vitro by testing the effects on the frequency of beating in synchronously beating myocyte cultures. While an HSV mutant in which the essential immediate-early gene IE2 had been deleted gave high efficiency gene transfer to the cardiac myocytes in vitro and the rat heart in vivo, viruses in which ICP34.5 or ICP34.5 and VMW65 were inactive (and which were also unable to replicate in these cells) gave a much lower efficiency of gene transfer, mirroring the degree of cytopathic effect observed in the beating myocyte cultures. Gene transfer to the vascular smooth muscle cells was considerably less efficient than to the myocytes in all cases. These results indicate that while HSV may be inappropriate for highly efficient gene transfer to the arterial wall, efficient gene transfer can be achieved in the myocardium, and thus that HSV vectors may be suitable for the alteration of cardiac cell physiology in vivo.

Animals↗
PubMed1996

Gene delivery to the central and peripheral nervous systems of mice using HSV1 ICP34.5 deletion mutant vectors.

Herpes simplex virus 1 (HSV1) ICP34.5 deletion mutants are avirulent upon inoculation of both the central and peripheral nervous systems of mice, but they replicate to near wild-type titres in a number of non-neuronally derived cell lines in culture. Thus these mutants might be suitable for development as safe vectors for gene transfer to the nervous system. However, the mechanism of this avirulent phenotype in neuronal cells is at present poorly understood, although it has been suggested that nonpermissive cells infected with these mutants may undergo apoptosis, the function of ICP34.5 being to prevent this response and to allow continued virus replication. If this were the case ICP34.5 null mutants might be unsuitable for gene transfer as infected cells would quickly die, limiting the expression of a transgene. Here we have inserted a beta-galactosidase marker gene into a nonessential gene of an HSV1 strain 17+ mutant in which ICP34.5 has been deleted and also into a second mutant in which the virion transactivator protein VMW65 is also inactive. While all the mutants grew to high titre in tissue culture, mice inoculated by the foot-pad or intracranial route at high titre remained healthy until the end of the experiment. Moreover, beta-galactosidase was expressed either in the brain or in the dorsal root ganglia, depending on the site of inoculation. This suggests that in vivo the absence of ICP34.5 does not prevent the expression of a transgene in neuronal tissue and indicates that non-neurovirulent mutants lacking this gene may be suitable for further development as safe vectors for gene therapy in vivo.

Animals↗
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↗
PubMed1995

Orbital emphysema following remote skull trauma.

In an unusual case of orbital emphysema following nose blowing, a reliable patient history and examination demonstrated no direct trauma to the orbit. Blunt posterior skull trauma was sustained several hours before the development of the orbital emphysema. A "seismic" transmittal of force to the orbital walls is postulated.

Craniocerebral Trauma↗
PubMed1995

Replication, establishment of latent infection, expression of the latency-associated transcripts and explant reactivation of herpes simplex virus type 1 gamma 34.5 mutants in a mouse eye model.

The herpes simplex virus type 1 (HSV-1) gamma 34.5 gene is located within a region that is transcriptionally active during latent HSV-1 infection. To determine whether the gamma 34.5 gene deletion affects latency-associated transcript (LAT) gene expression or latent HSV-1 infection, a gamma 34.5 gene deletion mutant, 1716, and a stop codon insertion mutant, 1771, were studied in the mouse eye model. Although the gamma 34.5 gene is not essential, 1716 and 1771 replicated poorly in mouse eyes and trigeminal ganglia (TG). When mice were inoculated with 1716, infectious virus was detected in eyes only on the first day post-infection (p.i.), and was not detected at any time point in TG. Following inoculation with 1771, a small amount of virus was detected in the eyes on days 2 and 4 p.i., and in the TG of one animal on day 2 p.i. Reactivation of virus from mice latently infected with 1716 (0/30 TG) and 1771 (1/20 TG) was extremely low compared with the parental strain, 17+, and appropriate rescuants (80 to 100% reactivation), even though latent 1716 DNA was detected by PCR in 50% of TG. These results differ from those obtained following footpad inoculation; in the footpad there was limited 1716 replication and reactivatable latent infection was established in some dorsal root ganglia. The data support the hypothesis that the role of gamma 34.5 may be tissue and/or cell type specific. The synthesis, processing, and stability of the 2.0 kb LAT during 1716 and 1771 replication was not affected by these mutations in the gamma 34.5 gene. However, during latent infection of 1716 in mice the LATs were not detectable in TG by Northern blot, and were present in reduced amounts (approximately 10-fold less) during 1771 latency. The LATs from 1716 were barely detectable in a few neurons by in situ hybridization. Therefore, the gamma 34.5 gene might (i) affect replication in the eye, and reduce the amount of virus available to establish latent infection, be directly involved in (ii) establishment of latency, and/or (iii) the reactivation process.

