High incidence of elevated antibody titers to Epstein-Barr virus in patients with uveitis.

We assayed Epstein-Barr virus (EBV) antibody titers in patients' sera using indirect immunofluorescence and tested for the presence of antibody to EBV immediate-early BZLF1 protein ZEBRA by Western blotting to explore the association of EBV infection with uveitis. IgG and IgA antibodies to viral capsid antigen (VCA), IgG antibodies to early antigen (EA), and antibodies to EBV nuclear antigen were detected at higher titers in sera of patients with uveitis than in the sera of healthy controls. Neither IgM antibody to VCA nor EA was detected in the patients' sera. Anti-ZEBRA-IgG antibodies were detected in most patients' sera, but not in those of healthy controls. These results suggest that uveitis might be a disease accompanied by EBV reactivation.

An attempt to integrate Western and Chinese medicine: rationale for applying Chinese medicine as chronotherapy against cancer.

Current Western medical treatment lays its main emphasis on evidence-based medicine (EBM) and cure is assessed by quantifying the effects of treatment statistically. In contrast, in Chinese medicine, cure is generally assessed by evaluating the patient's "pattern" (Zheng) [cf. Glossary] and medicines are prescribed according to this. We believe that traditional Chinese medicine (TCM) cannot be evaluated precisely according to Western principles, in which a constant amount of the same medicine is given to a group of patients to be evaluated. When assessing cure using TCM, Zheng is more important than the determination of medical effects. This means that quantitative evaluation of TCM treatment can be very difficult. In this paper, we focused on the Yin-Yang [cf. Glossary]balance to determine Zheng, and at the same time attempted to determine the treatment effects by applying the concept of regulation of Yin-Yang according to chronotherapeutic principles. According to Zheng, advanced cancer patients generally lack both Yin and Yang. Chinese medical treatment therefore seeks to supplement both Yin and Yang. However, we divided patients into two groups and compared them with respect to survival. One group was administered a predominantly Yang (Qi) [cf. Glossary] tonic herbal treatment during the daytime, while the other group was administered Yin (Blood) [cf. Glossary] tonics during night time. A comparison of the results of treatment showed that the patients in the group receiving Yang (Qi) replenishment during the daytime lived longer than patients receiving Yin (Blood) nourishment during the night. Moreover, the patients in the daytime Yang (Qi) replenishment group also fared significantly better than patients treated solely by Western methods.

Spontaneous reduction in Epstein-Barr virus (EBV) DNA copy number in EBV-infected epithelial cell lines.

We found that spontaneous and 12-0-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus (EBV) reactivation occurred in short-term (ST)-cultured EBV-infected epithelial cell lines GT38 and GT39 after their establishment; however, it diminished in the long-term (LT)-cultured cells passaged for more than 2 years from ST-cultured cells. We hypothesized that the EBV reactivation may be related to the EBV DNA copy number in the cells. A higher level of EBV DNA content was detected in ST-cultured cells than in LT-cultured cells by Southern hybridization using an EBV DNA XhoI probe. Fluorescence in situ hybridization using EBV DNA BamHI W fragments showed that ST-cultured cells contained a higher EBV DNA copy number than that of LT-cultured cells. EBV DNA-negative cells were detected in small proportions in LT-cultured cells, but were undetected in ST-cultured cells. These results demonstrate that EBV genomes are not maintained stably in the cell lines, and some of them are lost in continuous passages of the cells. We discuss the mechanisms of reduction of EBV reactivation and EBV DNA in the cell lines.

[A case of esophageal small-cell carcinoma responding to chemotherapy with CTP-11].

After examination of a 64-year-old man with dysphagia who consulted our hospital, a diagnosis was made an esophageal small-cell carcinoma. This tumor had infiltrated the aorta and upon further examination another carcinoma, trachial adenosquamous cell carcinoma was found. We judged this case to be inoperable. At first, 2 courses of systemic chemotherapy were administered by CDDP but this therapy had little effect. We know that CPT-11 is effective in the treatment of lung small-cell carcinoma, leading us to a decision to use CPT-11 for the second systemic chemotherapy. As a result of 8 courses, the tumor minification rate was 64% with PR. After completing 13 courses, the patient could ingest any type of food. We observed grade 2 leukopenia as the only side effect. The patient survived for 1 year and 10 months after beginning the initial treatment. He transferred to another hospital for laser therapy for his trachial carcinoma. The patient died of brain metastasis. To date the treatment of patients with esophageal small-cell carcinoma with chemotherapy using CPT-11 has not been reported. We suggest that CPT-11 is an effective therapy in improving the condition of patients QOL with esophageal small-cell carcinoma.

