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Biomedical subjects

J M Nicholls

Publications and source records attributed to J M Nicholls.

At least 19 recordsLinked to original sources
PubMed2007

Tropism of avian influenza A (H5N1) in the upper and lower respiratory tract.

Poor human-to-human transmission of influenza A H5N1 virus has been attributed to the paucity of putative sialic acid alpha2-3 virus receptors in the epithelium of the human upper respiratory tract, and thus to the presumed inability of the virus to replicate efficiently at this site. We now demonstrate that ex vivo cultures of human nasopharyngeal, adenoid and tonsillar tissues can be infected with H5N1 viruses in spite of an apparent lack of these receptors.

Cells, Cultured↗
PubMed2006

Avian influenza H5N1 in viverrids: implications for wildlife health and conservation.

The Asian countries chronically infected with avian influenza A H5N1 are 'global hotspots' for biodiversity conservation in terms of species diversity, endemism and levels of threat. Since 2003, avian influenza A H5N1 viruses have naturally infected and killed a range of wild bird species, four felid species and a mustelid. Here, we report fatal disseminated H5N1 infection in a globally threatened viverrid, the Owston's civet, in Vietnam, highlighting the risk that avian influenza H5N1 poses to mammalian and avian biodiversity across its expanding geographic range.

Animals↗
PubMed2005

Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells.

BACKGROUND: Fatal human respiratory disease associated with influenza A subtype H5N1 has been documented in Hong Kong, and more recently in Vietnam, Thailand and Cambodia. We previously demonstrated that patients with H5N1 disease had unusually high serum levels of IP-10 (interferon-gamma-inducible protein-10). Furthermore, when compared with human influenza virus subtype H1N1, the H5N1 viruses in 1997 (A/Hong Kong/483/97) (H5N1/97) were more potent inducers of pro-inflammatory cytokines (e.g. tumor necrosis factor-a) and chemokines (e.g. IP-10) from primary human macrophages in vitro, which suggests that cytokines dysregulation may play a role in pathogenesis of H5N1 disease. Since respiratory epithelial cells are the primary target cell for replication of influenza viruses, it is pertinent to investigate the cytokine induction profile of H5N1 viruses in these cells. METHODS: We used quantitative RT-PCR and ELISA to compare the profile of cytokine and chemokine gene expression induced by H5N1 viruses A/HK/483/97 (H5N1/97), A/Vietnam/1194/04 and A/Vietnam/3046/04 (both H5N1/04) with that of human H1N1 virus in human primary alveolar and bronchial epithelial cells in vitro. RESULTS: We demonstrated that in comparison to human H1N1 viruses, H5N1/97 and H5N1/04 viruses were more potent inducers of IP-10, interferon beta, RANTES (regulated on activation, normal T cell expressed and secreted) and interleukin 6 (IL-6) in primary human alveolar and bronchial epithelial cells in vitro. Recent H5N1 viruses from Vietnam (H5N1/04) appeared to be even more potent at inducing IP-10 than H5N1/97 virus. CONCLUSION: The H5N1/97 and H5N1/04 subtype influenza A viruses are more potent inducers of proinflammatory cytokines and chemokines in primary human respiratory epithelial cells than subtype H1N1 virus. We suggest that this hyper-induction of cytokines may be relevant to the pathogenesis of human H5N1 disease.

Bronchi↗
PubMed2004

Re-emergence of fatal human influenza A subtype H5N1 disease.

Human disease associated with influenza A subtype H5N1 re-emerged in January, 2003, for the first time since an outbreak in Hong Kong in 1997. Patients with H5N1 disease had unusually high serum concentrations of chemokines (eg, interferon induced protein-10 [IP-10] and monokine induced by interferon gamma [MIG]). Taken together with a previous report that H5N1 influenza viruses induce large amounts of proinflammatory cytokines from macrophage cultures in vitro, our findings suggest that cytokine dysfunction contributes to the pathogenesis of H5N1 disease. Development of vaccines against influenza A (H5N1) virus should be made a priority.

