Biomedical subjects

G J Atkins

Publications and source records attributed to G J Atkins.

At least 55 records · Page 3Linked to original sources

PubMed1999

Calcitonin receptor-mediated growth suppression of HEK-293 cells is accompanied by induction of p21WAF1/CIP1 and G2/M arrest.

We investigated the mechanisms by which calcitonin (CT) suppresses cellular proliferation, using HEK-293 cells stably transfected with either the rat C1a CT receptor (CTR) or the insert-negative form of the human CTR. CT treatment of clonal cell lines expressing either receptor type, but not untransfected HEK-293 cells, strongly suppressed cell growth in a concentration-dependent manner. The reduction in cell growth with CT treatment could not be attributed to cellular necrosis or apoptotic cell death, the latter assessed by both DNA fragmentation analysis and caspase 3 (CPP-32) assay. Growth inhibition was associated with an accumulation of cells in the G2 phase of the cell cycle. CT treatment of the human and rat CTR-expressing cell lines resulted in a rapid and sustained induction of mRNA encoding the cyclin-dependent kinase inhibitor, p21WAF1/CIP1, increased levels of which were maintained at least 48 h after initiation of treatment. Western blot analysis showed a rapid corresponding increase in p21WAF1/CIP1 protein, whereas protein levels of another member of the cyclin-dependent kinase inhibitor family, p27kip1, were unchanged. In parallel with the induction of p21, CT treatment reduced levels of p53 mRNA and protein. CT treatment resulted in a specific cell cycle block in G2, which was associated with inhibition of Cdc2/cyclin B kinase activity as measured by histone H1 phosphorylation. There was no evidence for p21 association with this complex despite the inhibition of Cdc2 activity. Evidence that p21 induction was causative of cell growth suppression was obtained from p21 antisense oligonucleotide experiments. Treatment with a p21 antisense oligonucleotide blocked induction of p21 expression and significantly reduced the CT-mediated growth inhibition. These observations suggest that p21 is required for the G2 arrest in response to CT, but argue against a direct role of p21 in the inhibition of Cdc2 activity. These studies suggest a novel regulation of cell cycle progression by CT and will provide a basis for detailed examination of the molecular mechanisms involved.

Animals↗

PubMed1998

The Semliki Forest virus vector induces p53-independent apoptosis.

Three deletion mutants of the structural protein region of the Semliki Forest virus (SFV) genome, including one which encompassed all the viral structural protein genes, induced apoptosis in BHK cells at 48 h after transfection, as shown by DNA laddering and TUNEL staining, as did the wild-type SFV4 RNA. A similar result was obtained for the SFV1 expression vector, which has a multicloning site inserted in place of the structural protein genes. However, in cells transfected with viral RNA containing a deletion of the nsP2 gene, neither viral RNA synthesis nor the induction of apoptosis occurred. Both SFV1 vector and wild-type SFV4 RNA induced apoptosis in human H358a lung carcinoma cells, which have a homozygous deletion of the p53 gene. It is concluded that the SFV vector encodes a function in the nonstructural coding region which induces p53-independent apoptosis and is dependent on viral RNA synthesis.

Animals↗

PubMed1997

Hepatitis B virus binding to leucocyte plasma membranes utilizes a different region of the preS1 domain to the hepatocyte receptor binding site and does not require receptors for opsonins.

A quantitative assay of hepatitis B virus (HBV) binding to hepatocyte plasma membranes was adapted to show that leucocyte plasma membranes bind serum-derived HBV saturably, and that this binding is inhibited using synthetic peptides representative of the large envelope protein of HBV. Using a panel of ligand-blocking monoclonal antibodies (mAb) to opsonin receptors, it was shown that the three classes of Fc gamma R and CR3 are not major receptors for HBV on leucocytes or hepatocytes. It was also shown that HBV does not utilize the receptor for IgA, Fc alpha R, for attachment to leucocytes, despite reported sequence homology between the large envelope protein of HBV and the Fc portion of human IgA. Evidence is presented that the receptor for HBV on leucocytes may differ from the hepatocyte receptor(s), based on synthetic peptide inhibition assays of HBV binding. Furthermore, it was observed that glycosaminoglycans influence the HBV-liver and leucocyte interactions, providing evidence that HBV attachment may be a multi-stage process.

Adult↗

PubMed1997

Sequence analysis of the avirulent, demyelinating A7 strain of Semliki Forest virus.