Effect of transforming growth factor-beta1 on the cell growth and Epstein-Barr virus reactivation in EBV-infected epithelial cell lines.

Transforming growth factor (TGF)-beta1 is a multifunctional cytokine that plays important roles in regulating cell growth and differentiation in many biological systems. In this study, we found that gastric tissue-derived Epstein-Barr virus (EBV)-infected epithelial cell lines GT38 and GT39 had resistance to TGF-beta1-mediated growth inhibition and apoptosis compared to a TGF-beta1-susceptible gastric carcinoma cell line HSC-39. However, TGF-beta1 partially induced EBV reactivation in GT38 and GT39 cells, as shown by the induction of EBV immediate-early BZLF1 RNA and its protein product ZEBRA and early antigen-D. The expressions of TGF-beta receptor I and II were detected in GT38 and GT39 cells by Northern and Western blot analyses. Both cell lines spontaneously produced the TGF-beta1, which was sufficient for inhibiting cell growth of HSC-39 cells. Taken together, these data suggest that TGF-beta1 may be a key factor for EBV reactivation and selective growth of EBV-infected epithelial cells in vivo.

12-O-tetradecanoylphorbol-13-acetate induces Epstein-Barr virus reactivation via NF-kappaB and AP-1 as regulated by protein kinase C and mitogen-activated protein kinase.

Signaling pathway components mediating Epstein-Barr virus (EBV) reactivation by 12-O-tetradecanoylphorbol-13-acetate (TPA) were characterized in terms of induction and modification of specific transacting factors. The consequences of protein kinase C (PKC) activation by TPA in inhibiting inducible nitric oxide synthase (iNOS) mRNA expression were analyzed in the EBV-infected gastric epithelial cell line GT38. Spontaneous expression of the EBV BZLF1 gene product ZEBRA became undetectable upon long-term culturing of GT38 cells, while iNOS mRNA expression increased. In such cells the PKC inhibitors 1-(5-isoquinolinesulphonyl)-2,5-dimethylpiperazine (H7) and staurosporine inhibited TPA-induced expression of BZLF1 and BRLF1 and reversed TPA-mediated inhibition of iNOS gene expression. The mitogen-activated protein kinase inhibitor PD98059 inhibited TPA-induced BZLF1 expression. Electrophoretic mobility shift assays demonstrated that transcription factors NF-kappaB and AP-1 were also activated by TPA in a time-dependent manner. The TPA-induced NF-kappaB activation was inhibited by prior treatment of the cells with the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC). TPA-induced BZLF1 expression was also inhibited by the treatment with PDTC. Northern blot analyses characterized changes in levels of the c-jun and junB expressions of the AP-1 family. These results show that TPA induces EBV reactivation via NF-kappaB and AP-1 and that PKC is an important mediator in regulating gene expression leading to EBV reactivation after TPA treatment of GT38 cells.

A case of exaggerated mosquito-bite hypersensitivity with Epstein-Barr virus-positive inflammatory cells in the bite lesion.

We describe a unique patient with mosquito-bite hypersensitivity who had extremely high titres of Epstein-Barr virus antibodies. For many years he developed intractable ulcers on the sites of mosquito-bite. Epstein-Barr virus infection was detected in almost all inflammatory cells in the ulcers and in the peripheral blood lymphocytes by using in situ hybridization to Epstein-Barr virus-encoded small ribonucleic acids and by polymerase chain reaction to Epstein-Barr virus DNA. The inflammatory cells in the ulcers were positive for T-cell marker. Our results suggest that the Epstein-Barr virus infection in T cells may participate in the pathogenesis of exaggerated mosquito hypersensitivity and in delayed healing of ulcers on the sites of mosquito-bite.

Tumorigenesis of Epstein-Barr virus-positive epithelial cell lines derived from gastric tissues in the SCID mouse.

