Mechanisms of Action of Epstein-Barr Virus Nuclear Antigen 1 as an Oncogene

Epstein-Barr Virus (EBV) is a human herpes virus associated with several malignancies including endemic Burkitt's lymphoma (eBL), nasopharyngeal carcinoma (NPC) and polyclonal B-cell lympho-proliferations in immunosuppressed individuals. Epstein-Barr virus nuclear antigen 1 (EBNA 1) plays a key role in the life cycle of the virus and is consistently expressed in all these tumour types. However, no oncogenic activities of EBNA 1 have been identified in cell cultures. Nevertheless, EBNA 1 is a viral oncogene when expressed in vivo in transgenic mice which succumb to monoclonal B cell lymphoma (Wilson and Levine, 1992, Wilson et al., 1996). The experiments described in this thesis were designed to explore how EBNA 1 may act as an oncogene in vivo. EBNA 1 was found to be redundant in terms of tumour latency with Bcl2 in transgenic crossbreeding experiments. Therefore, the ability of EBNA 1 to inhibit cell death or differentiation was examined by cell culture techniques. Moreover, cell surface markers examined by FACS. Flow cytometric analysis of spleen and bone marrow (BM) cells from EmuEBNA 1 transgenic mice revealed that the percentage of slg+ (and possibly CD23+) cells was statistically significantly higher when compared to the wild type control littermates. EmuEBNA 1 spleen and bone marrow (BM) cell survival cultures showed no survival advantage over the wt cultures. However, a proliferation/survival assay revealed that EmuEBNA 1 transgenic positive BM cells have statistically significant higher proliferation/survival rate than the wt BM cells. These results may indicate that EBNA 1 drives cells to differentiate and supports their survival/proliferation leading to increased sig. Since EBNA 1 is a DNA-binding protein acting as a transcriptional transactivator the expression levels of cellular genes involved in cell death and differentiation were examined in transgenic mice at pre- and post-tumour stages. The antiapoptotic gene BclxL was found up-regulated and the pro-apoptotic gene Bad was found downregulated. This strongly supports the earlier phenotypic observation that EBNA 1 may act in a similar fashion or through Bcl2 family genes. Moreover, RagJ and Rag2 genes were also found to be up-regulated in EmuEBNA 1 transgenic mouse spleens and this could lead to increased recombination, genome instability and possibly tumour development. Further studies, using a macroarray for differential gene expression, suggest that several immediate early response genes such as Jun and Fos family members and Egrl may be affected by EBNA 1. If EBNA 1 does up-regulate these genes this action could lead to increased differentiation and/or proliferation of EBNA 1 positive B cells. Thus, there are several lines of evidence suggesting that EBNA 1 supports B cell survival/proliferation and differentiation through de-regulation of genes responsible for the development of B cells. This may be the mechanism by which EBNA 1 causes tumourigenesis

Antibacterial surface modification of titanium implants in orthopaedics

The use of biomaterials in orthopaedics for joint replacement, fracture healing and bone regeneration is a rapidly expanding field. Infection of these biomaterials is a major healthcare burden, leading to significant morbidity and mortality. Furthermore, the cost to healthcare systems is increasing dramatically. With advances in implant design and production, research has predominately focussed on osseointegration; however, modification of implant material, surface topography and chemistry can also provide antibacterial activity. With the increasing burden of infection, it is vitally important that we consider the bacterial interaction with the biomaterial and the host when designing and manufacturing future implants. During this review, we will elucidate the interaction between patient, biomaterial surface and bacteria. We aim to review current and developing surface modifications with a view towards antibacterial orthopaedic implants for clinical applications

Genomic analysis of the role of transcription factor C/EBPδ in the regulation of cell behaviour on nanometric grooves

C/EBPδ is a tumour suppressor transcription factor that induces gene expression involved in suppressing cell migration. Here we investigate whether C/EBPδ-dependent gene expression also affects cell responses to nanometric topology. We found that ablation of the C/EBPδ gene in mouse embryonal fibroblasts (MEFs) decreased cell size, adhesion and cytoskeleton spreading on 240 nm and 540 nm nanometric grooves. ChIP-SEQ and cDNA microarray analyses demonstrated that many binding sites for C/EBPδ, and the closely related C/EBPβ, exist throughout the mouse genome and control the upregulation or downregulation of many adjacent genes. We also identified a group of C/EBPδ-dependent, trans-regulated genes, whose promoters contained no C/EBPδ binding sites and yet their activity was regulated in a C/EBPδ-dependent manner. These genes include signalling molecules (e.g. SOCS3), cytoskeletal components (Tubb2, Krt16 and Krt20) and cytoskeletal regulators (ArhGEF33 and Rnd3) and are possibly regulated by cis-regulated diffusible mediators, such as IL6. Of particular note, SOCS3 was shown to be absolutely required for efficient cell spreading and contact guidance on 240 nm and 540 nm nanometric grooves. C/EBPδ is therefore involved in the complex regulation of multiple genes, including cytoskeletal components and signalling mediators, which influence the nature of cell interactions with nanometric topology

