PubMed Health⌕ Search

Biomedical subjects

A J Stevenson

Publications and source records attributed to A J Stevenson.

At least 19 recordsLinked to original sources
PubMed2006

Analysis of changes in optical fibers during arc-fusion splicing by use of quantitative phase imaging.

A non-interferometric imaging technique in conjunction with Abel inversion is used to directly and quantitatively examine the changes in optical fibers due to the heating produced during arc-fusion splicing as a function of fusion arc parameters. Phase images in the vicinity of a fusion splice are obtained using Quantitative Phase Microscopy, allowing the refractive-index change to be reconstructed with high spatial resolution. This simple, nondestructive method confirms that, for a fixed arc current, while the fusion time increases, the refractive-index of both fiber cores within the fusion region decreases in magnitude, the core region broadens, and the axial gradient decreases.

Journal Article↗
PubMed2000

A herpesvirus saimiri-based gene therapy vector with potential for use in cancer immunotherapy.

The herpesvirus saimiri (HVS) genome has the capacity to incorporate large amounts of heterologous DNA and can be maintained episomally in many different human cell types. To evaluate the efficacy of HVS-mediated gene transfer into human hemopoietic cells, we investigated the ability of an HVS-based construct, carrying the enhanced green fluorescent protein (EGFP) and neomycin resistance genes, to transduce a variety of human hemopoietic cell lines and primary CD34+ cells. As measured by flow cytometry, the numbers of EGFP+ cells at 2 days postinfection differed between various cell types ranging, from 1.3% for KG1 cells to 56.8% for THP-1 cells. In addition, the expression of EGFP in Jurkat cells was retained at >95% per round of cell division over a period of 6 weeks (comparable with Epstein-Barr virus-derived gene therapy systems). Although the virus was not specifically disabled, no lytic viral mRNAs could be detected in transduced Jurkat cells, and infectious virus could not be detected by sensitive virus recovery assay. We also describe a simple centrifugation method that increases the efficiency of transduction by >100% in some cases and may be generally applicable to other herpesvirus-based vectors for ex vivo gene delivery. Using this technique, we were able to demonstrate a tropism for CD34+/CD14+ cells, transducing 30% of the population. These cells are known to give rise to dendritic cells (the most potent of the antigen-presenting cells), suggesting that the vector could be used to deliver DNA sequences encoding tumor antigens for cancer immunotherapy.

Antigens, CD34↗
PubMed2000

Herpesvirus saimiri-based gene delivery vectors maintain heterologous expression throughout mouse embryonic stem cell differentiation in vitro.

In order to achieve a high efficiency of gene delivery into rare cell types like stem cells the use of viral vectors is presently without alternative. An ideal stem cell gene therapy vector would be able to infect primitive progenitor cells and sustain or activate gene expression in differentiated progeny. However, many viral vectors are inactivated when introduced in developing systems where cell differentiation occurs. To this end, we have developed a mouse in vitro model for testing herpesvirus saimiri (HVS)-based gene therapy vectors. We demonstrate here for the first time that HVS is able to infect totipotent mouse embryonic stem (ES) cells with high efficiency. We have transduced ES cells with a recombinant virus carrying the enhanced green fluorescent protein (EGFP) gene and the neomycin resistance gene (NeoR) driven by a CMV promoter and the SV40 promoter, respectively. ES cells maintain the viral episomal genome and can be terminally differentiated into mature haematopoietic cells. Moreover, heterologous gene expression is maintained throughout in vitro differentiation. Besides its obvious use in gene therapy, this unique expression system has wide ranging applications in studies aimed at understanding gene function and expression in cell differentiation and development.

Animals↗
PubMed2000

Specific oncolytic activity of herpesvirus saimiri in pancreatic cancer cells.

