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Biomedical subjects

R J Shaw

Publications and source records attributed to R J Shaw.

At least 19 recordsLinked to original sources
PubMed2006

Promoter methylation of P16, RARbeta, E-cadherin, cyclin A1 and cytoglobin in oral cancer: quantitative evaluation using pyrosequencing.

Methylation profiling of cancer tissues has identified this mechanism as an important component of carcinogenesis. Epigenetic silencing of tumour suppressor genes through promoter methylation has been investigated by a variety of means, the most recent of which is pyrosequencing. We have investigated quantitative methylation status in oral squamous cell carcinoma patients. Fresh tumour tissue and normal control tissue from resection margin was obtained from 79 consecutive patients undergoing resection of oral squamous cell carcinoma. DNA was extracted and bisulphite treated. PCR primers were designed to amplify 75-200 bp regions of the CpG rich gene promoters of p16, RARbeta, E-cadherin, cytoglobin and cyclinA1. Methylation status of 4-5 CpG sites per gene was determined by pyrosequencing. Significant CpG methylation of gene promoters within tumour specimens was found in 28% for p16, 73% for RARbeta, 42% for E-cadherin, 65% for cytoglobin and 53% for cyclinA1. Promoter methylation was significantly elevated in tumours compared to normal tissue for p16 (P = 0.048), cytoglobin (P = 0.002) and cyclin A1 (P = 0.001) but not in RARbeta (P = 0.088) or E-cadherin (P = 0.347). Concordant methylation was demonstrated in this tumour series (P = 0.03). Significant differences in degree of methylation of individual CpG sites were noted for all genes except RARbeta and these differences were in a characteristic pattern that was reproduced between tumour samples. Cyclin A1 promoter methylation showed an inverse trend with histological grade. Promoter methylation analysis using pyrosequencing reveals valuable quantitative data from several CpG sites. In contrast to qualitative data generated from methylation specific PCR, our data demonstrated p16 promoter methylation in a highly tumour specific pattern. Significant tumour specific methylation of cyclin A1 promoter was also seen. Cytoglobin is a novel candidate tumour suppressor gene highly methylated in upper aero-digestive tract squamous cancer.

Adult↗
PubMed2005

The transfer of maternal IgE and other immunoglobulins specific for Trichostrongylus colubriformis larval excretory/secretory product to the neonatal lamb.

The transference of immunoglobulins from six New Zealand Romney ewes to their lambs was examined. Immunoglobulin levels were determined in ewe plasma, colostrum and lamb plasma shortly after birth and before the lambs fed, in lamb plasma 2 days after birth, and lamb plasma, ewe plasma and milk 30 days after parturition. Levels of total IgE, and IgE, IgG1, IgG2, IgM, and IgA with specificity for Trichostronglus colubriformis third stage larval secretory/excretory products (TcL3E/S) were determined. Mean levels of total IgE were three times higher in colostrum than in parturient ewe plasma while only trace amounts were detected in milk at 30 days after birth (107.7, 34.3, and 0.2U ml(-1), respectively, differences between means P< or =0.01). Mean total IgE in lamb plasma rose from being undetectable before suckling to levels comparable to those of the ewes by 2 days after birth (21.7U ml(-1)) and then declined to low levels by 30 days (0.4U ml(-1)). Total IgE levels in lamb plasma were significantly correlated with levels in ewe plasma and colostrum (r=0.91, P< or =0.01; r=0.96, P< or =0.003, respectively). The transference of TcL3E/S-specific IgE, IgG1 and IgA was substantial with mean levels of these antibodies in lamb plasma at 2 days comparable to that in parturient ewe plasma (absorbance levels in lamb plasma of 0.283, 0.537, and 0.334, respectively). Proportionally less maternal IgM and IgG2 appeared to be transferred to the lambs (absorbance of 0.112 and 0.081, respectively). Levels of TcL3E/S-specific IgE and IgG1 in lamb plasma at 2 days were significantly correlated with levels in parturient ewe plasma and colostrum (r=0.89 and 0.82, 0.85 and 0.96; all P< or =0.05, respectively). These results indicate that IgE is concentrated in ewe colostrum and that substantial amounts of maternal IgE are transferred to lambs via colostrum. Further, the results suggest that humoral immunity against gastro-intestinal nematode parasites and potentially other parasites in colostrum-fed lambs may approximate that of the ewe. The implications of the transference of humoral immunity through colostrum in ruminants for the passive protection and the development of active immunity against parasites remains to be fully explored.