Lymphoid Hyperplasia and Lymphoma in Transgenic Mice Expressing the Small Non-Coding RNA, EBER1 of Epstein-Barr Virus

Non-coding RNAs have critical functions in diverse biological processes, particularly in gene regulation. Viruses, like their host cells, employ such functional RNAs and the human cancer associated Epstein-Barr virus (EBV) is no exception. Nearly all EBV associated tumours express the EBV small, non-coding RNAs (EBERs) 1 and 2, however their role in viral pathogenesis remains largely obscure.To investigate the action of EBER1 in vivo, we produced ten transgenic mouse lines expressing EBER1 in the lymphoid compartment using the mouse immunoglobulin heavy chain intronic enhancer Emicro. Mice of several of these EmicroEBER1 lines developed lymphoid hyperplasia which in some cases proceeded to B cell malignancy. The hallmark of the transgenic phenotype is enlargement of the spleen and mesenteric lymph nodes and in some cases enlargement of the thymus, liver and peripheral lymph nodes. The tumours were found to be of B cell origin and showed clonal IgH rearrangements. In order to explore if EBER1 would cooperate with c-Myc (deregulated in Burkitt's lymphoma) to accelerate lymphomagenesis, a cross-breeding study was undertaken with EmicroEBER1 and EmicroMyc mice. While no significant reduction in latency to lymphoma onset was observed in bi-transgenic mice, c-Myc induction was detected in some EmuEBER1 single transgenic tumours, indicative of a functional cooperation.This study is the first to describe the in vivo expression of a polymerase III, non-coding viral gene and demonstrate its oncogenic potential. The data suggest that EBER1 plays an oncogenic role in EBV associated malignant disease

Osteoblast response to disordered nanotopography

The ability to influence stem cell differentiation is highly desirable as it would help us improve clinical outcomes for patients in various aspects. Many different techniques to achieve this have previously been investigated. This concise study, however, has focused on the topography on which cells grow. Current uncemented orthopaedic implants can fail if the implant fails to bind to the surrounding bone and, typically, forms a soft tissue interface which reduces direct bone contact. Here, we look at the effect of a previously reported nanotopography that utilises nanodisorder to influence mesenchymal stromal cell (as may be found in the bone marrow) differentiation towards bone and to also exert this effect on mature osteoblasts (as may be found in the bone). As topography is a physical technique, it can be envisaged for use in a range of materials such as polymers and metals used in the manufacture of orthopaedic implants

Material-driven fibronectin assembly for high-efficiency presentation of growth factors

Growth factors (GFs) are powerful signaling molecules with the potential to drive regenerative strategies, including bone repair and vascularization. However, GFs are typically delivered in soluble format at supraphysiological doses because of rapid clearance and limited therapeutic impact. These high doses have serious side effects and are expensive. Although it is well established that GF interactions with extracellular matrix proteins such as fibronectin control GF presentation and activity, a translation-ready approach to unlocking GF potential has not been realized. We demonstrate a simple, robust, and controlled material-based approach to enhance the activity of GFs during tissue healing. The underlying mechanism is based on spontaneous fibrillar organization of fibronectin driven by adsorption onto the polymer poly(ethyl acrylate). Fibrillar fibronectin on this polymer, but not a globular conformation obtained on control polymers, promotes synergistic presentation of integrin-binding sites and bound bone morphogenetic protein 2 (BMP-2), which enhances mesenchymal stem cell osteogenesis in vitro and drives full regeneration of a nonhealing bone defect in vivo at low GF concentrations. This simple and translatable technology could unlock the full regenerative potential of GF therapies while improving safety and cost-effectiveness

Nanotopographical effects on mesenchymal stem cell morphology and phenotype

There is a rapidly growing body of literature on the effects of topography and critically, nanotopography on cell adhesion, apoptosis and differentiation. Understanding the effects of nanotopography on cell adhesion and morphology and the consequences of cell shape changes in the nucleus, and consequently, gene expression offers new approaches to the elucidation and potential control of stem cell differentiation. In the current study we have used molecular approaches in combination with immunohistology and transcript analysis to understand the role of nanotopography on mesenchymal stem cell morphology and phenotype. Results demonstrate large changes in cell adhesion, nucleus and lamin morphologies in response to the different nanotopographies. Furthermore, these changes relate to alterations in packing of chromosome territories within the interphase nucleus. This, in turn, leads to changes in transcription factor activity and functional (phenotypical) signalling including cell metabolism. Nanotopography provides a useful, non-invasive tool for studying cellular mechanotransduction, gene and protein expression patterns, through effects on cell morphology. The different nanotopographies examined, result in different morphological changes in the cyto- and nucleo-skeleton. We propose that both indirect (biochemical) and direct (mechanical) signalling are important in these early stages of regulating stem cell fate as a consequence of altered metabolic changes and altered phenotype. The current studies provide new insight on cell-surface interactions and enhance our understanding of the modulation of stem cell differentiation with significant potential application in regenerative medicine