Animals↗
PubMed2004

The aetiology, origins, and diagnosis of severe acute respiratory syndrome.

Severe acute respiratory syndrome (SARS) is a new infectious disease that first emerged in Guangdong province, China, in November, 2002. A novel coronavirus was later identified in patients with SARS. The detection of the virus in these patients, its absence in healthy controls or other patients with atypical pneumonia, and the reproduction of a similar disease in a relevant animal model fulfilled Koch's postulates for implicating this coronavirus as the causal agent of SARS. The full genome sequence was determined within weeks of the virus's identification. The rapid progress in the aetiology, the development of laboratory diagnostic tests, and the defining of routes of viral transmission were facilitated through a unique WHO-coordinated virtual network of laboratories, which shared information on a real-time basis through daily teleconferences. Subsequent studies have indicated that the SARS coronavirus is of animal origin, that its precursor is still present in animal populations within the region, and that live-animal markets in southern China may have provided the animal-human interphase that allowed this precursor virus to adapt to human-human transmission. These findings underscore the potential for the re-emergence of SARS and the need for laboratory tests for early diagnosis. However, the low viral load in the respiratory tract makes early diagnosis of SARS a diagnostic challenge, although improvements in the sensitivity of molecular diagnostic methods continue to be made.

Animals↗
PubMed2001

Expression of cytokine and chemokine genes in Epstein-Barr virus-associated nasopharyngeal carcinoma: comparison with Hodgkin's disease.

Nasopharyngeal carcinoma (NPC) and Hodgkin's disease (HD) are characterized by their association with Epstein-Barr virus (EBV) and the presence of an intense lymphoid stroma, consisting of T lymphocytes and other reactive cells. In both entities, the tumour cells express viral proteins known to provide target epitopes for cytotoxic T-cells (CTLs), yet in vivo, the tumour cells appear to escape CTL recognition. A comparative in situ hybridization study of cytokine and chemokine gene expression in NPC and HD has been undertaken, focusing on cytokines which are known to be inducible by EBV in vitro. Hodgkin and Reed-Sternberg (HRS) cells expressed interleukin (IL)-6, IL-8, and IL-10, and the thymus and activation regulated chemokine (TARC) in 15/22, 0/22, 5/22, and 16/21 cases, respectively. In NPC, the epithelial tumour cells showed expression of IL-6 in 3/43 cases and of IL-8 in 2/40 cases. There was no detectable expression of IL-10 and TARC in these cases. These data confirm that HRS cells frequently express cytokine and chemokine genes and suggest that this may enable HRS cells to modulate the immune response in their microenvironment and to escape CTL detection. In contrast, NPC tumour cells show only rare expression of IL-6 and IL-8 and no detectable expression of IL-10 and TARC. Thus, the results suggest that the mechanisms employed by the EBV-positive tumour cells to escape immune recognition and destruction differ between HD and NPC.

Carcinoma↗
PubMed2001

Analytic validation of a competitive polymerase chain reaction assay for measuring Epstein-Barr viral load.

Epstein-Barr virus (EBV) is associated with several benign and malignant diseases, and blood tests for EBV viral load show promise as markers of disease burden in affected patients. A commercial quantitative PCR method (BioSource International) was recently introduced to facilitate measuring viral load. It relies on coamplification of EBV DNA and a spiked competitor in plasma or serum, followed by semiautomated product detection on enzyme-linked immunosorbent assay (ELISA) plates. In the current study, analytic performance characteristics were assessed, and the authors describe several methodologic improvements to facilitate laboratory implementation. Rapid DNA extraction was accomplished using commercial silica spin columns, heat-labile uracil-N-glycosylase was used to inhibit amplicon contamination, and inexpensive agarose gels were used to screen for polymerase chain reaction products requiring ELISA plate quantitation. Accuracy and precision were verified using EBV DNA standards derived from two cell lines and plasmid containing viral sequences. The assay was sensitive to as few as five template copies per polymerase chain reaction and was linear across four orders of magnitude (correlation coefficient 0.995). When applied to matched plasma and serum samples from 15 patients with nasopharyngeal carcinoma, both sample types yielded similar viral load results. This commercial EBV viral load assay provides sensitive and quantitative detection of EBV DNA using equipment already available in many molecular diagnostic laboratories.

