mSphere of Influence: 3-D Culture Models Influence Studies on Epstein-Barr Virus Molecular Pathogenesis in the Epithelium.

Kathy Shair works in the field of Epstein-Barr virus (EBV)-associated cancers, with emphasis on nasopharyngeal carcinoma (NPC). In this mSphere of Influence article, she reflects on how the paper "Efficient replication of Epstein-Barr virus in stratified epithelium in vitro" by Temple et al. (R. M. Temple, J. Zhu, L. Budgeon, N. D. Christensen, et al., Proc Natl Acad Sci U S A 111:16544-16549, 2014, https://doi.org/10.1073/pnas.1400818111) has influenced her work on EBV molecular pathogenesis in the nasopharynx by highlighting the importance of using three-dimensional (3-D) culture models to study epithelial infection.

Epstein-Barr virus LMP1 induces focal adhesions and epithelial cell migration through effects on integrin-α5 and N-cadherin.

Epstein-Barr virus (EBV) is a γ-herpesvirus associated with human epithelial and B-cell malignancies. The EBV latent membrane protein (LMP) 1 is expressed in nasopharyngeal carcinoma (NPC) and promotes oncogenic intracellular signaling mechanisms. LMP1 also promotes a pro-migratory phenotype through potential effects on cell surface proteins, as expression of LMP1 induces an epithelial-mesenchymal transition (EMT) in epithelial cell lines. In this study, LMP1 was examined for potential effects on cadherin and integrin surface interactions, and assessed for biological effects on adhesion and motility to fibronectin. Expression of LMP1 in the non-tumorigenic epithelial cell line MCF10a induced an EMT-associated cadherin switch. The induced N-cadherin was ligated and localized to the cell surface as determined by triton-solubility and immunofluorescence assays. In addition, LMP1 induced the assembly of focal adhesions (FAs) with increased production of fibronectin in MCF10a and NP460hTERT-immortalized nasopharyngeal cells. Biochemical enrichment of fibronectin-associated proteins indicated that LMP1 selectively promoted the recruitment of integrin-α5 and Src family kinase proteins to FA complexes. Neutralizing antibodies to N-cadherin and integrin-α5, but not integrin-αV, blocked the adhesion and transwell motility of MCF10a cells to fibronectin induced by LMP1. LMP1-induced transwell motility was also decreased by Src inhibition with the PP2 kinase inhibitor and short hairpin RNAs. These studies reveal that LMP1 has multiple mechanisms to promote the adhesive and migratory properties of epithelial cells through induction of fibronectin and modulation of cell surface interactions involving integrin-α5 and N-cadherin, which may contribute to the metastatic potential of NPC.

Development, dimensions, reliability and validity of the novel Manchester COPD fatigue scale.

INTRODUCTION: Fatigue is a prominent symptom in chronic obstructive pulmonary disease (COPD) and it has distinctive features; however, there is a need for a robust scale to measure fatigue in COPD. METHODS: At baseline, 122 patients with COPD (forced expiratory volume in 1 s (FEV(1)) 52%, women 38%, mean age 66 years) completed a pilot fatigue scale covering a pool of 57 items and underwent a range of tests, including indicators of mood and a short general fatigue questionnaire. All patients responded to the 57-item scale and it was readministered to a subset of 30 patients. The pilot scale was first subjected to constructive validated shortening steps and then to a principal components analysis. RESULTS: The Manchester COPD fatigue scale (MCFS) consists of 27 items, loading into three dimensions: physical, cognitive and psychosocial fatigue. Internal consistency (Cronbach's alpha = 0.97) and test-retest repeatability (r = 0.97, p<0.001) were tested. It had significant convergent validity, correlating with the FACIT (Functional Assessment of Chronic Illness Therapy) fatigue scale and the fatigue in Borg scale at baseline and after a 6 minute walk distance (6MWD) test (r = -0.81, 0.53 and 0.63, respectively, p<0.001). Its scores were associated with BODE, SGRQ (St George's Respiratory Questionnaire) and MRC (Medical Research Council) dyspnoea scores (r = 0.46, 0.8 and 0.51, respectively, p<0.001). The scale demonstrated meaningful discriminating ability; patients who walked <350 m in a 6MWD test as well as depressed patients (>or=16 scores in the Center for Epidemiologic Study on Depression (CES-D) scale) had nearly twice as high fatigue scores as those who walked >or=350 m or were not depressed (p<0.001). CONCLUSION: The MCFS provides a simple, reliable and valid measurement of total and dimensional fatigue in moderate stable COPD.

LMP1 signaling and activation of NF-kappaB in LMP1 transgenic mice.

Transgenic mice expressing Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) under the control of an immunoglobulin heavy-chain promoter and enhancer develop lymphoma at a threefold higher incidence than LMP1-negative mice. In vitro, LMP1 activates numerous signaling pathways including p38, c-Jun N terminal kinase (JNK), phosphatidylinositol 3 kinase (PI3K)/Akt, and NF-kappaB through interactions with tumor necrosis receptor-associated factors (TRAFs). These pathways are frequently activated in EBV-associated malignancies, although their activation cannot be definitively linked to LMP1 expression in vivo. In this study, interactions between LMP1 and TRAFs and the activation of PI3K/Akt, JNK, p38, and NF-kappaB were examined in LMP1 transgenic mice. LMP1 co-immunoprecipitated with TRAFs 1, 2, and 3. Akt, JNK, and p38 were activated in LMP1-positive and -negative splenocytes as well as LMP1-positive and -negative lymphomas. Multiple forms of NF-kappaB were activated in healthy splenocytes from LMP1 transgenic mice, in contrast to healthy splenocytes from LMP1-negative mice. However, in both LMP1-positive and -negative lymphomas, only the oncogenic NF-kappaB c-Rel, was specifically activated. Similarly to EBV-associated malignancies, p53 protein was detected at high levels in the transgenic lymphomas, although mutations were not detected in the p53 gene. These data indicate that NF-kappaB is activated in LMP1-positive healthy splenocytes; however, NF-kappaB c-Rel is specifically activated in both the transgenic lymphomas and in the rare lymphomas that develop in negative mice. The LMP1-mediated activation of NF-kappaB may contribute to the specific activation of c-Rel and lead to the increased development of lymphoma in the LMP1 transgenic mice.