[High inducibility of Epstein-Barr virus replication in B lymphocytes in Vogt-Koyanagi-Harada disease].

PURPOSE: The role of Epstein-Barr virus (EBV) reactivation in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease was examined. MATERIAL AND METHODS: Using B lymphocytes obtained from 8 patients with VKH disease and 10 patients with other types of uveitis, immortarized lymphoblast lines were established and infected with EBV. The degree of EBV activation in each lymphoblast line, in the presence and absence of various stimuli, was assessed by measuring the expression of 3 different antigens involved in replication by immunofluorescent staining and western blot analysis. Quantification of EBV DNA in cell culture supernatants was done by polymerase chain reaction. RESULT: Cell lines established from VKH patients expressed more viral antigens that those established from patients with other types of uveitis. There were greater amounts of EBV DNA in the VKH cell lines. CONCLUSION: B lymphocytes from VKH patients may be more susceptible to EBV activation, and the reactivation of EBV may be involved in the pathogenesis of VKH.

[Clinical, phenotypic and genotypic aspects of nasal T-cell lymphoma and its causal association with Epstein-Barr virus].

The authors reviewed the clinical, phenotypic, genotypic, and Epstein-Barr virus (EBV)-findings of 18 patients with nasal T-cell lymphoma(NTL). The clinical features were characterized as prolonged fever, widespread dissemination into distant sites, and poor prognosis with median survival of only 6 months. EBV-encoded small nuclear early region(EBER) transcripts were identified in 16 of 18 patients. Monoclonal EBV genome, EBV-encoded nuclear antigen(EBNA)-1, and latent membrane protein(LMP)-1 were also detected in all EBER-positive cases tested. All EBV-positive NTL showed coexpression of natural killer(NK) cell phenotype CD56 and CD2. Of 9 EBV-positive NTL, seven cases expressed T-cell receptor (TCR)-delta chain with rearranged beta-, gamma- and/or delta- genes. These data suggest that some cases of EBV-positive NTL may be derived from the lineage of NK-like T-cells or gamma delta T-cells, and that EBV may play a role in the lymphomagenesis.

Breast milk is not a significant source for early Epstein-Barr virus or human herpesvirus 6 infection in infants: a seroepidemiologic study in 2 endemic areas of human T-cell lymphotropic virus type I in Japan.

In order to evaluate the possibility of Epstein-Barr virus (EBV) and human herpesvirus 6 (HHV-6) transmission via breast milk, a total of 331 serum specimens collected from bottle-fed and breast-fed children and their mothers, in 2 endemic areas of human T-cell lymphotropic virus type I (HTLV-I) in Japan, were assayed for antibodies to EBV and HHV-6. The seroprevalences of EBV and HHV-6 were over 95% both in the mothers of bottle-fed children and in those of breast-fed children. The seroprevalence of EBV at 12-23 months of age was 54.5% (36/66) and 55.8% (24/43) in breast-fed children and bottle-fed children, respectively. The seroprevalence of HHV-6 at 12-23 months of age was 90.9% (60/66) and 93.0% (40/43) in breast-fed children and bottle-fed children, respectively. No difference was observed between the seroprevalences of EBV and HHV-6 in breast-fed and bottle-fed children at 12-23 months of age. Our seroepidemiologic data indicate that breast milk is not a significant source of early EBV or HHV-6 infection in infancy.

Hodgkin disease occurring in a patient with extremely high serum antibody titers to Epstein-Barr virus--associated antigens without chronic illness.

PURPOSE: We described for the first time a patient with long-lasting, extremely high serum antibody titer against Epstein-Barr virus (EBV) viral capsid antigen and early antigen without clinical symptoms suggestive of active EBV infection; the patient finally developed Hodgkin disease (HD) after 7 years of follow-up. PATIENT AND METHODS: High serum EBV antibody titers were noted at 2 years of age. Immunological evaluation was performed at the age of 7 years. EBV-specific cytotoxic T-lymphocyte activity was normal. None of the other results showed any significant abnormalities except for the abnormal antibody titers against EBV-associated antigens. RESULTS: The patient developed HD at the age of 9 years. In addition, EBV genomes were found in the nuclei of Hodgkin and Reed-Sternberg cells in the lymph node. CONCLUSIONS: This case suggests that (a) a patient with extremely high serum antibody titers against EBV-associated antigens may develop HD after a prolonged period, even though no clinical symptom suggestive of active EBV infection is observed; (b) EBV may play an important role in the occurrence of HD.

