Lawrence S. Young

Natural history of cervical human papillomavirus infection in young women: a longitudinal cohort study

BACKGROUND Laboratory and epidemiological research suggests an association between human papillomavirus (HPV) and cervical intraepithelial neoplasia (CIN). We studied the natural history of incident cervical HPV infection and its relation to the development of CIN. METHODS We recruited 2011 women aged 15-19 years who had recently become sexually active. We took a cervical smear every 6 months and stored samples for virological analysis. We immediately referred all women with any cytological abnormality for colposcopic assessment, but postponed treatment until there was histological evidence of progression to high-grade CIN. FINDINGS In 1075 women who were cytologically normal and HPV negative at recruitment, the cumulative risk at 3 years of any HPV infection was 44% (95% CI 40-48): HPV 16 was the most common type. The cumulative risk at 3 years of detecting an HPV type not present in the first positive sample was 26% (20-32). 246 women had an abnormal smear during follow-up, of whom 28 progressed to high-grade CIN. The risk of high-grade CIN was greatest in women who tested positive for HPV 16 (risk ratio 8.5 [3.7-19.2]); this risk was maximum 6-12 months after first detection of HPV 16. All HPV types under consideration were associated with cytologically abnormal smears. Although abnormality was significantly less likely to be associated with low-viral-load samples, the cumulative risk at 3 years of a high-viral-load sample after a low-viral-load sample was 45% (95% CI 35-56). Five women who progressed to high-grade CIN consistently tested negative for HPV. INTERPRETATION Our findings suggest that attempts to exploit the association between cervical neoplasia and HPV infection to improve effectiveness of cervical screening programmes might be undermined by the limited inferences that can be drawn from the characterisation of a womans HPV status at a single point in time, and the short lead time gained by its detection.

Expression of Epstein–Barr Virus Transformation–Associated Genes in Tissues of Patients with EBV Lymphoproliferative Disease

Epstein-Barr virus (EBV) has been associated with serious or fatal lymphoproliferative disease in immunocompromised patients. EBV nuclear protein 2 and latent membrane protein are characteristically expressed in B lymphocytes proliferating in vitro in response to growth transformation by EBV. These two proteins are thought to be effectors of lymphocyte growth since they increase the expression of B-lymphocyte activation (CD23) and cell-adhesion (LFA 3 and ICAM 1) molecules in vitro. Using monoclonal antibody-immune microscopy, we have demonstrated that these two EBV proteins and their associated B-lymphocyte activation or adhesion molecules are expressed in the infiltrating B lymphocytes in immunocompromised patients with EBV lymphoproliferative disease. These monoclonal antibodies should be useful in the early diagnosis of EBV lymphoproliferative disease and in distinguishing it from other B-lymphocyte cancers associated with EBV, such as Burkitts lymphoma. The finding of EBV nuclear protein 2 and latent membrane protein and their associated activation or adhesion molecules provides a further pathophysiologic link between EBV and the proliferation of B lymphocytes in immunocompromised patients.

The natural history of cervical HPV infection: unresolved issues

The identification of high-risk human papillomavirus (HPV) types as a necessary cause of cervical cancer offers the prospect of effective primary prevention and the possibility of improving the efficiency of cervical screening programmes. However, for these opportunities to be realized, a more complete understanding of the natural history of HPV infection, and its relationship to the development of epithelial abnormalities of the cervix, is required. We discuss areas of uncertainty, and their possible effect on disease prevention strategies.

Expression of Epstein-Barr virus latent gene products in tumour cells of Hodgkin's disease

The Epstein-Barr virus (EBV)-encoded latent gene products, latent membrane protein (LMP) and EBV nuclear antigen 2 (EBNA 2), seem to have important roles in EBV-induced cell transformation in vitro, and have been implicated as important effector molecules in EBV-associated lymphomagenesis. Because up to 35% of Hodgkins disease (HD) samples have been reported to contain EBV genomes, the expression of LMP and EBNA 2 in these tumours was investigated. 84 cases of HD were studied with monoclonal antibodies and immunohistochemical labelling of acetone-fixed cryostat sections. LMP, but not EBNA 2, was demonstrated in Reed-Sternberg (RS) cells of 40 cases (48%); the two proteins were easily detected in transformed lymphocytes of positive control acute infectious mononucleosis tonsils. LMP expression in RS cells varied according to the histological subtype of HD (1/10 cases [10%] of lymphocyte predominance subtype, 16/50 cases [32%] of nodular sclerosis, 23/24 [96%] cases of mixed cellularity type). That the LMP antibodies showed no substantial cross-reactivity with negative control tissues shows that they are useful probes for the diagnosis of latent EBV infection in tissue sections. The findings suggest that EBV is associated with more cases of HD than was previously recognised, that in positive cases RS cells express a latent infection protein phenotype (LMP+, EBNA 2-) which differs from that of other EBV-associated lymphomas, and that LMP expression is related to histologically aggressive subtypes of HD.