The nonstructural region of the genome of the avirulent A7 strain of Semliki Forest virus (SFV) has been sequenced, so that the complete nucleotide sequence is available. Compared to the virulent SFV4 strain (produced from the infectious clone pSP6-SFV4), A7 contains 226 nucleotide changes in the translated region, which result in 47 amino acid changes. The 5' nontranslated region has two nucleotide changes, and the 3' nontranslated region is longer in A7 than SFV4, and contains divergent and repeated sequences. Chimeras containing SFV4 and A7 sequences and an infectious clone of A7, pSP6-CA7, were constructed. The virulence of these was tested by intraperitoneal and intranasal infection of adult BALB/c mice. It was shown that determination of the avirulent phenotype of A7 was polygenic, and required the additive effect of sequences from both the structural and non-structural regions of the SFV genome.

Animals↗

PubMed1997

Death mechanisms in cultured cells infected by Semliki Forest virus.

We have investigated the induction of cell death in cultured cells by the virulent SFV4 and avirulent A7 strains of Semliki Forest virus (SFV). In BHK cells, death occurred by a typical apoptotic mechanism, as did the death of oligodendrocytes in glial cell cultures. For cerebellar neuron cultures, virus-induced death was due to necrosis. Although the SFV4 and A7 strains did not differ in the mechanism of induction of cell death, the virulent SFV4 strain did multiply to a higher titre in cultured neurons than the avirulent A7 strain. This is consistent with previous animal studies which indicate that the virulence of SFV strains is controlled by rapidity of multiplication in the CNS, leading to a lethal threshold of damage, rather than differential cell tropism or cell death mechanisms. The immune-mediated demyelination induced by avirulent strains may be triggered by apoptosis of oligodendrocytes, the consequences of which are obscured by death for virulent strains.

Cell Death↗

PubMed1997

Long-term effects of Semliki Forest virus infection in the mouse central nervous system.

Semliki Forest virus (SFV) infection of mice is used as a model to study pathogenic processes occurring in viral encephalitis and demyelinating disease. In this study, the long-term effects of infection by the avirulent M9 mutant of SPV on the central nervous system (CNS) of BALB/c and SJL mice were determined. The presence of infectious virus, viral RNA and cytokine mRNA in the brains of individual mice and the presence of lesions in the spinal cords of the same mice up to 360 days post-infection (d.p.i.) were analysed in order to detect any correlation between these parameters of pathogenesis. Infectious virus could not be detected beyond 7 d.p.i. for either mouse strain. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the presence of the E2 and nsP1 regions of the virus genome and mRNA for interferon-gamma and tumour necrosis factor-alpha. Viral RNA could be detected up to 90 d.p.i. for both mouse strains. Cytokine mRNA could be detected up to 28 d.p.i. for BALB/c mice but up to 360 d.p.i. for SJL mice. Inflammatory lesions, which were associated with cytokine mRNA expression, were not detected in BALB/c mice beyond 28 d.p.i. but were detected in two SJL mice at 90 d.p.i. It is concluded that M9-SFV infection induces long-term prolonged expression of pro-inflammatory cytokines in the CNS of the majority of SJL (but not BALB/c) mice which is not associated with persistence of the virus genome. M9-SFV infection of SJL mice may be a relevant model for the pathogenesis of multiple sclerosis in man.

Alphavirus Infections↗

PubMed1996

Manipulation of the Semliki Forest virus genome and its potential for vaccine construction.

The Semliki Forest virus (SFV) expression vector consists of a plasmid based on the SFV infectious clone. Foreign genes may be inserted into the structural coding region, transcribed as RNA, and expressed in cell culture after transfection. RNA containing inserted sequences may be packaged into virions using a helper systems. This allows efficient infection and expression without chemical transfection, but only one round of multiplication is possible. The biosafety of the system has been increased by the introduction of multiple mutations, specifying a maturation defect, into the helper. Potential vaccines can be constructed by insertion of genes coding for antigenic proteins into the vector. Following insertion of the influenza virus nucleoprotein (NP) into the SFV vector, immunity was induced following injection of packaged or naked RNA into mice. The SFV vector is a "suicide" expression vector that has great potential for the construction of vaccines for both human and veterinary use.

Animals↗

PubMed1996

A rapid microwave-in situ hybridization method for the definitive diagnosis of oral hairy leukoplakia: comparison with immunohistochemistry.