Transcriptional analysis of Epstein-Barr virus gene expression in EBV-positive gastric carcinoma: unique viral latency in the tumour cells.

Although case-oriented evidence for an association of Epstein-Barr virus (EBV) with gastric carcinoma has been accumulating recently, the interaction(s) between EBV and gastric epithelial cells is/are largely unknown. In this study, we examined seven EBV-positive gastric carcinoma tissues for viral gene expression at the mRNA level, from which studies on the EBV oncogenicity in human epithelial cells will benefit. Reverse transcription-PCR analysis showed that all seven EBV-positive tumour tissues constitutively expressed EBV nuclear antigen (EBNA) 1 mRNA, but not EBNA2 mRNA. The EBNA transcription was initiated from one of three EBNA promoters, Qp: by contrast, both Cp and Wp were silent, thus resulting in the lack of EBNA2 mRNA. Latent membrane protein (LMP) 2A mRNA was detected in three of seven cases; however, neither LMP1 nor LMP2B mRNA was detected in any of the tumours tested. Transcripts from the BamHI-A region of the viral genome were detectable in all cases. BZLF1 mRNA and the product, an immediate-early gene for EBV replication, was not expressed in any of them, thereby suggesting that the tumour cells carried EBV genomes in a tightly latent form. These findings further extended our previous data regarding EBV latency in gastric carcinoma cells at the protein level, and have affirmed that the programme of viral gene expression in the tumour more closely resembles 'latency I' represented by Burkitt's lymphoma than 'latency II' represented by the majority of nasopharyngeal carcinomas.

Epstein-Barr virus and gastric carcinoma.

Epstein-Barr virus (EBV) has been found in most cases of rare gastric lymphoepithelioma-like carcinomas and a small but significant proportion of common gastric adenocarcinomas. The presence of EBV in gastric cancer has been detected by polymerase chain reaction and in-situ hybridization, the latter technique demonstrating EBV in every malignant epithelial cell. The carcinoma cells express EBNA1 but not the other EBNAs or LMP1. A single fused terminal fragment of the EBV genome was detected in each of the EBNA1-expressing tumours suggesting that the virus-positive gastric carcinomas represent a clonal proliferation of EBV-infected cells. EBV antibody titres were elevated in patients with virus-positive carcinomas and also in those destined to develop EBV-positive carcinomas. EBV-specific cytotoxic T-lymphocyte activity was retained in patients with EBV-positive carcinomas suggesting that the tumours can evade immunosurveillance possibly by only expressing the EBNA1 protein which is not able to be processed and presented. These results implicate EBV as one of the factors contributing to the development of gastric cancer.

Nasal T-cell lymphoma causally associated with Epstein-Barr virus: clinicopathologic, phenotypic, and genotypic studies.

BACKGROUND: The authors have previously demonstrated nasal T-cell lymphoma (NTL) associated with Epstein-Barr virus (EBV). The detailed clinical, phenotypic, and genotypic features and the role of EBV in lymphomagenesis remain to be clarified. METHODS: The study group consisted of 18 patients with NTL. The phenotype was determined by immunoperoxidase staining with various monoclonal antibodies. Genotypic study was done using Southern blot hybridization. The presence of EBV-encoded small nuclear early region (EBER) RNA and EBV DNA were determined by in situ hybridization. The expression of EBV-encoded nuclear antigen (EBNA) and latent membrane protein (LMP1) were identified by immunohistologic methods. Clonotypic analysis of EBV genomes was performed by Southern blot hybridization with EBV termini fragment probe. RESULTS: The clinical features of NTL were characterized as prolonged fever (16 patients), widespread dissemination into distant sites (13 patients), and poor prognosis with a median survival of only 6 months. EBER transcripts were identified in 16 of 18 patients. Monoclonal EBV genomes EBNA1 and LMP1 were also detected in all EBER-positive cases tested. All 18 patients expressed pan-T antigens such as MT1, CD45RO, and/or CD2. The rearrangements of T-cell receptor (TCR)-beta, -gamma, and/or -delta genes were shown in all 11 patients tested. The natural killer (NK) cell phenotype CD56 was expressed in all EBV-positive cases tested, and was not detected in EBV-negative cases. Seven EBV-positive cases expressed a TCR-delta chain with rearranged TCR-gamma or -delta genes whereas both EBV-negative cases corresponded to alpha beta T-cell lymphoma, which expressed a TCR-beta chain with a rearranged TCR-beta gene. CONCLUSIONS: These data suggest that EBV-positive NTL may be derived from the lineage of NK-like T-cells or gamma delta T-cells, and that EBV may play a role in lymphomagenesis. Therefore, we propose that NTL which has peculiar clinical and histologic features could be classified as a new lymphoma entity.