Animals↗
PubMed2005

Total and nematode-specific IgE responses in intestinal lymph of genetically resistant and susceptible sheep during infection with Trichostrongylus colubriformis.

Total and antigen-specific IgE responses in afferent (AIL) and efferent (EIL) intestinal lymph of sheep with a nematode resistant (R) or susceptible (S) genotype during challenge infection with the intestinal nematode parasite Trichostrongylus colubriformis were examined. Within each sheep line, lambs with a nematode naive or nematode field-primed pre-challenge status were used. Total IgE level in AIL and EIL was dependent on nematode infection and was further influenced by genotype or the immune phenotype (nematode immune mean FEC+/-SDM=77+/-179 or non-immune mean FEC+/-SDM=4016+/-4318) of the animal. During T. colubriformis challenge immune animals had higher levels of total IgE in lymph than non-immune sheep, R line sheep had higher concentrations of total IgE than S line sheep, and field-primed animals had higher total IgE levels than nematode naive animals. Concentrations of total IgE were consistently higher in AIL than EIL or serum and were higher in lymph draining the proximal than the distal jejunum demonstrating that polyclonal IgE in AIL was largely derived from the intestinal mucosa of the anatomical compartment where the nematodes reside. The consistently higher concentration of total IgE in AIL was dependent on phenotype or genotype and in S genotype sheep also on the pre-challenge status. Concentrations of nematode specific IgE were significantly higher in EIL than AIL indicating a preference for the production of IgE reacting with excretory secretory products of the infective T. colubriformis larvae in the regional lymph node.

Animals↗
PubMed2005

Standards for gene therapy clinical trials based on pro-active risk assessment in a London NHS Teaching Hospital Trust.

Conducting gene therapy clinical trials with genetically modified organisms as the vectors presents unique safety and infection control issues. The area is governed by a range of legislation and guidelines, some unique to this field, as well as those pertinent to any area of clinical work. The relevant regulations covering gene therapy using genetically modified vectors are reviewed and illustrated with the approach taken by a large teaching hospital NHS Trust. Key elements were Trust-wide communication and involvement of staff in a pro-active approach to risk management, with specific emphasis on staff training and engagement, waste management, audit and record keeping. This process has led to the development of proposed standards for clinical trials involving genetically modified micro-organisms.

Bacteria↗
PubMed2003

Changes in brain intracellular pH and membrane phospholipids on oxygen therapy in hypoxic patients with chronic obstructive pulmonary disease.

Chronic hypoxia due to chronic obstructive pulmonary disease (COPD) constitutes a stress to cerebral metabolic homeostasis. Previous studies using phosphorus-31 magnetic resonance spectroscopy (31P MRS) have suggested that the brains of such patients utilize anaerobic glycolysis, which in neonatal, animal, and in vitro studies is associated with a protective intracellular alkalosis. To identify such a compensatory intracellular alkalosis in hypoxic COPD patients, in vivo cerebral 31P MRS was performed in eight patients and eight controls. The mean intracellular pH (pHi) in patients with COPD was similar to that of age-matched controls, but decreased in the patients with COPD by a mean pHi of 0.02 (p = 0.04), following supplemental oxygen. There was no change in cerebral pHi in normal subjects following oxygen administration. The broadband component of the MR spectrum increased in all the patients with COPD (p = 0.01), suggesting altered phospholipid membrane fluidity in the brain associated with the change in pHi following oxygen administration. The change in the broadband resonance was strongly correlated with the change in pHi (r = -0.68, p = 0.014). This study suggests that patients with COPD exhibit a compensatory change in pHi and abnormalities in cerebral membrane phospholipid conformation in the face of chronic hypoxia.

Adult↗
PubMed2003

Immune rejection of Trichostrongylus colubriformis in sheep; a possible role for intestinal mucus antibody against an L3-specific surface antigen.