DNA, Viral↗
PubMed2001

Activity of the EBNA1 promoter associated with lytic replication (Fp) in Epstein-Barr virus associated disorders.

BACKGROUND/AIMS: In Epstein-Barr virus (EBV) positive cell lines that are stably infected, three different promoters are known to direct the transcription of EBV nuclear antigen 1 (EBNA1). These are located in the BamHI-C, BamHI-Q, and BamHI-F regions of the viral genome (Cp, Qp, and Fp, respectively). Fp is activated upon induction of the viral lytic cycle. The aim of this study was to investigate the activity of Fp in EBV associated diseases. METHODS: Using reverse transcriptase polymerase chain reaction, a qualitative analysis of EBNA1 promoter usage in various EBV associated diseases was performed. RESULTS: Fp driven transcription was detected in the context of primary infection and/or lytic replication; at least a portion of the Fp driven transcripts encoded EBNA1. Qp driven EBNA1 transcripts were detected in most samples across the range of disorders tested. Cp driven EBNA1 transcripts were detected in the context of immune suppression and in samples containing EBV positive (non-neoplastic) lymphoid cells. CONCLUSIONS: These results confirm the previously proposed "housekeeping" function of the Qp promoter.

Cell Line↗
PubMed2000

Cytotoxic effect of gossypol on colon carcinoma cells.

Gossypol, a male contraceptive drug extracted from cottonseeds, has been found to have antiproliferative activity on tumour cells and is thought to be a potential anticancer drug. The aim of this study was to investigate the mechanisms of gossypol-induced cell death on two colon carcinoma cell lines, HT29 and LoVo. Firstly, we studied the effect of gossypol on the colony forming ability of these tumour cells, which is the main target of chemotherapeutic drugs. Using clonogenic assays, flow cytometry and DNA gel electrophoresis techniques, we have found that gossypol not only inhibited colony forming ability of these tumour cells, but we also observed cellular internucleosomal DNA fragmentation in the cells treated with 3 doses of gossypol and this was accompanied by the appearance of a sub-G1 apoptotic peak and morphological characteristics of apoptosis. Our results suggest that the gossypol induced cell death is via an apoptotic pathway and the effect of gossypol may not be cell cycle specific. Using Western blotting analysis, we found that the gossypol-induced apoptosis may not be involved in the regulation of p53 but possibly associated with the regulation of bcl-2 and Bax expression. Our evidence indicates that gossypol may provide a potential therapeutic benefit for the treatment of colon carcinoma and understanding the mechanisms of gossypol-induced cytotoxicity on tumour cells is essential for including this drug in clinical use.

Cell Cycle↗
PubMed2000

High level expression of deltaN-p63: a mechanism for the inactivation of p53 in undifferentiated nasopharyngeal carcinoma (NPC)?

