Yukio Satoh

Detection of Epstein-Barr virus in salivas and throat washings in healthy adults and children.

It is well known that Epstein-Barr virus (EBV) is excreted from oral regions in the patients with infectious mononucleosis. We analyzed the prevalence of EBV in saliva and throat washings from healthy people in Japan by the polymerase chain reaction assay. EBV DNA was detected in 43 (90%) of the 48 throat washings from healthy adults (21 to 57 years old) and in 35 (38%) of the 93 salivas from healthy children (0 to 6 years old). The percentages of the EBV DNA-positive ratio in salivas increased in proportion relative to the increase of the childrens ages. EBV type 1 was predominant and was detected in 86 and 94% of adults and children, respectively. Umbilical cord lymphocytes were transformed by some throat washings from EBV seropositive donors. EBV DNA was detected in throat washings from two healthy adults whose EBV antibody was not detected. In both cases, higher amounts of EBV DNA were detected in their peripheral blood mononuclear cells than in those of other, EBV antibody-positive donors. These results demonstrated the incidence of EBV excretion in oral regions of healthy individuals in Japan and defined a novel type of EBV infection in healthy adults.

The Interaction of Mitogen-Activated Protein Kinases to Epstein-Barr Virus Activation in Akata Cells

To understand the mechanism by which Epstein-Barr virus (EBV) is activated in Akata cells by cross-linking of surface immunoglobulin, the interaction between mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and EBV activation was investigated. Immunoblotting using an anti-phosphoMAPK antibody (Ab) revealed that anti-IgG Ab induced rapid phosphorylation of MAPK in the cells. The phosphorylation was inhibited by MAPK/ERK kinase specific inhibitor, PD98059. The expressions of the EBV immediate early BZLF1 mRNA and its protein product ZEBRA, and early antigen were also inhibited by the inhibitor. These results indicate that MAPK is involved in the pathways of EBV activation.

The vIL-10 gene of the Epstein-Barr virus (EBV) is conserved in a stable manner except for a few point mutations in various EBV isolates.

A gene of the Epstein-Barr virus (EBV), BamHI-C fragment rightward reading frame 1 (BCRF1), codes viral interleukin-10 (vIL-10), which is a close homolog to human IL-10. EBV strain variations are known at EBV latent membrane protein 1 (LMP1), and the distinct forms of LMP1 have been identified. In order to further elucidate the variations of EBV strains, the BCRF1 (vIL-10) gene was analyzed using PCR-direct sequencing in African Burkitt’s lymphoma (BL) cell lines Raji, P3HR-1, EB1 and Daudi, Japanese BL cell line Akata, lymphoblastoid cell line OB and 22 wild EBV isolates from eight gastric carcinoma tissues and 14 throat washes. We found only five variations of the vIL-10 gene in them with one silent mutation and three non-silent mutations. Raji had no mutation to the prototype gene of B95-8. EB1 and P3HR-1 had non-silent mutations in the sequences leading to the arginine/serine and threonine/proline interchanges at residues 4 and 166, respectively. The silent mutation was detected at valine 102 in Daudi and also in the Japanese cell lines Akata, OB and 20 (90.9%) of the wild EBV isolates. The type of variations in the vIL-10 gene had a common relationship with those in the LMP1 gene. All of the variants of valine 102 had China1-type LMP1 sequences except for Daudi with Med-type LMP1 and other minorities with B95-8 type LMP1. The conservativeness of vIL-10 with a few variations suggests the indispensability of the vIL-10 gene in EBV and that the variations of the vIL-10 gene may depend upon the geographical prevalence of the EBV strains. This is the first report regarding the variations of the vIL-10 gene in cell lines and other wild isolates.

Differential effect of TPA on cell growth and Epstein-Barr virus reactivation in epithelial cell lines derived from gastric tissues and B cell line Raji.