Sheep that have been immunized by multiple truncated infections with the parasitic nematode Trichostrongylus colubriformis contain anti-larval activity in their intestinal mucus and high-speed mucus supernatants. This activity induces T. colubriformis L3 to clump in vitro and causes a significant reduction in larval establishment in naive sheep after infusion of larvae and mucus into the intestinal lumen via a duodenal cannula. In this report, we provide evidence that one factor contributing to the anti-larval activity of immune mucus is antibody against a 35-kDa L3-specific cuticular antigen. The anti-larval activity in mucus is > 100 kDa by membrane filtration, is heat labile and sensitive to either protease digestion or reduction with DTT. Immunoblotting showed that mucus and supernatants of ultracentrifuged mucus from immune sheep contained IgG1 and IgA antibodies that recognized predominantly a larval antigen with an estimated molecular weight of 35 kDa on SDS-PAGE. Antibodies eluted from the surface of washed larvae that had been incubated in immune mucus also reacted specifically with the 35 kDa antigen on blots of larval homogenate. Immunofluorescence and immunogold electron microscopy showed that the 35 kDa antigen is present on the epicuticle of L3 and is shed during the moult to L4. The antigen is not present in eggs, L1, L2, L4 or adult worms and is found only in extracts of sheaths and L3 before infection and up to 4 days after infection. We hypothesize that the binding of antibody to the larval surface prevents larvae from establishing at their preferred site, causing them to be eliminated from the intestine. Monoclonal antibody PAB-1 recognizes the 35 kDa T. colubriformis larval antigen and also cross-reacts with antigens of similar molecular weight on blots of L3 extracts of the parasitic nematodes Haemonchus contortus and Ostertagia circumcincta; and with a 22-kDa antigen on blots of L3 extracts from Cooperia curticei and Nematodirus spathiger. This indicates that an antigenically related surface antigen with immunizing potential is present on several nematode species and can be identified by mAb PAB-1. The 35 kDa T. colubriformis larval antigen and related molecules in other nematodes are potential novel targets for stimulating host-protective immunity against nematode infections.

Animals↗
PubMed2002

The molecular epidemiology of tuberculosis in inner London.

The study used DNA fingerprint typing (spoligotyping and Heminested-lnverse-PCR) of Mycobacterium tuberculosis from all culture-confirmed inner London patients over a 12-month period to describe transmission. The methodology was evaluated by comparison with standard IS6110 typing and by examining its ability to identify known household clusters of cases. Isolates sharing indistinguishable typing patterns using both techniques were defined as clustered. Clusters were investigated to identify epidemiological links. The methodology showed good discriminatory power and identified known household clusters of cases. Of 694 culture-confirmed cases, 563 (81%) were typed. Eleven (2%) were due to laboratory cross-contamination and were excluded. Of the remaining 552 isolates 148 (27%) were clustered. Multivariate analysis indicated that clustering was more common in those with pulmonary smear positive disease (P < 0.02); those born in the United Kingdom (P < 0.0003) and in patients living in south London (P = 0.02). There was also a trend towards clustering being more common in those not known to have HIV infection (P = 0.051). The results suggest that in inner London, recent local transmission makes an important contribution to notification rates.

Adolescent↗
PubMed2002

The role of small airways in lung disease.

The small airways constitute one of the least understood areas of the lungs. They play a role in many lung diseases, and small airway pathology results in significant morbidity New approaches to their evaluation may provide insights into this major area of lung disease. Asthma is well recognized as a disease of both large and small airways. Physiological and pathological evidence, from techniques such as post-mortem tissue histological analysis, induced sputum and transbronchial biopsies, has reinforced the concept of the involvement of the entire bronchial tree n the inflammatory process in asthma, In addition to describing the airway pathology in asthma, th s review focuses on the pathogenesis and role of small airway obstruction n other diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), sarcoidosis and obliterative bronchiolitis (OB). COPD is characterized by the presence of airflow obstruction resulting from lesions in the small airways. In addition, features compatible with small airways disease are common in IPF, sarcoidosis and OB. Recent advances in pulmonary imaging, such as high-resolution computed tomography (HRCT) and magnetic resonance imaging (MRI) with hyperpolarized 3He, have allowed non-invasive reproducible measurements of structure-function relationships to be made for the small airways. These techniques have great potential for diagnosing changes in small airway function and for assessing responses to treatment. New insights into the contribution of small airways to a range of lung diseases may lead to the development of therapies targeted at this part of the bronchial anatomy.