Animals↗
PubMed1995

Identification and characterization of the protein product of gene 67 in equine herpesvirus type 1 strain Ab4.

Equine herpesvirus type 1 (EHV-1) strain Ab4 gene 67 has no counterpart in any herpesvirus sequenced to date. To identify and characterize the product of EHV-1 gene 67, we have expressed the putative amino acids 11 to 260 encoded by gene 67 as a beta-galactosidase fusion protein in Escherichia coli. The expressed fusion protein has been used to generate an antiserum raised against the gene 67 product. Immunoblotting and immunoprecipitation experiments have revealed that the anti-67 serum specifically recognizes a polypeptide with an M(r) of 36,000 (the 36K polypeptide) in infected cell extracts. The gene 67 protein is regulated as an early polypeptide in EHV-1 strain Ab4 infected cells and post-translational modification experiments have revealed that the protein is phosphorylated, but not glycosylated. The gene 67 protein has been transiently expressed in BHK-21/C13 cells using plasmid pCMV67, which contains the putative gene 67 ORF under the control of the cytomegalovirus immediate early promoter. Immunoblotting experiments with anti-67 have shown that the 36K protein is expressed at high levels in transfected cells. From both immunofluorescence and cellular fractionation experiments it is concluded that the gene 67 protein is associated with intracellular membranes and produces novel ribbon or filament-like structures within the cytoplasm of infected cells. We have demonstrated that the gene 67 product is a component of the virion nucleocapsid/tegument.

Animals↗
PubMed1995

Cloning and characterization of the katB gene of Pseudomonas aeruginosa encoding a hydrogen peroxide-inducible catalase: purification of KatB, cellular localization, and demonstration that it is essential for optimal resistance to hydrogen peroxide.

Pseudomonas aeruginosa is an obligate aerobe that is virtually ubiquitous in the environment. During aerobic respiration, the metabolism of dioxygen can lead to the production of reactive oxygen intermediates, one of which includes hydrogen peroxide. To counteract the potentially toxic effects of this compound, P. aeruginosa possesses two heme-containing catalases which detoxify hydrogen peroxide. In this study, we have cloned katB, encoding one catalase gene of P. aeruginosa. The gene was cloned on a 5.4-kb EcoRI fragment and is composed of 1,539 bp, encoding 513 amino acids. The amino acid sequence of the P. aeruginosa katB was approximately 65% identical to that of a catalase from a related species, Pseudomonas syringae. The katB gene was mapped to the 71- to 75-min region of the P. aeruginosa chromosome, the identical region which harbors both sodA and sodB genes encoding both manganese and iron superoxide dismutases. When cloned into a catalase-deficient mutant of Escherichia coli (UM255), the recombinant P. aeruginosa KatB was expressed (229 U/mg) and afforded this strain resistance to hydrogen peroxide nearly equivalent to that of the wild-type E. coli strain (HB101). The KatB protein was purified to homogeneity and determined to be a tetramer of approximately 228 kDa, which was in good agreement with the predicted protein size derived from the translated katB gene. Interestingly, KatB was not produced during the normal P. aeruginosa growth cycle, and catalase activity was greater in nonmucoid than in mucoid, alginate-producing organisms. When exposed to hydrogen peroxide and, to a greater extent, paraquat, total catalase activity was elevated 7- to 16-fold, respectively. In addition, an increase in KatB activity caused a marked increase in resistance to hydrogen peroxide. KatB was localized to the cytoplasm, while KatA, the "housekeeping" enzyme, was detected in both cytoplasmic and periplasmic extracts. A P. aeruginosa katB mutant demonstrated 50% greater sensitivity to hydrogen peroxide than wild-type bacteria, suggesting that KatB is essential for optimal resistance of P. aeroginosa to exogenous hydrogen peroxide.

Aerobiosis↗
PubMed1995

Therapy of experimental human brain tumors using a neuroattenuated herpes simplex virus mutant.