We characterized the cell growth and Epstein-Barr virus (EBV) reactivation for EBV infected epithelial cell lines, GT38, GT39, and GTC-4 using 12-O-tetradecanoylphorbol-13-acetate (TPA). These cell lines grew similarly in liquid medium, and formed colonies in soft agar. The cell growth was inhibited with TPA, dose-dependently in liquid medium. The colony formation was enhanced with low concentrations of TPA, but was inhibited with high concentrations. The latent EBV was reactivated with high concentrations of TPA as shown by the expression of EBV BZLF1 gene product ZEBRA. The effects of TPA on GTC-4 were compared with a Burkitts lymphoma cell line Raji. The mode of actions of TPA in GTC-4 was different from Raji in terms of cell growth and EBV reactivation. The effective concentrations of TPA for cell growth inhibition and EBV reactivation were higher in Raji than GTC-4. Cell cycle analysis showed that TPA (20 ng/ml) induced cell cycle arrest to Raji but not to GTC-4; however, the rate of trypan blue stained cells increased in the TPA treated GTC-4 but not Raji. These results demonstrated that TPA affects differentially for the stimulation and inhibition of cell growth, and also EBV reactivation depends on TPA concentrations and cell types.

Difference of Epstein-Barr virus isolates from Japanese patients and African Burkitt's lymphoma cell lines based on the sequence of latent membrane protein 1.

Sequence variations in the Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) gene have been described in many EBV-isolates. To characterize the genomic relationship between Japanese EBV and the EBV isolates of other countries, we analyzed the LMP1 nucleotide sequences in EBV positive cell lines and clinical specimens, including five African Burkitt’s lymphoma (BL) cell lines, a Japanese BL cell line, a B-lymphoblastoid cell line, a nasopharyngeal carcinoma hybrid cell line, six gastric carcinoma tissues, two peripheral blood mononuclear cells, and a B95-8 cell line, which contained the prototype EBV genome. We determined the C-terminal nucleotide sequences of LMP1 by PCR-direct sequencing analysis and characterized the sequence variation of Japanese isolates, made a phylogenetic tree from the sequence patterns of LMP1 by a neighbor-joining method. The results indicate that the Japanese EBV isolates are greatly different from the African BL isolates but are closely related to the China 1, which is a strain of Chinese EBV isolates.

Carboxyl-terminal sequence variation of latent membrane protein 1 gene in Epstein-Barr virus-associated gastric carcinomas from Eastern China and Japan.

Objectives: To elucidate variations of latent membrane protein 1 (LMP1) in Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) and explore the LMP1 variations of neighboring countries, China and Japan. Methods: In 12 and 8 EBVaGCs from eastern China and Japan, respectively, the C-termini of LMP1 were analyzed using PCR and sequencing. The sequences were compared with previously published strains and were characterized on a phylogenetic tree. The difference between Chinese and Japanese isolates was characterized. Results: Ten of 12 Chinese GC isolates (83.3%) and all of the 8 (100%) Japanese GC isolates belonged to the China 1 strain. Also, B95-8 type isolates were found in 2 of 12 Chinese GC. In the 18 China 1 type isolates, additional mutations outside the signature sequence changes were found. All Japanese isolates (100%) had two or more additional mutations, whereas only 5 of 10 (50%) Chinese isolates had two or more additional mutations. The difference was statistically significant (p = 0.0359). Conclusions: China 1 is the dominant strain in GC from eastern China and Japan. The similarity to that of nasopharyngeal carcinoma (NPC) from China supports the view that China 1 strain represents a geographic-associated polymorphism rather than an NPC-associated polymorphism. Japanese isolates show more mutations than Chinese isolates, suggesting a geographic difference between Chinese and Japanese isolates in GC.

