Sang Taek Oh

Expression of Viral MicroRNAs in Epstein-Barr Virus-Associated Gastric Carcinoma

ABSTRACT Epstein-Barr virus (EBV) is associated with about 6 to 16% of gastric carcinoma cases worldwide. Expression of the EBV microRNAs (miRNAs) was observed in B cells and nasopharyngeal carcinoma cells infected with EBV. However, it is not clear if the EBV miRNAs are expressed in EBV-associated gastric carcinomas (EBVaGCs). We found that BART miRNAs but not BHRF1 miRNAs were expressed in EBV-infected gastric carcinoma cell lines and the tumor tissues from patients as well as the animal model. The expression of viral miRNAs in EBVaGCs suggests that these EBV miRNAs may play important roles in the tumorigenesis of EBVaGCs.

Patients with systemic lupus erythematosus have abnormally elevated Epstein–Barr virus load in blood

Various genetic and environmental factors appear to be involved in systemic lupus erythematosus (SLE). Epstein–Barr virus (EBV) is among the environmental factors that are suspected of predisposing to SLE, based on the characteristics of EBV itself and on sequence homologies between autoantigens and EBV antigens. In addition, higher titers of anti-EBV antibodies and increased EBV seroconversion rates have been observed in SLE patients as compared with healthy control individuals. Serologic responses do not directly reflect EBV status within the body. Clarification of the precise status of EBV infection in SLE patients would help to improve our understanding of the role played by EBV in this disease. In the present study we determined EBV types in SLE patients (n = 66) and normal control individual (n = 63) by direct PCR analysis of mouthwash samples. We also compared EBV load in blood between SLE patients (n = 24) and healthy control individuals (n = 29) using semiquantitative PCR assay. The number of infections and EBV type distribution were similar between adult SLE patients and healthy control individuals (98.5% versus 94%). Interestingly, the EBV burden in peripheral blood mononuclear cells (PBMCs) was over 15-fold greater in SLE patients than in healthy control individuals (mean ± standard deviation: 463 ± 570 EBV genome copies/3 μg PBMC DNA versus 30 ± 29 EBV genome copies/3 μg PBMC DNA; P = 0.001), suggesting that EBV infection is abnormally regulated in SLE. The abnormally increased proportion of EBV-infected B cells in the SLE patients may contribute to enhanced autoantibody production in this disease.

Prolonged gene expression in primary porcine pancreatic cells using an Epstein–Barr virus-based episomal vector

Epstein-Barr virus (EBV)-based plasmids containing the origin of replication (oriP) and EBV nuclear antigen 1 (EBNA-1) are well known for the stable episomal maintenance in human cells. In order to clarify whether an EBV-based plasmid can be maintained stably in the porcine pancreatic cells which are the primary candidate sources of islet xenotransplantation, we constructed pEBVGFP encoding the green fluorescent protein (GFP). Monolayer culture of the porcine neonatal pancreatic cells was lipofected with pEBVGFP or pGFP which was derived from pEBVGFP by deleting out oriP and EBNA-1. pEBVGFP significantly prolonged GFP expression not only in human cell lines but also in the primary porcine pancreatic cells compared with pGFP. Interestingly, the duct cells that are believed as the pancreatic precursor cells were preferentially transfected and conveniently enriched among the mixed primary cell populations using a hygromycin B selection. To our knowledge, this is the first report suggesting the potential application of an EBV-based plasmid for the extended gene expression in the primary porcine pancreatic duct cells.

Long-term Outcome of Extranodal NK/T Cell Lymphoma Patients Treated With Postremission Therapy Using EBV LMP1 and LMP2a-specific CTLs

Extranodal NK/T-cell lymphoma (ENKTCL) is associated with latent Epstein-Barr virus (EBV) infection and frequent relapse even after complete response (CR) to intensive chemotherapy and radiotherapy. The expression of EBV proteins in the tumor provides targets for adoptive immunotherapy with antigen-specific cytotoxic T cells (CTL). To evaluate the efficacy and safety of EBV latent membrane protein (LMP)-1 and LMP-2a-specific CTLs (LMP1/2a CTLs) stimulated with LMP1/2a RNA-transferred dendritic cells, we treated 10 ENKTCL patients who showed complete response to induction therapy. Patients who completed and responded to chemotherapy, radiotherapy, and/or high-dose therapy followed by stem cell transplantation (HDT/SCT) were eligible to receive eight doses of 2 × 107 LMP1/2a CTLs/m2. Following infusion, there were no immediate or delayed toxicities. The 4-year overall survival (OS) and progression-free survival (PFS) were 100%, and 90% (95% CI: 71.4 to 100%) respectively with a median follow-up of 55·5 months. Circulating IFN-γ secreting LMP1 and LMP2a-specific T cells within the peripheral blood corresponded with decline in plasma EBV DNA levels in patients. Adoptive transfer of LMP1/2a CTLs in ENKTCL patients is a safe and effective postremission therapeutic approach. Further randomized studies will be needed to define the role of EBV-CTLs in preventing relapse of ENKTCL.