As a diagnostic technique, in situ hybridization requires a long processing time, a degree of expertise and may be difficult to handle routinely in some laboratories. To simplify the in situ hybridization method, we have modified a microwave in situ hybridization technique and applied it to oral hairy leukoplakia (OHL) biopsies obtained from 10 HIV-seropositive patients (definitively diagnosed by a conventional in situ hybridization technique) with appropriate controls. It was necessary to design a novel chamber to avoid drying of sections during the hybridization step. This modified microwave in situ hybridization technique was equispecific and equisensitive to the conventional technique and it shortens the hybridization time from overnight incubation to 14 minutes. To determine the sensitivity of our microwave in situ hybridization method we applied it to previously documented tongue tissue obtained from an AIDS autopsy without clinical evidence of OHL, but found to contain Epstein-Barr virus (EBV) by conventional in situ hybridization. This tissue specimen acted as a low EBV copy number, positive control. The sensitivity of immunohistochemistry using three different commercial detection kits was compared to that of in situ hybridization on the same tissues, following optimisation steps. This included the use of 2 cycles of primary and biotinylated secondary antibodies (antibody double cycling). Clearly positive signals for EBV were detected in all OHL biopsies with the Vectastain Elite ABC and the Histostain-SP kits. The sensitivity of the three commercial detection kits was evaluated at immunohistochemistry level by their application to the low-EBV copy number positive control specimen. Signals for EBV antigen in the low copy number positive control specimen were obtained only with the Vectastain Elite ABC kit. This indicates that, in this application, use of the Vectastain Elite ABC kit gives comparable sensitivity for immunohistochemistry to that found by in situ hybridiation.

AIDS-Related Opportunistic Infections↗

PubMed1995

Detection of Epstein-Barr virus DNA in tongue tissues from AIDS autopsies without clinical evidence of oral hairy leukoplakia.

Epstein-Barr virus (EBV) DNA was detected by in situ hybridization at 3 sites of 30 samples taken from clinically normal lateral border of tongue mucosa from 15 AIDS autopsies and in none of 20 samples from 10 controls. The first positive case showed a thin layer of parakeratosis correlated with positive signals for EBV in one area and an adjacent area without obvious parakeratosis was also positive for EBV. These findings were present on both sides of the tongue. The second case was unilaterally positive for EBV and parakeratosis was absent. The hybridization signals were localised to koilocyte-like cells in the stratum spinosum, as in oral hairy leukoplakia (OHL). These observations suggest that the in situ hybridization technique can detect very early or subclinical OHL, and supports the role of EBV in the pathogenesis of this lesion.

Acquired Immunodeficiency Syndrome↗

PubMed1994

A single amino acid change in the E2 spike protein of a virulent strain of Semliki Forest virus attenuates pathogenicity.

The virulent strain SFV4 of Semliki Forest virus (SFV), produced from the infectious clone pSP6-SFV4, is lethal after intranasal (i.n.) infection of adult mice and for pregnant mice after intraperitoneal (i.p.) infection. In contrast, the A7 strain of SFV is avirulent when given i.n. to adult mice, but induces fetal death in pregnant mice after i.p. infection. The nucleotide and deduced amino acid sequences of part of the core and all of the envelope region of A7-SFV were determined and compared to those of SFV4. A7 differed from SFV4 at 80 nucleotides (nt) in the coding sequence, 15 of which were associated with amino acid differences and seven of which (two in the E2 protein and five in E1) were non-conservative. The 3' non-coding sequence of A7 was longer (415 nt) than that of SFV4 (263 nt) and a divergent sequence of 181 nt was present adjacent to the end of the E1 coding region. The effects on virulence of two mutations in the E2 gene of SFV4, resulting in the non-conservative amino acid substitutions present in A7, were analysed. One mutation (mut 8729 a/c) resulted in only slight attenuation, whereas the other (mut 8902 a/g) resulted in avirulence for pregnant mice. However, mut 8902 a/g was lethal for the majority of developing fetuses after i.p. infection of the mother.

Alphavirus Infections↗

PubMed1994

Analysis of the molecular basis of neuropathogenesis of RNA viruses in experimental animals: relevance for human disease?