To study the tumorigenesis of Epstein-Barr virus (EBV)-positive epithelial cell lines GT38 and GT39 derived from human gastric tissues, we inoculated these cells under the skin of severe combined immunodeficient (SCID) mice. The development of tumors was observed in each of the mice about 2 months after the inoculation. The tumors were diagnosed with undifferentiated carcinoma by hematoxylin/eosin staining. EBV-encoded small RNA1 was detected in the paraffin-embedded tumor sections. The tumor cells had human chromosome. The circular, but not linear, EBV DNA was detected in the tumors. The molecular sizes of EBV DNA termini were the same as that of the inoculated GT38 or GT39 cells. The expressions of EBV nuclear antigen 2 and latent membrane protein 1 reduced in the tumors. Transcripts of BamHI C and W promoters in latency III were detected in the tumors and the cultured cells in vitro. The tumor cells were passaged from one SCID mouse to other SCID mice and to cultures in vitro. This is the first evidence that the EBV-positive epithelial cell lines produced tumors in the SCID mouse.

Inhibition of cell growth and Epstein-Barr virus reactivation by CD40 stimulation in Epstein-Barr virus-transformed B cells.

The CD40 molecule plays important roles in B cell activation, proliferation, and immunoglobulin (Ig) class switching. In Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL), CD40 mediates growth inhibition and EBV reactivation via the CD40 signaling pathways. CD40 cross-linking with a monoclonal antibody arrests cell growth in G1 and induces expression of p27kip1 cyclin-dependent kinase inhibitor. CD40 cross-linking also induces EBV reactivation, as detected by the induction of EBV-specific early antigen, immediate early BZLF1 RNA, and its protein product ZEBRA. These results support hypotheses that the proliferation of EBV-infected B cells in vivo can be inhibited by interactions with the CD40 ligand on activated helper T cells, and latent EBV is reactivated via the signaling pathways controlled by CD40 interactions.

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 Burkitt's 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.

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 children's 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.

[A case of advanced gastric cancer with lung and liver metastasis responding remarkably to combination chemotherapy with CDDP and CPT-11].

A 56-year-old male patient with upper epigastric discomfort was introduced to our hospital from the previous clinic due to gastric cancer on July 8, 1998. Several examinations showed massive lung and liver metastases from Type-I gastric cancer beneath the esophagogastric mucosal junction. First we tried transcatheter arterial embolization (TAE) with a single agent, CDDP 20 mg/day for 4 days, but there was no change in the metastatic lesions. We then tried combination chemotherapy of CPT-11 40 mg/day a day with CDDP 15 mg/day for 4 days. After a 6-week interval, we added 4 courses at the same doses. The primary stomach lesion was reduced and was visible as a small nodular flat mass. Moreover, we found that the lung and liver metastatic lesions were already reduced. Three months have passed since CDDP-CPT-11 combination therapy, and we have not found any recurrent tumors so far.

Epstein-Barr virus (EBV) infection and gastric carcinoma: the approach through EBV infected epithelial cell lines.

Epstein-Barr virus (EBV) infection is found in 7% of Japanese gastric carcinoma. Our strategy is to establish the EBV infected epithelial cell lines from EBV infected gastric carcinomas and to characterize the cell lines on the bases of cellular and molecular biology to define the etiological role of EBV. We have characterized two EBV positive cell lines, GT38 and GT39 from gastric tissues. The both cell lines were EBV latency type III and produced the virus spontaneously. The character of tumor cells was demonstrated by the colony formation in soft agar and the tumorigenesis in severe combined immunodeficient mice.

Nitric oxide down-regulates Epstein-Barr virus reactivation in epithelial cell lines.

Nitric oxide (NO), a mediator of biological functions, has an antimicrobial activity against a variety of pathogens including viruses. In this study, we found that a constitutive, low level of inducible NO synthase (iNOS) mRNA was expressed in the EBV-infected gastric tissue-derived GT38 and GT39 cell lines, by analysis with the reverse transcription-polymerase chain reaction (RT-PCR) and Southern blotting. Treatment of these cells with a specific NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA), induced the immediate-early, EBV transactivator gene BZLF1 protein ZEBRA, suggesting a significant increase in EBV reactivation by L-NMMA. Northern blotting demonstrated that BZLF1 and BRLF1 transcripts were also induced by 12-O-tetradecanoylphorbol-13 acetate (TPA). Meanwhile, constitutive expression of iNOS mRNA was inhibited by TPA. L-NMMA also enhanced TPA-induced expression of the BZLF1 gene. On the other hand, a NO donor, S-nitroso-N-acetylpenicillamine (SNAP), which releases NO in an aqueous solution, inhibited the TPA-induced BZLF1 gene expression in a dose-dependent manner at both mRNA and protein levels. These results demonstrated that NO is a regulatory factor in maintaining virus latency via inhibiting EBV reactivation in the infected epithelial cells.