Induction of Efficient Differentiation and Survival of Porcine Neonatal Pancreatic Cell Clusters Using an EBV-based Plasmid Expressing HGF

Porcine neonatal pancreatic cell clusters (NPCCs) have been actively studied as a source of pancreatic stem cell transplantation for the treatment of diabetes. In this study, the hepatocyte growth factor (HGF) gene was cloned in an Epstein-Barr virus (EBV)-based plasmid vector (pEBVHGF) and the effects of the HGF expression on the survival and differentiation of NPCCs were analysed. For comparison, pHGF was constructed by deleting EBNA-1 and OriP from pEBVHGF. The expression of HGF, as measured by ELISA, lasted longer when pEBVHGF was used than when pHGF was used. C-Met phosphorylation co-related with the expression of HGF in the transfected NPCCs. Immunocytochemistry experiments showed that NPCCs showed a higher and longer expression of insulin when they were transfected with pEBVHGF than with pHGF. Moreover, a greater number of NPCCs survived for a longer period after they were transfected with pEBVHGF than when they were transfected with pHGF. Taken together, these results indicate that transfecting NPCCs with the HGF gene using an EBV-based plasmid is a more effective method of inducing differentiation to beta cells and enhancing survival than using a conventional plasmid. Therefore, it may be possible to use EBV-based plasmids to modify pancreatic stem cells for xenotransplantation.

Mechanism of prolonged gene expression by Epstein–Barr virus-based plasmid in porcine cells

Abstract:  Background:  We previously showed that an Epstein‐Barr virus (EBV)‐based plasmid, pEBVGFP, exerts prolonged gene expression in porcine neonatal pancreatic cell clusters (NPCCs). In this study, the mechanism underlying this was investigated.

Maintenance of the viral episome is essential for the cell survival of an Epstein-Barr virus positive gastric carcinoma cell line

While Epstein Barr virus (EBV) is associated with about 10% of gastric carcinomas worldwide, the role of the virus in the tumorigenesis of EBV-associated gastric carcinoma (EBVaGC) is unclear. Previously, we reported that a gastric cancer cell line, SNU-719, that is naturally infected with EBV closely resembles EBVaGC. Here, we attempted to eliminate the EBV genome from SNU-719 cells to ascertain the influence of EBV in EBVaGC. Southern blotting and fluorescence in situ hybridization (FISH) showed that EBV genomes were maintained as episomes in SNU-719 cells. To remove EBV episomes, SNU-719 cells were first cultured in a hydroxyurea (HU)-containing medium for up to 6 months. Real-time polymerase chain reaction and FISH results revealed no evidence of HU-mediated EBV genome reduction, although cell growth was reduced by acute HU treatment in dose- and time-dependent manners. Two small interfering RNAs against Epstein Barr nuclear antigen 1 (EBNA1) abrogated over 90% of the ectopic EBNA1 expression in HeLa cells, but only 40% of endogenous EBNA1 expression in SNU-719 cells. Together, our data suggest that maintenance of latent EBV infection is essential for the viability of EBVaGC cells, avoiding elimination of EBV episomes from the cells.

TGF-β1 Expression by Proliferated Keratinocytes in the Skin of E-Irradiated Mice

In this study, we established a radiodermatitis animal model and investigated the change in immune cell proportions in the secondary lymphoid organs. The cells responsible for the increased transforming growth factor-β1 (TGF-β1) and interleukin-10 (IL-10) production in the lesions following irradiation were also investigated. The radiodermatitis model was constructed by locally exposing the posterior dorsal region of hairless-1 (HR-1) mice to 10 Gy electron (E)-ray/day for six consecutive days. The change in immune cell proportions was analyzed by FACS. Immunohistochemistry was carried out to detect the expression of cytokines and cell-specific markers in the skin. The proportions of antigen-presenting cells, T cells, and B cells in the lymph nodes and spleen were affected by E-irradiation. After irradiation, TGF-β1 and IL-17 were co-localized in the papillary region of the dermis with keratin-14 (K-14)-positive cells rather than with regulatory T cells (Treg). IL-10 was not co-stained with Treg, T helper 17 (Th17) cells, dendritic cells, or macrophages. Our data indicate that TGF-β1 is over-expressed mainly by proliferated keratinocytes in the lesions of a radiodermatitis animal model.