BACKGROUND: Engineered herpes simplex virus (HSV) strains previously have been shown to offer a potential therapeutic alternative to conventional treatment modalities for brain tumors. Because HSV Type 1 strain 1716 has a deletion in the gamma 34.5 neurovirulence gene that renders it avirulent in the mouse central nervous system, we have assessed its potential to induce selective lysis of tumor cells versus neurons in vitro and in vivo. EXPERIMENTAL DESIGN: To do this, we studied parental HSV-1 strain 17+ and strain 1716 using human embryonal carcinoma cells (NT2 cells). These cells resemble neuronal progenitor cells and can be induced to differentiate into neurons (NT2N) with retinoic acid. Intracerebral grafts of NT2 cells into the brains of nude mice resulted in lethal brain tumors, and grafts of NT2N cells resulted in the integration of NT2N cells. RESULTS: In vitro studies showed that strain 1716 replicates in and spreads on monolayers of NT2 cells but not in NT2N cells. In vivo, strain 1716 replicated preferentially in NT2 tumors as evidenced by immunohistochemical staining for viral antigens, by in situ hybridization for HSV-specific transcripts, and by titration of virus from brains with tumor after intracranial injection of the virus into these mice. The temporal regression of NT2 tumors in mice treated with strain 1716 was demonstrated in vivo by magnetic resonance imaging. Electron microscopy and studies of DNA fragmentation suggested that regression of NT2 brain tumors in strain 1716-treated mice was mainly caused by a nonapoptotic, lytic mode of cell death. Finally, strain 1716-treated NT2 tumor-bearing mice survived more than twice as long as mock-treated tumor-bearing mice, and these differences in survival (25 vs. 9 weeks) were statistically significant (p < 0.03). CONCLUSIONS: We conclude from these studies that strain 1716 induces regression of human neural tumors established in the brains of nude mice, resulting in their prolonged survival.

Animals↗
PubMed1995

Deletions and duplication in internal inverted repeat sequence of long region/unique sequence of long region (IRL/UL) of herpes simplex virus type-1 (HSV-1) genome are not evidently associated with intracranial and foot-pad pathogenicity in mouse model.

The biological properties of three deletion variants (1704, 1705 and 1706) of herpes simplex virus type-1 (HSV-1) strain 17 syn+, were studied by establishing a base line pathogenicity of nine individual plaques from the parental 17 syn+ elite stock. Restriction enzyme analysis of deoxyribonucleic acid (DNA) from each of the nine plaque stocks and intracranial inoculation into three weeks old BALB/c mice showed no difference in the size of fragments and distribution of the sites or their 50% lethal dose (LD50) values [plaque forming units (pfu)/mouse] as compared to the parental 17 syn+ stock. Inoculation of the variants into three weeks old BALB/c mice showed that 1705 was not different in pathogenicity from the wild type following intracranial and footpad inoculations. On the other hand variants 1704 and 1706, when compared to the wild type virus were less virulent on intracranial inoculation i.e. the difference in LD50 values was approximately one log and two logs respectively and both the variants failed to kill any of the animals following footpad inoculation even at the dose of 1 x 10(7) pfu/mouse. During in vivo replication experiment in the peripheral nervous system of mice, 1704 and 1706 grew very poorly.

Amino Acid Sequence↗
PubMed1995

Latent phenotype analysis of three deletion variants of herpes simplex virus type 1 (HSV-1) in mouse model.

Latency analysis of three herpes simplex virus type 1 (HSV-I) strain 17 syn+ deletion variants (1704, 1705 and 1706) showed that they established, maintained and reactivated from latency. The kinetics of reactivation of 1705 and 1706 were similar to the parent HSV-1, 17 syn+, in which reactivation occurred 5-6 days post-explanation, but 1704 reactivated with delayed kinetics i.e. on the 12th day post-explantation. Since 1704 has deleted both copies of the latency associated transcripts (LATs) promoter region and one copy of the LAT coding region in internal inverted repeat sequence of long region (IRL), it was concluded that the LATs play a part in latency reactivation of 1704 from dorsal root ganglia (DRG) of spinal cord in mouse model. Restoration of the deleted sequences in the variant 1704 by marker rescue with the wild type BamHI b fragment resulted in a wild type genotype. This virus was designated as 1704R. Latency studies of 1704R revealed that the rate and frequency of reactivation was intermediate between 17 syn+ and 1704, suggesting a secondary undetected mutation affecting latency phenotype. Isolation of 1704LP-, during the same marker rescue experiment in which both copies of promoter region of the LAT are deleted and reactivation of this virus from latency with delayed kinetics confirms that the LATs play a role in reactivation from latency.