The potential use of oncolytic viruses in the treatment of cancer has been investigated for some time. A variety of agents have been studied, including some which appear to be selectively replication-competent in cancer cell lines. In this study, we have investigated the ability of herpesvirus saimiri to specifically lyse selected human cancer cell lines. Upon infection with a replication-competent virus carrying the EGFP reporter gene and a neomycin resistance marker, the pancreatic cancer lines MIAPACA and PANC-1 exhibited definite cytopathic effects. In contrast, the colonic carcinoma cell lines SW480 and HCT116 were phenotypically unaltered. In addition, stable cell lines could not be generated from PANC-1 infected cultures, in marked contrast to cultures of cells from other human tissues. Virus recovery assays demonstrated that all of the cell lines produced a small amount of virus post-infection, but that virus replication was minimal after 1 week in culture. In addition, treatment with acyclovir inhibited virus replication but paradoxically increased cytopathic effect. These data suggest that herpesvirus saimiri may have potential as an oncolytic agent for the treatment of pancreatic cancer.

Genetic Therapy↗
PubMed2000

Stable marker gene transfer into human bone marrow stromal cells and their progenitors using novel herpesvirus saimiri-based vectors.

We have evaluated the ability of new herpesvirus saimiri (HVS)-based vectors to deliver a marker gene green fluorescent protein (GFP) into human bone marrow (BM) stromal cells and their progenitors. Stromal cells expanded from adherent layers of long-term BM cultures (LTC) were susceptible to HVS-based infection in a dose-dependent manner, and the efficiency of 94.8 +/- 2.0% was achieved using single exposure with HVS/EGFP vector at multiplicity of infection (moi) of approximately 50. Colony-forming unit-fibroblast (CFU-F) assay established the ability of HVS-based vectors to infect progenitors for bone marrow stroma fibroblasts and transfer the marker gene over multiple cell divisions in the absence of selective pressure. HVS was not toxic for stromal cells and progenitors and no viral replication was detected upon growth of modified stroma. On the basis these data, we believe that HVS-based constructs can offer a new opportunity for selective gene delivery into bone marrow stromal cells and progenitors. The ability of HVS to infect nondividing cells can be considered advantageous in the development of both ex vivo and in vivo strategies.

Bone Marrow Cells↗
PubMed2000

Analysis of gene expression in a human cell line stably transduced with herpesvirus saimiri.

Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus; it has significant homology to the human gammaherpesviruses Kaposi's sarcoma-associated virus and Epstein-Barr virus and the murine gammaherpesvirus murine herpesvirus 68. HVS causes a persistent asymptomatic infection in its natural host, the squirrel monkey. Both subgroups A and C possess the ability to immortalize common marmoset T lymphocytes to interleukin-2-independent proliferation. However, only subgroup C is capable of transforming human, rabbit, and rhesus monkey lymphocytes in vitro. In addition, HVS can stably transduce a variety of human cell lines where the virus persists as a nonintegrating circular episome. In this study, we have developed a system in which the HVS DNA is stably maintained as a nonintegrated circular episome in the human lung carcinoma cell line A549. Virus production can be reactivated using chemical inducing agents, including tetradecanoyl phorbol acetate and n-butyrate, suggesting that the infection in human A549 cells is latent. To analyze virus gene expression in these stably transduced cells, Northern blot analysis was performed using a series of probes produced from restriction fragments spanning the entire coding region of the HVS genome. This demonstrated that an adjacent set of genes containing open reading frames (ORFs) 71 to 73 are expressed in this stably transduced cell line. Moreover, these genes are transcribed as a polycistronic mRNA species produced from a common promoter upstream of ORF 73. This model may serve as a useful tool in the further analysis of the role of ORFs 71 to 73 in gamma-2 herpesvirus latency.

Animals↗
PubMed2000

Rates of spontaneous reactivation and aging of acetylcholinesterase in human erythrocytes after inhibition by organophosphorus pesticides.