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.

Identification of an alternative form of caspase-9 in human gastric cancer cell lines: a role of a caspase-9 variant in apoptosis resistance.

Overcoming apoptosis resistance to chemotherapy and radiation may lead to a reduction in gastric cancer death. We hypothesize that the apoptotic machinery in gastric cancer cells is dependent upon specific cellular conditions. In the course of our study of the expression of apoptosis-related genes in human gastric cancer cell lines, we have identified a cDNA clone which predicts an alternative form of caspase-9. The caspase-9 variant, which we designated as caspase-9 beta, retained a truncated structure of native caspase-9 without its catalytic domain and was expressed in seven cell lines from human gastric cancer. Among the cell lines examined, MKN-28 cells, which exhibited the most resistance against apoptotic stimuli, expressed the highest level of caspase-9 beta. The induction of apoptosis by staurosporine or actinomycin D was markedly suppressed in caspase-9 beta-transfected HeLa cells. These results are consistent with our hypothesis that the caspase-9 beta may be an endogenous dominant-negative molecule which attenuates apoptotic activity in human gastric cancer cells.

Pretreatment with restriction enzyme or bovine serum albumin for effective PCR amplification of Epstein-Barr virus DNA in DNA extracted from paraffin-embedded gastric carcinoma tissue.

An association between Epstein-Barr virus (EBV) and gastric carcinoma has been studied through the EBV genome present in the carcinoma cells. Recently, we found that EBV DNA in paraffin-embedded gastric carcinoma tissue was detected effectively by PCR after pretreatment of the extracted DNA with a restriction enzyme, BamHI or EcoRI. Here, we show that the PCR amplification was also enhanced by pretreatment of the DNA with other restriction enzymes or with bovine serum albumin and several other proteins. Treatment with these proteins may remove a PCR inhibitor(s) in the DNA samples extracted from the paraffin blocks.

Productive infection of Epstein-Barr virus (EBV) in EBV-genome-positive epithelial cell lines (GT38 and GT39) derived from gastric tissues.

We characterized the expression of Epstein-Barr virus (EBV) on two epithelial cell lines, GT38 and GT39, derived from human gastric tissues. The EBV nuclear antigen (EBNA) was detected in all cells of both cell lines. The EBV immediate-early BZLF 1 protein (ZEBRA), the early antigen diffuse component (EA-D), and one of the EBV envelope proteins (gp350/220) were expressed spontaneously in small proportions in the cells. EBNA 1, EBNA2, latent membrane protein 1, ZEBRA, and EA-D molecules were then observed by Western blotting in the cells. The lytic cycle was enhanced with treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or n-butyrate. The virus particles were observed in the TPA treated GT38 cells by electron microscopy. Infectious EBV was detected with the transformation of cord blood lymphocytes and also with the induction of early antigen to Raji cells by the supernatants of both cells lines. A major single and minor multiple fused terminal fragments and a ladder of smaller fragments of the EBV genome were detected with a Xhol probe in both cell lines. These epithelial cells lines and viruses will be useful in studying their association with EBV in gastric epithelial cells.

Evaluation of antibodies to the Epstein-Barr virus immediate early gene product ZEBRA by a new enzyme-linked immunosorbent assay.

For the serodiagnosis of Epstein-Barr virus (EBV) infections, we have developed a new enzyme-linked immunosorbent assay (ELISA) for antibodies to the ZEBRA product of EBV immediate early gene BZLF1. ZEBRA protein fused with glutathione-S-transferase (GST) was expressed in Escherichia coli and purified by affinity chromatography with glutathione-Sepharose 4B. An ELISA sandwich capture system was constructed with the GST-ZEBRA immobilized on plastic microtiter plates which had been coated with a mouse monoclonal antibody to GST. ZEBRA-IgG antibodies in patients' sera with chronic active EBV infection (CAEBV) and infectious mononucleosis (IM) had, respectively, very high and high titers. Anti-ZEBRA antibodies were also detected at low titers in sera of some healthy controls. ZEBRA-IgM antibodies were detected in sera of patients with IM and CAEBV but not in sera of healthy controls. In sera of patients with CAEBV, the titers of IgG antibodies to ZEBRA correlated with the antibody titers to early antigens obtained with an immunofluorescence assay, but not to EBV nuclear antigens. This ELISA is a useful diagnostic and prognostic test for EBV infection.