RNA viruses with segmented genomes were the first model used for molecular analysis of viral neuropathogenesis, since they could be analysed genetically by reassortment. Four viruses with non-segmented genomes have been used as models of neurovirulence and demyelinating disease: JHM coronavirus, Theiler's virus, Sindbis virus and Semliki Forest virus (SFV). Virus gene expression in the central nervous system of infected animals has been measured by in situ hybridization and immunocytochemistry. Cell tropism has been analysed by neural cell culture. Infectious clones have been constructed for Theiler's virus, Sindbis virus and SFV, and these allow analysis of the sequences involved in the determination of neuropathogenesis, through the construction of chimeric viruses and site-specific mutagenesis. Measles and rubella viruses have been studied in animal systems because of their importance for human disease. The importance of two recently discovered mechanisms of neuropathogenesis, antibody-induced modulation of virus multiplication, and persistence of virus in the absence of multiplication, remains to be assessed.

Animals↗

PubMed1994

In situ hybridization and the polymerase chain reaction (PCR) in the analysis of biopsies and exfoliative cytology specimens for definitive diagnosis of oral hairy leukoplakia (OHL).

The definitive diagnosis of oral hairy leukoplakia (OHL) demands that Epstein-Barr virus (EBV) is demonstrated in the lesional tissue, since the histopathological features on conventional light microscopy are not pathognomonic. We have investigated the possible use of polymerase chain reaction (PCR) technology in reaching a definitive diagnosis of this lesion by its application to ten biopsy specimens with definitive diagnoses of OHL determined by in situ hybridization. EBV DNA was demonstrated by PCR in all ten OHL biopsy specimens analysed, and none of ten control specimens. Furthermore, we have investigated the role of PCR in analysis of exfoliative cytology samples collected from the lateral border of the tongue by a minimally-invasive scraping technique. EBV DNA was not only detected in all OHL lesional scrapings but also in more than one-third of healthy controls, due to viral presence in saliva at the time of sampling. In this application, the highly sensitive PCR technique has low specificity and cannot be recommended.

Adolescent↗

PubMed1993

Semliki Forest virus expression system: production of conditionally infectious recombinant particles.

In the recently developed Semliki Forest virus (SFV) DNA expression system, recombinant RNA encoding the viral replicase, and helper RNA molecules encoding the structural proteins needed for virus assembly are cotransfected into cells. Since the helper RNA lacks the sequence needed for its packaging into nucleocapsids, only recombinant RNAs should be packaged. We have found, however, that small amounts of replication-proficient SFV particles can still be produced. Here we describe the construction of a helper variant with a mutation in the gene encoding the viral spike protein such that its product cannot undergo normal proteolytic processing to activate viral entry functions. Hence, the recombinant stock is noninfectious, but may be activated by cleavage with chymotrypsin. When recombinant virus produced with the new helper was examined in a variety of assays, including sensitive animal tests, we were unable to detect any replication-competent SFV particles. We therefore conclude that this conditional expression system meets extremely stringent biosafety requirements.

Amino Acid Sequence↗

PubMed1993

Virulent and avirulent strains of Semliki Forest virus show similar cell tropism for the murine central nervous system but differ in the severity and rate of induction of cytolytic damage.

The pathogenicity of the avirulent, demyelinating A7 strain of Semliki Forest virus (SFV) and the virulent SFV4 strain (derived from an infectious clone) for the central nervous system of adult BALB/c mice following intranasal infection was compared. The techniques used included immunocytochemistry using anti-SFV antibody and antibodies to cell markers, in situ hybridization (ISH) using a biotinylated cDNA probe specific for SFV, and immunocytochemistry/ISH double labelling. Whereas SFV4 was lethal at 4 days post-infection, A7-infected mice appeared normal at all times. Neuronal necrosis in the pyriform cortex was present in both infections, but developed sooner and was more severe following infection with SFV4 than with A7. Intact neurons and putative oligodendrocytes contained viral RNA and virus-specific antigen in SFV4 infected mice; viral RNA but not virus-specific antigen was detected in similar cells in A7-infected mice. These results confirm that SFV4 and A7 share similar cell tropisms for the murine central nervous system, but differ in the severity and rate of development of cytolytic damage. Intranasal infection is an efficient monitoring system for studies of the molecular basis of pathogenicity of SFV infection in mice.

Animals↗

PubMed1993

Effect of infection with rubella virus on the development of rat cerebellar cells in culture.