The in vitro rates of spontaneous reactivation and aging in human erythrocyte acetylcholinesterase were studied after inhibition by a dimethoxy (R1R2) and diethoxy substituted (R1R2) organophosphate pesticide (OP) of general structure R1R2P(O)X. These have been compared with data for human plasma cholinesterase previously reported using a similar methodology. A significantly slower rate of aging for erythrocyte acetylcholinesterase was found compared to plasma cholinesterase, whether inhibited by dimethoxy or diethoxy substituted OPs. For diethoxy OPs the rate of spontaneous reactivation of the inhibited plasma enzyme was significantly slower than for the inhibited red cell enzyme. This acetylcholinesterase, and previously published plasma cholinesterase, data suggest that in practise a blood sample taken 30-40 h after significant acute OP exposure will still show inhibition in either plasma or erythrocyte cholinesterase when analysed, but that any inhibited plasma enzyme is more likely to be in the aged form. In contrast a substantial proportion of the erythrocyte acetylcholinesterase is found unaged and therefore sensitive to reactivation by oximes. Samples from an occupational exposure where depressions in plasma or erythrocyte cholinesterase activity from baseline measurements were reactivated ex vivo using the oxime 2-PAM support this hypothesis. These data also confirm that the plasma enzyme is a more sensitive than erythrocyte acetylcholinesterase as an indicator of OP exposure and thus the potential value of ex vivo oxime reactivation of erythrocyte acetylcholinesterase in a blood sample to indicate subclinical OP exposure may be limited. However, this study is too small to draw conclusions on the sensitivity of ex vivo oxime reactivation of acetylcholinesterase as a novel biomarker of excessive OP absorption. Given that there is a better relationship between anticholinergic symptoms and red cell acetylcholinesterase inhibition, and that the slower resynthesis rate of any aged or inhibited red cell enzyme may be interpretatively useful when venepuncture is delayed, it is suggested that red cell acetylcholinesterase activity does have a place in monitoring potential OP exposure.

Acetylcholinesterase↗
PubMed1999

The gene product encoded by ORF 57 of herpesvirus saimiri regulates the redistribution of the splicing factor SC-35.

The herpesvirus saimiri (HVS) gene product encoded by ORF 57 shares limited C-terminal similarity with herpes simplex virus 1 ICP27, a protein that has been demonstrated to be involved in the inhibition of host-cell splicing and is responsible for the redistribution of components of the spliceosome. It has previously been shown that ORF 57 can either activate or repress viral gene expression by a post-transcriptional mechanism. Furthermore, repression of gene expression by ORF 57 is dependent on the presence of an intron within the target gene coding region. In this report, it is shown that HVS infection results in the redistribution of the SC-35 splicing factor in the infected cell nucleus. Furthermore, the redistributed SC-35 colocalized with the ORF 57 protein product and expression of the protein alone was sufficient to cause the redistribution of the spliceosome components. These results suggest that the mechanism by which ORF 57 down-regulates expression of intron-containing genes involves the redistribution of the spliceosome complex.

Animals↗
PubMed1999

The open reading frame 57 gene product of herpesvirus saimiri shuttles between the nucleus and cytoplasm and is involved in viral RNA nuclear export.

The herpesvirus saimiri open reading frame (ORF) 57 is homologous to genes identified in all classes of herpesviruses. It has previously been shown to regulate gene expression through a posttranscriptional mechanism. We demonstrate in this report that the expression of the ORF 57 protein leads to the cytoplasmic accumulation of glycoprotein B and capsid mRNAs. We also demonstrate that ORF 57 has the ability to specifically bind viral RNA transcripts. Utilizing an interspecies heterokaryon assay, we show that ORF 57 has the ability to shuttle between the nucleus and the cytoplasm. Furthermore, we show that ORF 57 contains a relatively leucine-rich sequence which shares some homology with nuclear export signals (NES) found in a number of proteins with the ability to shuttle between the nucleus and the cytoplasm. Moreover, we demonstrate that the ORF 57 NES enables the nuclear export of a heterologous protein and that mutation of the conserved leucine residues contained within the ORF 57 NES signal abrogates the ability of the ORF 57 protein to shuttle between the nucleus and cytoplasm. These results suggest that ORF 57 is involved in mediating the nuclear export of viral transcripts.

Animals↗
PubMed1999

The activation domain of herpesvirus saimiri R protein interacts with the TATA-binding protein.