Epstein-Barr virus (EBV)-carrying and -expressing T-cell lines established from severe chronic active EBV infection.

Four novel Epstein-Barr virus (EBV)-carrying T-cell lines, designated SIS, AIK-T8, AIK-T4, and SKN, were established from peripheral blood lymphocytes (PBL) of patients with severe chronic active EBV infection, in the presence of interleukin-2 and 4-deoxyphorbol ester. AIK-T8 and -T4 were derived from a single patient. Cell marker and genotype analyses showed that SIS, AIK-T8, and AIK-T4 had mature T-cell phenotypes with clonally rearranged T-cell receptor (TCR) genes, whereas SKN had an immature T-cell phenotype without TCR gene rearrangement. None of the cell lines expressed B, natural killer, or myeloid antigens or had Ig gene rearrangement. All lines carried EBV genomes in a single episomal form. SIS, AIK-T8, and SKN showed the same phenotype, TCR gene configuration, and/or EBV clonotype as their source or biopsied materials; therefore, they represented EBV-infected T cells proliferating in the patients. TCR gene and EBV episomal structures similar to those of AIK-T4 were not found in its source PBL, probably due to the few parental clones in vivo. All lines expressed EBV-encoded small RNA (EBER) 1, nuclear antigen (EBNA) 1, and latent membrane protein (LMP) 1, -2A, and -2B, but not other EBNAs that could be recognized by EBV-specific immune T cells. EBV replicative antigens were rarely expressed or induced. Such EBV latency reflects the in vivo situation, in which the T cells may evade immune surveillance and be insensitive to antiherpesvirus drugs. Collectively, the data suggest that EBV can target and latently infect T cells at any stage of differentiation in vivo, thus potentially causing uncontrolled T-cell proliferation. These cell lines will facilitate further analyses of possible EBV-induced oncogenicity in T cells.

Immunological role of human palatine tonsil in Epstein-Barr virus persistence.

To determine whether EBV-specific cellular immunity persists and controls EBV in the palatine tonsil, we compared EBV-specific cellular immune responses in the palatine tonsil and peripheral blood. In 32 EBV-seropositive donors, we performed in vitro growth inhibition assays of EBV-induced B-lymphocyte transformation (regression assay). The results showed that EBV-specific cytotoxic T lymphocytes (CTL) precursor frequency was higher in the palatine tonsil than in the peripheral blood. After a first stimulation with autologous lymphoblastoid cell line (LCL), the EBV-specific cytotoxicity of reactivated CTL was higher in the palatine tonsil than in the peripheral blood. Furthermore EBV-specific CTL lines established from the palatine tonsil had significantly higher EBV-specific cytotoxicity in comparison with those from the peripheral blood. Both the EBV-specific CTLs from tonsils and peripheral blood were restricted by HLA class-I determinants. These data indicate that the palatine tonsil, which is frequently exposed to EBV, plays an important role in controlling EBV infection in the oropharynx.

Intact antigen presentation for Epstein-Barr virus (EBV)-specific CTL by a lymphoblastoid cell line established from a patient with severe chronic active EBV infection.