Lymphocyte deficiency limits Epstein-Barr virus latent membrane protein 1 induced chronic inflammation and carcinogenic pathology in vivo

Background: The importance of the malignant cell environment to its growth and survival is becoming increasingly apparent, with dynamic cross talk between the neoplastic cell, the leukocyte infiltrate and the stroma. Most cancers are accompanied by leukocyte infiltration which, contrary to an anticipated immuno-protective role, could be contributing to tumour development and cancer progression. Epstein-Barr virus (EBV) associated cancers, including nasopharyngeal carcinoma and Hodgkin's Disease, show a considerable leukocyte infiltration which surrounds the neoplastic cells, raising the questions as to what role these cells play in either restricting or supporting the tumour and what draws the cells into the tumour. In order to begin to address this we have studied a transgenic model of multistage carcinogenesis with epithelial expression of the EBV primary oncoprotein, latent membrane protein 1 (LMP1). LMP1 is expressed particularly in the skin, which develops a hyperplastic pathology soon after birth. Results: The pathology advances with time leading to erosive dermatitis which is inflamed with a mixed infiltrate involving activated CD8+ T-cells, CD4+ T-cells including CD4+/CD25+/FoxP3+ Treg cells, mast cells and neutrophils. Also significant dermal deposition of immunoglobulin-G (IgG) is observed as the pathology advances. Along with NF-kappaB activation, STAT3, a central factor in inflammation regulation, is activated in the transgenic tissue. Several inflammatory factors are subsequently upregulated, notably CD30 and its ligand CD153, also leukocyte trafficking factors including CXCL10, CXCL13, L-selectin and TGF beta 1, and inflammatory cytokines including IL-1 beta, IL-3 and the murine IL-8 analogues CXCL1, CXCL2 and CXCL5-6, amongst others. The crucial role of mature T- and/or B-lymphocytes in the advancing pathology is demonstrated by their elimination, which precludes mast cell infiltration and limits the pathology to an early, benign stage. Conclusions: LMP1 can lead to the activation of several key factors mediating proliferation, angiogenesis and inflammation in vivo. With the initiation of an inflammatory programme, leukocyte recruitment follows which then itself contributes to the progressing pathology in these transgenic mice, with a pivotal role for B-and/or T-cells in the process. The model suggests a basis for the leukocyte infiltrate observed in EBV-associated cancer and its supporting role, as well as potential points for therapeutic intervention

Osteogenic and bactericidal surfaces from hydrothermal titania nanowires on titanium substrates

Nanotopographical cues on Ti have been shown to elicit different cell responses such as cell differentiation and selective growth. Bone remodelling is a constant process requiring specific cues for optimal bone growth and implant fixation. Moreover, biofilm formation and the resulting infection on surgical implants is a major issue. Our aim is to identify nanopatterns on Ti surfaces that would be optimal for both bone remodelling and for reducing risk of bacterial infection. Primary human osteoblast/osteoclast co-cultures were seeded onto Ti substrates with TiO2 nanowires grown under alkaline conditions at 240 °C for different times (2, 2.5 or 3 h). Cell growth and behaviour was assessed by scanning electron microscopy (SEM), immunofluorescence microscopy, histochemistry and quantitative RT-PCR methods. Bacterial colonisation of the nanowire surfaces was also assessed by confocal microscopy and SEM. From the three surfaces tested the 2 h nanowire surface supported osteoblast and to a lesser extent osteoclast growth and differentiation. At the same time bacterial viability was reduced. Hence the 2 h surface provided optimal bone remodeling in vitro conditions while reducing infection risk, making it a favourable candidate for future implant surfaces

Biomimetic oyster shell–replicated topography alters the behaviour of human skeletal stem cells

The regenerative potential of skeletal stem cells provides an attractive prospect to generate bone tissue needed for musculoskeletal reparation. A central issue remains efficacious, controlled cell differentiation strategies to aid progression of cell therapies to the clinic. The nacre surface from Pinctada maxima shells is known to enhance bone formation. However, to date, there is a paucity of information on the role of the topography of P. maxima surfaces, nacre and prism. To investigate this, nacre and prism topographical features were replicated onto polycaprolactone and skeletal stem cell behaviour on the surfaces studied. Skeletal stem cells on nacre surfaces exhibited an increase in cell area, increase in expression of osteogenic markers ALP (p