Undifferentiated nasopharyngeal carcinoma (NPC) is an epithelial malignancy that is consistently associated with Epstein-Barr virus (EBV) but which very rarely has p53 gene mutations in primary tumours. Since the tumour suppressor p53 is mutated in most human cancers or the wild type protein is inactivated in a significant number of the remainder, here we have investigated cellular factors that could compromise p53 function in primary NPC. Twenty-five primary tumours were judged to carry only wild type p53 by SSCP analysis of all exons and sequence determination of exons 4-9. Only one tumour was found to express significant levels of hMdm2 and in 24/25 there were no detectable mutations or deletions in exons 1beta and 2 of the p14(ARF) gene. However, immunohistochemistry consistently revealed that all the tumour cells express substantial amounts of the p53-related protein p63. Semi-quantitative RT-PCR analysis of mRNA from tumour biopsies showed that the dominant species expressed was invariably the truncated deltaN-isotype. Since this can block p53-mediated transactivation, it is potentially a dominant-negative isoform. In normal nasopharyngeal epithelium the distribution of p63 was restricted to the proliferating basal and suprabasal layers. We suggest that deltaN-p63 is a good candidate as a suppressor of wild type p53 function in these tumours and also that it may prove to be a valuable diagnostic marker for undifferentiated NPC.

Carrier Proteins↗
PubMed2000

Effect of p16INK4a on chemosensitivity in nasopharyngeal carcinoma cells.

The p16INK4a tumor suppressor gene is frequently inactivated in nasopharyngeal carcinoma (NPC) and hence it may play an important role in the suppression of this tumor. In order to study the effect of p16INK4a restoration in NPC cells, full-length human p16INK4a gene was transfected into a NPC cell line, CNE1. Four individual clones with differential levels of p16INK4a protein expression, were selected for further studies. The introduction of p16INK4a into CNE1 cells induced growth suppression through G0/G1 cell cycle arrest; however, the cell growth rate was not correlated to the levels of p16INK4a protein expression. To study whether transfection of p16INK4a could protect NPC cells from radiation, cisplatin and 5-fluorouracil (5FU), the cellular sensitivity of p16INK4a transfectants and vector control were investigated. An increase in sensitivity to 5FU was observed (2-fold compared to IC50) in all 4 clones compared to vector-transfected control. P16INK4a transfection also resulted in increased sensitivity to cisplatin (1.5-1.8-fold) in 3 out of 4 cell lines. However, no difference in radiosensitivity was found between the p16INK4a transfectants and the control. These findings indicate that p16INK4a suppresses NPC cell growth through G0/G1 arrest and modulating cellular response to chemotherapeutic drugs in NPC cells. Therefore, restoration of p16INK4a may have a therapeutic purpose in the treatment of NPC.

Carrier Proteins↗
PubMed1999

Prenatal sonographic features of spondylocostal dysostosis and diaphragmatic hernia in the first trimester.

Spondylocostal dysostosis is a congenital disorder characterized by multiple malformations of the vertebrae and ribs. We describe the sonographic features of an affected fetus at 12 and 14 weeks of gestation. The fetus had thoracic scoliosis, multiple vertebral and rib malformations and a grossly dilated stomach that had herniated into the chest through a left-sided diaphragmatic hernia. The stomach spanned the whole length of the fetal trunk.

Adult↗
PubMed1999

Cisplatin-induced p53-independent growth arrest and cell death in cancer cells.

In a recent study, it was shown that DNA damaging agent cisplatin-induced growth arrest and cell death in cancer cells by a pathway sharing some of the characteristics of replicative senescence. The aim of this study was to determine the role of p53, p21WAF1 and p16INK4A proteins in this alternative route to cancer cell death in additional human cancer cell lines. After exposure to cisplatin, all the cell lines underwent growth arrest and expressed the senescence marker senescence-associated beta-galactosidase, but showed none of the features of apoptosis. However, there was no change in p53 protein expression, and neither p21WAF1 nor p16INK4A was expressed before or up to 4 days after cisplatin exposure. These findings provide further evidence that cells carrying mutations resulting in loss of function in the p53 gene can be killed by cisplatin via a p53-independent route with some similarities to replicative senescence, but not apoptosis.

Antineoplastic Agents↗
PubMed1998

Evidence of cisplatin-induced senescent-like growth arrest in nasopharyngeal carcinoma cells.