The herpesvirus saimiri open reading frame (ORF) 50 produces two transcripts. The first is spliced, contains a single intron, and is detected at early times during the productive cycle, whereas the second is expressed later and is produced from a promoter within the second exon. Analysis of their gene products has shown that they function as sequence specific transactivators. In this report, we demonstrate that the carboxy terminus of ORF 50b contains an activation domain which is essential for transactivation. This domain contains positionally conserved hydrophobic residues found in a number of activation domains, including the herpes simplex virus VP16 and the Epstein-Barr virus R proteins. Mutational analysis of this domain demonstrates that these conserved hydrophobic residues are essential for ORF 50 transactivation capability. Furthermore, this domain is required for the interaction between the ORF 50 proteins and the basal transcription factor TATA-binding protein.

Amino Acid Sequence↗
PubMed1999

Assessment of Herpesvirus saimiri as a potential human gene therapy vector.

Herpesvirus saimiri has characteristics that make it amenable to development as a gene therapy vector. The viral genome is thought to be capable of accommodating large quantities of heterologous DNA while the virus itself can infect many different cell types. Virus infection has been shown in many cases to be persistent by virtue of episomal maintenance in the target cell. In this article we examine the ability of nonselectable recombinant viruses expressing the beta-galactosidase gene product to infect a variety of human cells and demonstrate that this virus could be developed as an alternative hematopoietic stem cell gene therapy vector. In contrast to earlier observations, we demonstrate by a number of methods that the virus has the ability to replicate in many human cell types, suggesting the need for the development of a disabled virus for use as a gene therapy vector.

Blotting, Southern↗
PubMed1998

The use of power Doppler ultrasound in the diagnosis of isolated deep venous thrombosis of the calf.

While ultrasound has been well accepted as an accurate test for the diagnosis of both femoral and popliteal deep venous thrombosis (DVT), its role in the detection of calf DVT has been less clear. There have been variable results between studies in the accuracy of colour Doppler or compression sonographic techniques in demonstrating calf DVT. Many of these studies have suffered from high rates of technically inadequate examinations. Power Doppler (colour Doppler energy) has a higher sensitivity than conventional colour Doppler. It should therefore allow the detection of slow venous flow in patent vessels, that may not be demonstrated by conventional colour Doppler. This should decrease the number of technically inadequate examinations as well as decreasing the false-positive rate of the test. A prospective blinded reader study was undertaken comparing power Doppler to the gold standard venography, in the diagnosis of isolated calf deep venous thrombosis (DVT). Both examinations were successfully performed in 50 patients, in whom proximal DVT had been excluded by compression ultrasonography. Fifteen patients had positive evidence of an isolated calf DVT on the gold standard technique, venography. Power Doppler demonstrated a sensitivity of 100%, a specificity of 79%, a positive predictive value of 71% and a negative predictive value of 100%, in detecting an isolated calf DVT. There were no technically inadequate ultrasound examinations. Where venous flow is demonstrated, power Doppler is a highly accurate test in excluding a calf DVT. The specificity of the test, however, is limited, as the absence of flow does not always signify a DVT.

Humans↗
PubMed1998

Differences in the intracellular localization and fate of herpes simplex virus tegument proteins early in the infection of Vero cells.

The fate of herpes simplex virus 1 (HSV-1) tegument proteins during infection in Vero cells was investigated immunochemically. Input virion-associated VP13/14 and VP16 localized to the nucleus early in infection, while VP1/2 localized to the nuclear envelope of the cell and VP22 could not be detected using monoclonal antibody P43. Western blotting suggested that virion-associated VP13/14, VP16 and VP22 were stable in infected cells whereas VP1/2 appeared to be processed or modified. Further studies showed that P43 recognized a phosphorylation-sensitive epitope in VP22 and suggested that virion-associated VP22 was phosphorylated upon entry to the cell. VP13/14 and VP16 were easily extracted from cells early in infection whereas VP22 was largely insoluble. Phosphatase treatment of soluble extracts caused a shift in the molecular mass of VP16 showing it was phosphorylated. As infection progressed VP16 was observed in discrete nuclear compartments where it co-localized with ICP8 and the capsid-associated protein VP22a. VP13/14 was also observed in the nucleus. P43 immunostaining appeared around 6 h post-infection as punctate nuclear foci which often localized to the edge of VP16-immunoreactive areas. Punctate P43 cytoplasmic staining appeared around 12 h post-infection. By 18 h the nuclear pattern had disappeared and an extensive cytoplasmic stain was observed which closely overlapped that of other tegument proteins. On the basis of these data we suggest that virion-associated VP22 is phosphorylated upon entry of the virus into the cell and that unphosphorylated VP22, which is preferentially recognized by P43, becomes available later in infection, initially in the nucleus, for packaging into virions.