Immunogold-silver staining of mixed rat cerebellar neuron/glial cell cultures, using Ranscht monoclonal antibody, resulted in labelling of neuronal processes and oligodendrocytes. This antibody was originally believed to react with galactocerebroside alone, and it was concluded that labelling by it resulted from the presence of myelin [2]. Electron microscopic examination of the cultures did not reveal myelination. Infection of such cultures with rubella virus resulted in a cytopathic effect. This effect was initially characterized as a disaggregation of neuronal processes, and the disruption of a layer of membranous material covering both cell bodies and processes. The virus causes disruption of cultures of pure glial cells, but there is no evidence of virus multiplication or cytopathic effect in pure neuron cultures. The cytopathic effect in mixed neuron/glial cell cultures is concluded to be glial cell mediated.

Animals↗

PubMed1991

Two mutations in the envelope glycoprotein E2 of Semliki Forest virus affecting the maturation and entry patterns of the virus alter pathogenicity for mice.

The prototype strain of Semliki Forest virus (SFV) of known sequence and virus produced by the cDNA clone derived from it were lethal following intranasal (i.n.) infection of 40-day-old and intraperitoneal (i.p.) infection of pregnant BALB/c mice; this lethality was related to neuronal necrosis in the central nervous system (CNS). We conclude that the virulence of the prototype strain, and virus from the cDNA clone derived from it, is similar to that of L10 (the original SFV isolate). The effects of two mutations in the p62 envelope protein region of the clone were determined. Substitution of Glu for Lys at position 162 (mut64) extended the mean time of death following i.n. inoculation of 40-day-old mice. Pregnant mice infected with this virus survived but lethal infection of some fetuses did occur. Substitution of Leu for Arg at position 66 (mL), the cleavage site of the E2 and E3 proteins, results in the production of particles containing uncleaved p62. These particles were less virulent than the prototype strain when inoculated i.n. and induced immunity to virulent SFV challenge. The virus also induced the formation of multifocal glial nodules in the CNS of surviving mice. The differences in pathogenicity between the two mutants and the virulent parental virus are probably related to differences in the efficiency of virus multiplication in infected mice. The mut64 mutation attenuated the virus and allowed survival of pregnant mice infected i.p. so that the effects of fetal infection could be detected. The mL mutation allowed survival of i.n.-infected mice so that the later effects of virus multiplication in the CNS could be assessed. In the former case, this is probably a result of reduced virus release, whereas in the latter case it is due to inefficient entry of host cells. The results are consistent with our previous suggestion that lethality for virulent SFV infection results from a lethal threshold of damage to neurons in the CNS and that attenuating mutations may reduce neuronal damage below this threshold level.

Animals↗

PubMed1991

Multiplication of rubella and measles viruses in primary rat neural cell cultures: relevance to a postulated triggering mechanism for multiple sclerosis.

Rubella virus multiplied to low titre and produced a partial cytopathic effect in rat glial cell cultures. Anti-galactocerebroside staining showed that this cytopathic effect involved the disintegration of oligodendrocytes. A similar effect was produced following infection of myelinating neural cell cultures with rubella virus, but virus multiplication could not be detected in pure neuron cultures. Measles virus was found to multiply and produce a cytopathic effect in primary cultures of both neurons and glial cells. These results are discussed in relation to the ability of measles and rubella viruses to trigger human multiple sclerosis.

Animals↗

PubMed1990

Multiplication of virulent and demyelinating Semliki Forest virus in the mouse central nervous system: consequences in BALB/c and SJL mice.

The sites of multiplication in the mouse central nervous system (CNS) of the virulent L10 strain of Semliki Forest virus (SFV) and the L10-SFV-derived demyelinating M9 mutant were determined using both BALB/c and SJL mouse strains. In situ hybridization (ISH), using a cRNA probe to an SFV non-structural sequence, and immunogold-silver staining (IGSS), using polyclonal anti-SFV rabbit IgG, were the techniques utilized. For L10-SFV, viral RNA and antigen were detected in neurons and glial cells of both mouse strains. For BALB/c mice infected with M9-SFV, both neuronal and glial cell infection was less extensive than that obtained with L10. ISH or IGSS were generally not sensitive enough to detect viral RNA and antigen, respectively, in M9-SFV-infected SJL mice. M9-SFV multiplied to a similar titre in primary cultures of glial cells derived from either BALB/c or SJL mice. Following infection with M9-SFV, small plaques of demyelination in the CNS and occasional small aggregates of mononuclear leukocytes in the leptomeninges persisted for up to 12 months in SJL mice but not BALB/c mice. This was not associated with detectable persistence of infectious virus, viral antigen or viral RNA in the CNS.

Animals↗