Cellular senescence is a programmed cell response leading to growth arrest in human diploid fibroblasts. We have shown that a nasopharyngeal carcinoma cell line, CNE1, following treatment by the DNA-damaging agent cisplatin, can undergo cellular senescent-like growth arrest, similar to fibroblasts, judged by cellular morphological changes and the expression of senescence-associated beta-galactosidase (SA-beta-gal). This senescent-like change was dose related; at 0.5 microgram/ml, the percentage of cisplatin-induced SA-beta-gal-positive cells was high (40-96%), and the staining was intense. Higher doses (1.0 and 2.0 micrograms/ml) of cisplatin induced lower SA-beta-gal expression (30-70%), and the process was irreversible. This cisplatin-induced cellular senescent-like response was not due to the inhibition of telomerase activity. Our results indicate that cellular senescent-like pathways exist in nasopharyngeal carcinoma cells and can be induced by cisplatin. Our evidence suggests that cellular senescent-like responses may be a cellular protection mechanism that acts differently in response to different degrees of cellular damage.

Antineoplastic Agents↗
PubMed1998

Molecular characterization of antigen-processing function in nasopharyngeal carcinoma (NPC): evidence for efficient presentation of Epstein-Barr virus cytotoxic T-cell epitopes by NPC cells.

Potentiation of the EBV-specific CTL response by immunization with CTL epitopes has been proposed as a logical approach for immune-targeting nasopharyngeal carcinoma (NPC) cells in vivo. This approach will undoubtedly be influenced by the ability of these malignant cells to endogenously process and present target epitopes on their cell surface for immune recognition by CTLs. Analysis of NPC cells in fresh tumor biopsies and long-term, established NPC tumors in nude mice revealed normal expression of the MHC-encoded putative peptide transporters TAP1 and TAP2, as well as the proteasome components LMP2 and LMP7, which have been shown previously to be essential components of the class I processing pathway. Moreover, these tumor cells also showed high levels of HLA class I alleles on the cell surface, suggesting that peptides are available for binding to nascent MHC molecules in the endoplasmic reticulum. Using a recombinant vaccinia virus to transiently express the EBV nuclear antigens, we studied the antigen-processing efficiency of NPC cells. Our findings demonstrate that, in contrast to cells from another EBV-associated malignancy, Burkitt's lymphoma, NPC cells display normal antigen-processing function and are efficiently recognized by HLA class I-restricted, virus-specific CTLs. These studies also provide a rationale for focusing on strategies designed to activate CTLs specific for EBV antigens that are expressed in NPC cells in vivo.

ATP-Binding Cassette Transporters↗
PubMed1998

Epstein-Barr virus infection is associated with p53 accumulation in nasopharyngeal carcinoma.

Eighty-three cases of nasopharyngeal carcinoma were evaluated for the presence of Epstein-Barr virus (EBV) infection in tumor cells by in situ hybridization to EBER1 transcripts, and for p53 expression by immunostains using the D07 antibody which detects native and mutant forms of the p53 protein. A highly significant association was found between EBV infection and p53 overexpression (P = .0004), with 77% of cases coexpressing both markers. This newly discovered association suggests that EBV is not an innocent bystander with respect to p53 accumulation. One possible mediator of the interaction between EBV and p53, viral BZLF1, was not colocalized with p53 in these tumors, suggesting that BZLF1 is not the factor responsible for p53 accumulation. From an epidemiological standpoint, this series of cancers represents an international cohort in which cases from an endemic part of the world (Hong Kong) were examined alongside cases from the United States, where the disease is 50-fold less prevalent. The cancers from Hong Kong tended to be less differentiated and more frequently associated with EBV, suggesting that biological differences might underlie epidemiological variations in tumor prevalence. Finally, we examined 18 potential premalignant lesions of the surface epithelium of the nasopharynx. Although our numbers are small, our data suggest that p53 accumulation might precede EBV infection in the transition from metaplasia to carcinoma in situ. Further studies are needed to dissect the stepwise progression of nasopharyngeal carcinogenesis.

Carcinoma in Situ↗