Animals↗
PubMed1998

Influence of biological and analytical variation on urine measurements for monitoring exposure to cadmium.

OBJECTIVE: To define the mean intraindividual biological variation in urinary concentrations of cadmium and retinol binding protein (RBP) in untimed, random urine samples and the influence of creatinine or specific gravity correction on reducing this variation. The relation between biological variation and analytical variation in defining uncertainty in a single measurement and significant differences between successive measurements was explored. METHODS: Repeat measurement study in subjects with either high historical exposure to cadmium but without current exposure, or unexposed volunteers. Standard statistical tools used in clinical laboratory medicine were applied to define intraindividual biological and analytical variation. RESULTS AND CONCLUSION: Both creatinine and specific gravity correction of urinary cadmium measurements in random urine samples seem to reduce the intraindividual variability compared with uncorrected values. With a standard definition, acceptable long term analytical precision for measurements of cadmium and RBP combined with creatinine analyses should be < 9% and < 15% respectively. The mean intraindividual biological variation of cadmium and RBP, expressed as creatinine corrected, was 18% and 40% respectively in the subjects exposed to cadmium. With the analytical precision used, significant differences (p < 0.05) between consecutive measurements for creatinine corrected urinary cadmium and RBP would need to show changes of > 54% and > 110% respectively. The relation between significant differences in consecutive results and differences in the analytical precision of the method used to measure the samples is described.

Analysis of Variance↗
PubMed1997

Processing and intracellular localization of the herpes simplex virus type 1 proteinase.

The herpes simplex virus type 1 (HSV-1) capsid protein VP24 (encoded by UL26) was expressed as a GST-fusion protein and used to prepare a group of monoclonal antibodies. These were used to characterize the protein in capsids and virus infected cells and demonstrated that it exists as two polypeptide species. The nature of the relationship between these two species was investigated and found to be associated with disulphide bonding. Under non-reducing conditions a species corresponding to dimers of VP24 was identified in preparations of B capsids, the site of action of the proteinase. Biochemical subcellular fractionation studies suggested that only cleaved forms of UL26 and UL26.5 gene products could be detected in the nucleus of the infected cell at early times post-infection.

Animals↗
PubMed1997

Identification of a cis-acting element within the herpesvirus saimiri ORF 6 promoter that is responsive to the HVS.R transactivator.

We have previously demonstrated that two distinct transcripts are produced from ORF 50, the major transcriptional activating gene of herpesvirus saimiri. The products of these transcripts trans-activate the delayed-early ORF 6 promoter, though to different degrees. Deletion analysis demonstrated that the ORF 50 responsive elements are contained in a 132 bp fragment situated 127-259 bp from the transcription initiation site within the ORF 6 promoter. This fragment conferred ORF 50-responsiveness on an enhancerless simian virus 40. Gel retardation analysis further mapped the responsive elements to a 38 bp fragment.

Base Sequence↗
PubMed1997

Pulmonary artery pseudoaneurysm due to bronchial carcinoma.

A case of pseudoaneurysm of the pulmonary artery due to squamous cell bronchial carcinoma is reported. This complication of bronchial carcinoma has not been described previously. The patient presented with severe haemoptysis and the diagnosis was made using CT and pulmonary angiography. The pulmonary artery feeding the aneurysm was embolized with tungsten coils. Haemoptysis ceased following this intervention.

Aneurysm, False↗