Epstein–Barr virus and oncogenesis: from latent genes to tumours

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus associated with the development of both lymphoid and epithelial tumours. As a common virus infection, EBV appears to have evolved to exploit the process of B cell development to persist as a life-long asymptomatic infection. However, the virus can contribute to oncogenesis as evidenced by its frequent detection in certain tumours, namely Burkitts lymphoma (BL), post-transplant B cell lymphomas, Hodgkins disease (HD) and nasopharyngeal carcinoma (NPC), and by its unique ability to efficiently transform resting B cells in vitro into permanently growing lymphoblastoid cell lines (LCLs). These transforming effects are associated with the restricted expression of EBV genes such that only a subset of so-called latent virus proteins are expressed in virus infected tumours and in LCLs. Distinct forms of EBV latency are manifest in the different tumours and these appear to be a vestige of the pattern of latent gene expression used by the virus during the establishment of persistent infection within the B cell pool. This review summarises our current knowledge of EBV latent gene function and how this relates to the role of the virus in the aetiology of different tumours.

Epstein—Barr Virus Gene Expression in Nasopharyngeal Carcinoma

Epstein-Barr virus (EBV), an agent with growth transforming potential for human B cells, is associated with certain B cell lymphomas in man and also with an epithelial tumour, undifferentiated nasopharyngeal carcinoma (NPC). Since B cell growth transformation is associated with the constitutive expression of a small number of EBV-coded latent proteins, the nuclear antigens EBNA 1, EBNA 2, EBNA 3 and EBNA-LP and the latent membrane protein (LMP), the present work sought to determine whether this same pattern of virus gene expression occurred in NPC. Tumour biopsies were taken from NPC patients from three areas of differing tumour incidence (Kenya, Algeria, Britain) and immediately snap-frozen, as were biopsies of non-EBV-related carcinomas for controls. Immunoblotting of PAGE-separated proteins with selected human sera identified 24 NPC biopsies clearly expressing EBNA 1. When the analysis was extended using selected human sera with antibodies against the other EBNAs, there was no detectable expression of EBNA 2, EBNA 3 or EBNA-LP in any of these 24 biopsies; their EBNA 2-negative status was confirmed using a monoclonal antibody (MAb) PE2 which was reactive in immunoblotting and in immunoprecipitation with EBNA 2A and EBNA 2B proteins. Similar experiments with two different LMP-specific MAbs, CS1 to 4 and S12, revealed heterogeneity between NPC biopsies; 9/24 biopsies were demonstrably LMP-positive, the degree of expression varying considerably between individual tumours in a manner which was not related to the level of EBNA 1 expression. None of the 24 NPC biopsies expressed detectable amounts of EBV lytic cycle antigens. A nude mouse-passaged NPC cell line, C15, likewise expressed EBNA 1 and LMP but none of the other EBV latent proteins nor lytic cycle antigens. This work identifies a novel type of EBV-cell interaction in NPC cells which is distinct from that seen in in vitro transformed B cell lines and from that seen to date in EBV-positive B cell lymphomas.

Molecular Evolution of the Metaplasia-Dysplasia-Adenocarcinoma Sequence in the Esophagus

The incidence of adenocarcinoma of the esophagus has been increasing in developing countries over the last three decades and probably reflects a genuine increase in the incidence of its recognized precursor lesion, Barretts metaplasia. Despite advances in multimodality therapy, the prognosis for invasive esophageal adenocarcinoma is poor. An improved understanding of the molecular biology of this disease may allow improved diagnosis, therapy, and prognosis. We focus on recent developments in the molecular and cell biology of Barretts metaplasia, a heterogeneous lesion affecting the transitional zone of the gastro-esophageal junction whose associated molecular alterations may vary both in nature and temporally. Early premalignant clones produce biological and genetic heterogeneity as seen by multiple p53 mutations, p16 mutations, aneuploidy, and abnormal methylation resulting in stepwise changes in differentiation, proliferation, and apoptosis, allowing disease progression under selective pressure. Abnormalities in expression of growth factors of the epidermal growth factor family and cell adhesion molecules, especially cadherin/catenin complexes, may occur early in invasion. Exploitation of these molecular events may lead to a more appropriate diagnosis and understanding of these lesions in the future.

Activation of the p38 mitogen-activated protein kinase pathway by Epstein-Barr virus-encoded latent membrane protein 1 coregulates interleukin-6 and interleukin-8 production.

The Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) is a pleiotropic protein the activities of which include effects on gene expression and cell transformation, growth, and death. LMP1 has been shown to induce nuclear factor (NF)-κB and c-Jun NH2-terminal kinase/AP-1 activities in target cells, and in this study we demonstrate that LMP1 also engages the p38 mitogen-activated protein kinase cascade, leading to activation of the transcription factor ATF2. Mutational analysis of the LMP1 cytoplasmic COOH terminus revealed that p38 activation occurs from both the tumor necrosis factor receptor-associated factor (TRAF)-interacting, membrane-proximal COOH-terminal activating region (CTAR)1 domain (amino acids 186–231) and the extreme tumor necrosis factor receptor-associated death domain (TRADD) binding CTAR2 region (amino acids 351–386). Because LMP1 also engages signaling on the NF-κB axis through CTAR1 and CTAR2, we have examined whether these two pathways are overlapping or independent. We have found that inhibition of p38 by the highly specific inhibitor SB203580 did not affect NF-κB binding activity. Conversely, although the metabolic inhibitor D609 blocked NF-κB activation, it did not impair the ability of LMP1 to signal on the p38 axis, suggesting that these two LMP1-mediated pathways are primarily independent. Divergence of signals must, however, occur downstream of TRAF2 as a dominant negative TRAF2 mutant that blocks LMP1-induced NF-κB activation also inhibited p38 signaling. In addition, we have found that p38 inhibition significantly impaired LMP1-mediated interleukin-6 and -8 expression. Thus, p38 may play a significant cooperative role in regulating at least some of the pleiotropic activities of LMP1.

Viral gene therapy strategies: from basic science to clinical application

A major impediment to the successful application of gene therapy for the treatment of a range of diseases is not a paucity of therapeutic genes, but the lack of an efficient non‐toxic gene delivery system. Having evolved to deliver their genes to target cells, viruses are currently the most effective means of gene delivery and can be manipulated to express therapeutic genes or to replicate specifically in certain cells. Gene therapy is being developed for a range of diseases including inherited monogenic disorders and cardiovascular disease, but it is in the treatment of cancer that this approach has been most evident, resulting in the recent licensing of a gene therapy for the routine treatment of head and neck cancer in China. A variety of virus vectors have been employed to deliver genes to cells to provide either transient (eg adenovirus, vaccinia virus) or permanent (eg retrovirus, adeno‐associated virus) transgene expression and each approach has its own advantages and disadvantages. Paramount is the safety of these virus vectors and a greater understanding of the virus–host interaction is key to optimizing the use of these vectors for routine clinical use. Recent developments in the modification of the virus coat allow more targeted approaches and herald the advent of systemic delivery of therapeutic viruses. In the context of cancer, the ability of attenuated viruses to replicate specifically in tumour cells has already yielded some impressive results in clinical trials and bodes well for the future of this approach, particularly when combined with more traditional anti‐cancer therapies. Copyright

Activation of the cJun N-terminal kinase (JNK) pathway by the Epstein-Barr virus-encoded latent membrane protein 1 (LMP1).

Expression of the oncogenic Epstein-Barr virus (EBV)-encoded Latent Membrane Protein 1 (LMP1) activates signalling on the NF-κB axis through two distinct domains in the cytoplasmic C-terminus of the protein, namely CTAR1 (aa 187–231) and CTAR2 (aa 351–386). Whilst this effect is responsible for some of the functional consequences of LMP1 expression, additional LMP1-mediated signalling pathways may exist which contribute to the pleiotropic activities of this protein. In this study we provide evidence of a kinase cascade being activated by LMP1. Thus, we demonstrate that stable or transient expression of the LMP1 prototype from B95.8 in cells of epithelial or B cell origin activates the c-Jun N-terminal kinase (JNK, also known as the stress-activated protein kinase, SAPK) pathway, an effect which was found to be mediated through CTAR2 but not CTAR1. LMP1 from the Cao viral strain or LMP1 homologues from the simian EBV naturally infecting baboons and rhesus monkeys were also able to activate JNK. This phenomenon translates to induction of AP-1, a transcription factor which is readily activated by growth factors and mitogens. Interestingly, an LMP1/CD40 chimaera comprising of the N-terminus and transmembrane domain of LMP1 and the cytoplasmic tail of CD40 which shares a common TRAF binding motif with CTAR1, effectively induced JNK. As NF-κB and JNK are co-activated in LMP1-expressing cells, we investigated whether the two pathways are overlapping or independent. We have found that inhibition of NF-κB by metabolic inhibitors or a constitutively active mutated IκBα does not impair the ability of LMP1 to signal on the JNK axis. Conversely, whilst a dominant negative mutated SEK (JNKK) inhibited LMP1-induced JNK activation, it did not affect NF-κB suggesting that these two LMP1-mediated pathways are divergent.