Amino Acid Sequence↗
PubMed1995

Isolation and characterization of two herpes simplex virus type 1 variants containing duplication of sequences within the unique long component of their genomes.

This paper reports the first spontaneous isolation of two DNA duplication variants in the unique long (UL) component of herpes simplex virus type 1 (HSV-1) strain 17+ genome, one (1719) with a duplication of 7.5 kb DNA sequences centered around OriL and the other (1740In) with a 356 bp DNA duplication between the UL19 (MCP) and UL20 open reading frames (ORFs). The variant 1719 is stable with the rare isolation of a wild type (strain 17+) genome presumably generated by the excision of the duplicated sequences during homologous recombination. Because of the 7.5 kb duplication, UL29 (DBP) is diploid and UL30 (DNA pol) is present as one complete and one partial copy. Although duplication in the variant 1740In involved sequences from the UL20 ORF, the virus produces an intact UL20 gene product. Both variants show normal growth characteristics when compared with the parental viruses. DNA duplications in these variants suggest a link between replication and recombination in HSV-1.

Amino Acid Sequence↗
PubMed1994

Automatic data reduction and pattern recognition methods for analysis of 1H nuclear magnetic resonance spectra of human urine from normal and pathological states.

Multivariate data analysis techniques have been used to compare 600-MHz 1H nuclear magnetic resonance (NMR) spectra of urine obtained from patients with inborn errors of metabolism (IEM) and urine obtained from healthy subjects. These spectra are very complex; each contains many thousands of resonances with a high dynamic range. A consistent method of reducing this wealth of data to manageable proportions is presented as a two-stage process. Computer-based spectral descriptors are automatically generated and then reduced to two-dimensional maps for visualization of clustering. Data-scaling methodology has been developed to achieve complete separation between spectra from control adults and those from adult patients with independently diagnosed IEM. The methods were refined by relating IEM samples to the mean of the control samples and applying supervised learning techniques to identify descriptors contributing to class separation. This approach allowed separation of the various classes of IEM and achieved optimal separation of patients with cystinuria from those with oxalic aciduria; the principal metabolites responsible for this separation were determined as lysine and glyoxalate. The methods developed were then extended by application to the more subtle problem of classifying urine collected from healthy subjects under different physiological conditions (i.e., pre- and post-exercise and in different stages of hydration) where, unlike the IEM case, any underlying biochemical differences were not known at the outset. Fluid-loaded and fluid-deprived samples could be partially separated as well as fluid-deprived and fluid-restored samples. Partial classification of samples on the basis of subject was also observed. Therefore, intersubject differences were liable to obscure the separation by physiological state. However, by relating each sample to a mean of the normal daily urine samples for the same person and applying a form of "range scaling" to exclude data which contributed least to class separation, improved classification of the hydration states resulted, from which it was possible to deduce those biochemical substances which were altered. These novel techniques for the data reduction and classification of NMR spectra make comprehensive use of all of the NMR spectral information and have clear potential to assist in clinical diagnosis.

Adult↗
PubMed1994

A hookworm glycoprotein that inhibits neutrophil function is a ligand of the integrin CD11b/CD18.

The chronic survival of many endoparasites is dependent on the ability of these organisms to escape the host immune response. Identification of the molecular mechanisms by which these organisms evade this response may yield novel approaches in the development of anti-inflammatory agents. We describe here the discovery and characterization of a novel 41-kilodalton glycoprotein from the canine hookwork (Ancylostoma caninum) that potently inhibits CD11/CD18-dependent neutrophil function in vitro. Neutrophil inhibitory factor (NIF) blocks the adhesion of activated human neutrophils to vascular endothelial cells as well as the release of H2O2 from activated neutrophils, over a similar concentration range (IC50 10-20 nM). Studies aimed at determining the nature of the NIF binding site on neutrophils revealed selective, high affinity binding of this protein to the integrin CD11b/CD18. A cDNA encoding NIF was isolated from a canine hookworm cDNA library. NIF comprises a mature polypeptide of 257 amino acids, preceded by a 17-amino acid leader. The mature protein has 10 cysteines and has seven potential N-linked glycosylation sites. NIF has no significant sequence homologies to any previously reported protein. As such, NIF represents a prototype of a novel class of leukocyte function inhibitors.

Amino Acid Sequence↗