Bronchi↗
PubMed2001

The characterization of novel, dual ErbB-2/EGFR, tyrosine kinase inhibitors: potential therapy for cancer.

The type I receptor tyrosine kinases constitute a family of transmembrane proteins involved in various aspects of cell growth and survival and have been implicated in the initiation and progression of several types of human malignancies. The best characterized of these proteins are the epidermal growth factor receptor (EGFR) and ErbB-2 (HER-2/neu). We have developed potent quinazoline and pyrido-[3,4-d]-pyrimidine small molecules that are dual inhibitors of ErbB-2 and EGFR. The compounds demonstrate potent in vitro inhibition of the ErbB-2 and EGFR kinase domains with IC(50)s <80 nM. Growth of ErbB-2- and EGFR-expressing tumor cell lines is inhibited at concentrations <0.5 microM. Selectivity for tumor cell growth inhibition versus normal human fibroblast growth inhibition ranges from 10- to >75-fold. Tumor growth in mouse s.c. xenograft models of the BT474 and HN5 cell lines is inhibited in a dose-responsive manner using oral doses of 10 and 30 mg/kg twice per day. In addition, the tested compounds caused a reduction of ErbB-2 and EGFR autophosphorylation in tumor fragments from these xenograft models. These data indicate that these compounds have potential use as therapy in the broad population of cancer patients overexpressing ErbB-2 and/or EGFR.

Animals↗
PubMed2001

Analysis of an essential requirement for the poly(A) binding protein function using cross-species complementation.

Poly(A) binding protein (PABP) is an essential, well-conserved, multifunctional protein involved in translational initiation, mRNA biogenesis, and degradation [1--5]. We have used a cross-species complementation approach to address the nature of the essential requirement for PABP in yeast. The expression of Pab3p, a member of the Arabidopsis thaliana PABP multigene family, rescues the lethal phenotype associated with the loss of the yeast Pab1p. However, Pab3p neither protects the mRNA 5' cap from premature removal, nor does it support poly(A)-dependent translational initiation or the synergistic enhancement of translation by the poly(A) tail and 5' cap in yeast. However, Pab3p corrects the temporal lag prior to the entry of the mRNA into the degradation pathway characteristic of pab1 Delta yeast strains. Furthermore, this lag correction by Pab3p requires Pan3p, a subunit of poly(A) nuclease, an enzyme involved in the mRNA 3'-end processing. Importantly, the substitution of Pab3p for the yeast Pab1p is synthetically lethal with the PAN3 gene deletion. These results show that the function of PABP in mRNA biogenesis alone could be sufficient to support cell viability in yeast.

Arabidopsis↗
PubMed2001

Regulation of an IMP dehydrogenase gene and its overexpression in drug-sensitive transcription elongation mutants of yeast.

IMP dehydrogenase is a rate-limiting enzyme involved in the synthesis of GTP. In mammalian cells it is regulated with respect to growth rate and is the target of numerous therapeutic agents. Mutations in the RNA polymerase II elongation machinery render yeast sensitive to inhibitors of IMP dehydrogenase and defective in inducing transcription of one of the IMP dehydrogenase-encoding genes, IMD2. Here we show that loss of IMD2, but not IMD1, IMD3, or IMD4, conferred upon yeast the same drug sensitivity found in elongation mutants. We tested whether the drug sensitivity of elongation mutants is due to their inability to induce IMD2 by providing them with exogenous copies of the gene. In some elongation mutants, overexpression reversed drug sensitivity and a transcriptional defect. Overexpression in mutants with a more severe phenotype partially suppressed drug sensitivity but was inconsequential in reversing a defect in transcription. These findings suggest that the drug sensitivity of elongation mutants is largely but not solely attributable to defects in the ability to induce IMD2, because transcription is compromised even when IMD2 mRNA levels are adequate. We describe two DNA sequence elements in the promoter of the gene that regulate it. We also found that IMD2 mRNA abundance is coupled to cell growth rate. These findings show that yeast possess a conserved system that gauges nucleotide pools and cell growth rate and responds through a uniquely regulated member of the IMD gene family.