The evolution of Epstein-Barr virus inferred from the conservation and mutation of the virus glycoprotein gp350/220 gene

To study variations of Epstein-Barr virus (EBV), we analyzed the gp350/220 gene for several cell lines and Japanese wild isolates using direct sequencing. The N-terminal region was highly conserved in all EBVs except for Jijoye/P3HR-1 and a few isolates. The variation of the region coincided with EBV types A and B (also referred to as types 1 and 2) and were, respectively, designated as the types a and b. The type A/a was detected in most Japanese cell lines and wild isolates, and was classified as China1 type with latent membrane protein (LMP) 1 gene. The type B/b was detected in only a few wild isolates with the Med and China2 types. The C-terminus had more diversity than the N-terminus and lacked the divergence between types A/a and B/b. The phylogenetic analyses of the gp350/220 and LMP1 genes may suggest a mode of EBV evolution into types A/a and B/b and then to LMP1 subtypes.

The Role of p38 Mitogen-Activated Protein Kinase in Regulating Interleukin-10 Gene Expression in Burkitt's Lymphoma Cell Lines

In malignant B lymphoma cells interleukin‐10 (IL‐10) expression is frequently upregulated. This effect is thought to support to the malignant transformation of these cells and to be a potential target for pharmacotherapy. To define better the mechanism for upregulation of the IL‐10 gene, we tested the association between IL‐10 and p38 mitogen‐activated protein kinase (MAPK) in several Epstein‐Barr virus (EBV) infected and non‐infected Burkitts lymphoma (BL) cell lines. The all BL cell lines expressed IL‐10 and IL‐10 receptor mRNAs, and produced IL‐10. p38 MAPK was constitutively phosphorylated in the cytoplasm of the BL cell lines. We further analyzed molecular effects of p38 MAPK on IL‐10 expression in Akata cells. Exogenous IL‐10 lead rapidly to phosphorylation of Jak1 and Tyk2 as transducers of signals of IL‐10, and promoted growth of Akata cells in a dose‐dependent manner. The phosphorylation of cytoplasmic p38 MAPK in Akata cells was reduced by the serine/threonine kinase inhibitor, 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H7). A specific inhibitor of p38 MAPK, SB203580, blocked simultaneously STAT3 DNA‐binding activity, and IL‐10 mRNA expression, IL‐10 production, and then the cell growth was inhibited. These results indicate that the p38 MAPK pathway is functionally linked to IL‐10 gene expression and supports the view that the constitutive activation of cytoplasmic p38 MAPK in BL cells is a step in the upregulation of IL‐10 gene expression and lymphomagenesis.

Characterization of EBV-infected epithelial cell lines from gastric cancer-bearing tissues.

The long-term goal of our study is to explore how Epstein-Barr virus (EBV) associates with the pathogenesis of gastric carcinoma. The first goal is to establish EBV-positive epithelial cell lines from EBV-infected gastric carcinoma tissues and to characterize the cell lines and EBV infection in the cells. EBV, a ubiquitous human herpesvirus with oncogenic potential, is predominantly associated with the infection of two target tissues in vivo: (1) B lymphocytes, where the infection is largely nonproductive, and (2) the epithelium, in which virus replication occurs (Rickinson and Kieff 1996). Both target tissues are susceptible to EBV-associated malignant change, leading to tumors of B-cell origin, such as Burkitt’s lym-phoma, or of epithelial-cell origin, such as nasopharyngeal carcinoma (NPC) (Pathmanathan et al. 1995). Recently, EBV has also emerged as an etiologic agent implicated in gastric carcinoma (Min et al. 1991; Shibata et al. 1991; Shibata and Weiss 1992; Tokunaga et al. 1993a,b; Fukayama et al. 1994; Imai et al. 1994; Ohfuji et al. 1996; Iwasaki et al. 1998). EBV has been found in most cases of rare gastric lymphoepithelioma-like carcinomas (Min et al. 1991; Shibata et al. 1991; Ohfuji et al. 1996) and a small but significant proportion of common gastric adenocarcinomas (Shibata and Weiss 1992; Tokunaga et al. 1993a,b). EBV infection was found in approximately 7% of Japanese gastric carcinomas (Tokunaga et al. 1993a,b; Fukayama et al. 1994; Imai et al. 1994). The world distribution of gastric carcinomas with EBV infection is shown from 4% to 18% of total gastric carcinoma in different countries (Tashiro et al. 1998)