Severe chronic active Epstein-Barr virus (EBV) infection is a lymphoproliferative disease characterized by extremely high antibody titers to EBV, fever, lymphadenopathy, hepatosplenomegaly, and pancytopenia, without any prior immunological abnormality. A spontaneous lymphoblastoid cell line was established from a 4-year-old boy with severe chronic active EBV infection. Immunofluorescence and Western blotting analyses showed that the cell line was of B cell origin and expressed Epstein-Barr nuclear antigens 1, 2 3a, 3b and 3c, and latent membrane protein 1, which are reported to be targets for EBV-specific cytotoxic T lymphocytes (CTL). The cytotoxicity of peripheral blood mononuclear cells derived from the patient and his HLA-identical sister was assayed against the cell line. The cell line was recognized and killed by anti-EBV CTL derived from the HLA-identical sister, but the patient's peripheral blood mononuclear cells had no cytotoxicity. We conclude that antigen presentation in the EBV-infected cells from the patient is intact and sufficient for generation of an EBV-specific CTL response. These observations suggest that severe chronic active EBV infection may not be caused by impaired EBV-antigen presentation of the infected cells but by impaired cellular immune responses to the virus. Our results also suggest the therapeutic possibility that this disease may be treated by adoptive transfer of EBV-specific CTL or bone marrow transplantation from an HLA-matched donor whose immune response to EBV is intact.

Epstein-Barr virus (EBV) hypersensitivity of peripheral B lymphocytes in patients with EBV genome-positive Burkitt's lymphoma.

Peripheral blood mononuclear cells (PBMC) from four Japanese patients with Epstein-Barr virus (EBV) genome-positive Burkitt's lymphoma (BL) during remission were exposed to the B95-8 strain of EBV. Maximum concentrations of the EBV-determined nuclear antigen (EBNA) before cellular DNA synthesis were similar to those of healthy counterparts. Subsequently, EBV-immortalised cell lines were established. These immortalised lymphoblastoid cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and superinfected with the P3HR-1 strain of EBV. EBV early antigens (EA) and viral capsid antigen (VCA) were expressed in approximately 3-10 fold higher concentrations by these lymphoblastoid cells than by those from patients with other types of malignant neoplasia including EBV genome-negative BL and from healthy counterparts. Moderate to extremely high IgG antibody titres to EBV VCA as well as IgG antibodies to EA were demonstrated in these patients during the study. These results suggest that defective underlying cellular mechanisms for regulating the replication of EBV may be present in patients with EBV genome-positive BL.

Decreased cytoplasmic immunoglobulin A production during Epstein-Barr virus immortalization on lymphocytes from patients with ataxia-telangiectasia.

Previously, we have reported significantly lower immunoglobulin (Ig) A production in supernatants of cultured lymphoblastoid cells using enzyme-linked immunosorbent assay from patients with ataxia-telangiectasia (AT) when compared to that of age- and sex-matched healthy individuals. Here, we further assess the degree of cytoplasmic Ig production in these cells and also analyze it during the early phase of Epstein-Barr virus immortalization. All classes of cytoplasmic IgM, IgG, and IgA productions were demonstrated in cells from healthy controls. In contrast, cells from patients with AT showed only cytoplasmic IgM and IgG with low or nondetectable levels of IgA during and after the immortalizing process. These results suggest B lymphocytes bearing IgA are functionally immature and/or defective in patients with AT.

Prevalence of human T-cell leukemia virus type 1 (HTLV-I) in family members of adult T-cell leukemia (ATL) patients in non-ATL-endemic Hokkaido of Japan.

Family members of patients with adult T-cell leukemia (ATL) in non-ATL-endemic Hokkaido, the northernmost part of Japan, were assessed for the prevalence of HTLV-I infection. Immunofluorescence assay showed that 53 out of 133 (39.8%) healthy family members of 23 ATL patients were positive for antibodies to HTLV-I. When general inhabitants in Hokkaido were examined, 3 out of 18 (16.7%) family members of 5 seropositive healthy persons had HTLV-I antibodies. The overall seropositivity in Hokkaido was 0.7%. Of 26 family members of 6 patients with non-T-cell leukemia seroconverted by blood transfusion, none (0%) was seropositive.

Prevalence of human T-cell leukemia virus type 1 (HTLV-I) in general inhabitants in non-adult T-cell leukemia (ATL)-endemic Hokkaido, Japan.