Gene Expression Regulation, Enzymologic↗
PubMed2001

Contribution of Th1 and Th2 cells to protection and pathology in experimental models of granulomatous lung disease.

Mice that had received adoptive transfer of DO11.10 TCR transgenic T cells polarized toward a Th1 or a Th2 phenotype were challenged with Ag-coated beads or with recombinant Mycobacterium tuberculosis expressing the OVA determinant. The resulting bead-induced pulmonary granulomas reflected the phenotype of the adoptively transferred T cells, with the Th2 cells promoting a fibrotic reaction. Mice receiving Th1 cells mounted an epitope-specific protective response to challenge with recombinant M. tuberculosis. Th2 recipients were characterized by enhanced weight loss and lung fibrosis during acute high-dose infection. The combination of TCR transgenic T cells and epitope-tagged mycobacteria provides a novel experimental model for investigation of the pathogenesis of tuberculosis.

Adoptive Transfer↗
PubMed2001

The Nf2 tumor suppressor, merlin, functions in Rac-dependent signaling.

Mutations in the neurofibromatosis type II (NF2) tumor suppressor predispose humans and mice to tumor development. The study of Nf2+/- mice has demonstrated an additional effect of Nf2 loss on tumor metastasis. The NF2-encoded protein, merlin, belongs to the ERM (ezrin, radixin, and moesin) family of cytoskeleton:membrane linkers. However, the molecular basis for the tumor- and metastasis- suppressing activity of merlin is unknown. We have now placed merlin in a signaling pathway downstream of the small GTPase Rac. Expression of activated Rac induces phosphorylation and decreased association of merlin with the cytoskeleton. Furthermore, merlin overexpression inhibits Rac-induced signaling in a phosphorylation-dependent manner. Finally, Nf2-/- cells exhibit characteristics of cells expressing activated alleles of Rac. These studies provide insight into the normal cellular function of merlin and how Nf2 mutation contributes to tumor initiation and progression.

3T3 Cells↗
PubMed2001

The mere exposure effect in patients with schizophrenia.

The mere exposure effect refers to the development of an emotional preference for previously unfamiliar material because of frequent exposure to that material. This study compared schizophrenia subjects (n = 20) to normal controls (n = 21) to determine whether implicit memory, as demonstrated by the mere exposure effect, was intact. Patients with schizophrenia demonstrated a normal preference for both verbal and visual materials seen earlier relative to novel materials, despite impaired performance on a recognition task for explicit memory using similar materials. Previous studies of schizophrenia subjects have shown a dissociation between implicit and explicit memory on verbal tasks. We found a similar dissociation demonstrated by normal functioning on an implicit memory task and impaired functioning on an explicit memory task. Potential implications of these findings are discussed with regard to treatment and rehabilitation.

Adult↗
PubMed2001

Enhanced antimycobacterial response to recombinant Mycobacterium bovis BCG expressing latency-associated peptide.

With a view to exploring the role of transforming growth factor beta (TGF-beta) during mycobacterial infection, recombinant clones of bacillus Calmette-Guérin (BCG) were engineered to express the natural antagonist of TGF-beta, latency-activated peptide (LAP). Induction of TGF-beta activity was reduced when macrophages were infected with BCG expressing the LAP construct (LAP-BCG). There was a significant reduction in the growth of LAP-BCG in comparison to that of control BCG following intravenous infection in a mouse model. The enhanced control of mycobacterial replication was associated with an increase in the production of gamma interferon by splenocytes challenged during the acute stage of infection but with a diminished recall response assessed after 13 weeks. Organ weight and hydroxyproline content, representing tissue pathology, were also lower in mice infected with LAP-BCG. The results are consistent with the hypothesis that TGF-beta has a detrimental effect on mycobacterial immunity. While a reduction in TGF-beta activity augments the initial response to BCG vaccination, early bacterial clearance may adversely affect the induction of a long-term memory response by LAP-BCG.

Animals↗