In contrast to adult T-cell leukemia (ATL)-endemic southwestern Japan, the northernmost island Hokkaido has a small number of ATL patients annually. In this study, we surveyed 32,587 healthy inhabitants throughout Hokkaido for antibodies to human T-cell leukemia virus type 1 (HTLV-I). Only 244 individuals (0.8%) were seropositive as HTLV-I carriers; 0.6% (123 of 19,512) in males and 0.9% (121 of 13,075) in females. In some areas, however, the inhabitants had relatively high seropositivity (> 2%). The highest rate was 5.2% with a cluster of ATL patients in a certain town of the Hidaka area near the Pacific Ocean, in southeast Hokkaido.

Prevalence of human T-cell leukemia virus type 1 (HTLV-I) in adult T-cell leukemia (ATL) in non-ATL-endemic Hokkaido of Japan.

Sera and peripheral blood lymphocytes of 40 adult T-cell leukemia (ATL) patients in non-ATL-endemic Hokkaido were examined for the prevalence of human T-cell leukemia virus type 1 (HTLV-I). All patients had HTLV-I-specific antibodies. When the peripheral lymphocytes were assessed after short-term cultivation, HTLV-I antigens and virus particles were detected. The seroprevalence in 96 cases of non-T-cell leukemias and lymphomas and in 30,056 healthy individuals in Hokkaido were 3.1% and 0.7%, respectively. HTLV-I seropositive inhabitants of Hokkaido can be estimated at about 40,000, and one out of every few thousand HTLV-I carriers is likely to develop ATL.

Cryptic dysfunction of cellular immunity in asymptomatic human immunodeficiency virus (HIV) carriers and its actualization by an environmental immunosuppressive factor.

Peripheral blood lymphocytes from 4 asymptomatic HIV-1 carriers with normally retained EBV-specific cytotoxic T-cell activity were exposed to EBV and incubated with 0.2 ng/ml 4-deoxyphorbol ester, an immunosuppressive substance derived from an African plant Euphorbia tirucalli. The regression of EBV-induced B-cell transformation by EBV-specific cytotoxic T-cells was significantly impaired in the presence of a small amount of 4-deoxyphorbol ester, but not so in 5 HIV-1-seronegative healthy counterparts. When the EBV-specific cytotoxic T-cells from the asymptomatic carriers were exposed to 0.2ng/ml 4-deoxyphorbol ester and incubated with 51Cr-labeled autologous EBV-transformed B lymphocytes, the released radioactivity was significantly smaller than that of the healthy counterparts. The results suggest that the cellular immunity of the asymptomatic HIV-1 carriers is cryptically impaired, and the cryptic immunological dysfunction is actualized by exposure to a small amount of the immunosuppressive substance, a dose which does not affect the immunity of uninfected healthy individuals.

Gastric carcinoma: monoclonal epithelial malignant cells expressing Epstein-Barr virus latent infection protein.

In 1000 primary gastric carcinomas, 70 (7.0%) contained Epstein-Barr virus (EBV) genomic sequences detected by PCR and Southern blots. The positive tumors comprised 8 of 9 (89%) undifferentiated lymphoepithelioma-like carcinomas, 27 of 476 (5.7%) poorly differentiated adenocarcinomas, and 35 of 515 (6.8%) moderately to well-differentiated adenocarcinomas. In situ EBV-encoded small RNA 1 hybridization and hematoxylin/eosin staining in adjacent sections showed that the EBV was present in every carcinoma cell but was not significantly present in lymphoid stroma and in normal mucosa. Two-color immunofluorescence and hematoxylin/eosin staining in parallel sections revealed that every keratin-positive epithelial malignant cell expressed EBV-determined nuclear antigen 1 (EBNA1) but did not significantly express CD45+ infiltrating leukocytes. A single fused terminal fragment was detected in each of the EBNA1-expressing tumors, thereby suggesting that the EBV-carrying gastric carcinomas represent clonal proliferation of cells infected with EBV. The carcinoma cells had exclusively EBNA1 but not EBNA2, -3A, -3B, and -3C; leader protein; and latent membrane protein 1 because of methylation. The patients with EBV-carrying gastric carcinoma had elevated serum EBV-specific antibodies. The EBV-specific cellular immunity was not significantly reduced; however, the cytotoxic T-cell target antigens were not expressed. These findings strongly suggest a causal relation between a significant proportion of gastric carcinoma and EBV, and the virus-carrying carcinoma cells may evade immune surveillance.