[Recent progress on study of EB virus tumors].
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Biomedical subjects
Publications and source records attributed to T Osato.
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Four children with infectious mononucleosis (IM) and one with reactivated Epstein-Barr virus (EBV) infection had concomitant central nervous system disorders. Cerebrospinal fluid (CSF) samples from all five patients contained EBV genomic sequences and EBV-specific antibodies in the neurologic stage, but not during convalescence. Cerebrospinal fluid from two non-neurologic IM patients had neither EBV DNA nor EBV antibodies. The EBV-positive CSF of the five with neurological disorders were aseptic in culture and all negative for other human herpesvirus DNAs and antibodies: herpes simplex virus types 1 and 2, cytomegalovirus, varicella-zoster virus, and human herpesvirus 6. Epstein-Barr virus DNA and EBV antibodies were not detected in the CSF of 17 EBV-seropositive patients with mumps meningitis, rubella encephalitis, unknown febrile convulsion, or partial epilepsy. It is suggested that EBV plays a causal role in neurologic manifestations in patients with acute and reactivated EBV infections, through direct viral invasion and immunopathological reactions.
Six cases of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD)-like T cell lymphoma were analyzed by immunohistochemical staining, polymerase chain reaction (PCR) and Southern blot analysis. Five cases out of six showed gene rearrangements of the T cell receptor beta chain, indicating the existence of a clonal T cell proliferation. Epstein-Barr virus (EBV) DNA was detected in all six AILD type cases by PCR amplifying the sequence located in the internal repeat I, and confirmed by Southern blot hybridization, using a BamHI-W fragment as a probe. Detection of the EBV genome occurred more frequently as compared with other types of lymphoid disorders. Furthermore, EBV determined nuclear antigen (EBNA) was detected in UCHL-1 (CD45RO, pan-T cell marker) positive cells by immunostaining. These results suggest that a significant number of AILD type cases are T cell origin lymphomas and EBV infection may be relevant to the biological features of this type of lymphoma.
A Lymphoma cell line from the tumor tissue was established spontaneously from a Japanese patient with Epstein-Barr virus (EBV) genome-positive Burkitt's lymphoma (BL). Additionally lymphoblastoid cell lines from peripheral blood of this patient were established either spontaneously or by in vitro infection with B95-8 EBV. Lymphoma cells showed monoclonal surface immunoglobulins (kappa and gamma) with specific chromosomal translocations, t (8; 14). In contrast, lymphoblastoid cells expressed polyclonal surface immunoglobulins without specific chromosomal abnormalities. Lymphoma cells made colonies in soft agarose approximately 10 times more than those of the lymphoblastoid cells. When each cell line was cultured at lower temperature of 33 degrees C, treated with 12-O-tetradecanoyl- phorbol-13-acetate (TPA), and superinfected with P3HR-1 EBV, all cell lines expressed 5 to 10 times higher levels of EBV early antigens (EA) and viral capsid antigen (VCA) than lymphoblastoid cell lines from healthy controls. Furthermore, lymphoblastoid cell lines obtained from peripheral blood of this patient during the period of remission also exhibited high EA and VCA inducibility and superinfectibility. These findings suggested that the lymphoid cells in this patient were genetically highly susceptible to EBV infection, and this evidence possibly linked to the lymphomagenesis of EBV genome-positive BL.
The opportunistic herpesvirus infections in AIDS are often life-threatening and the patients have lowered Epstein-Barr virus (EBV)-specific T cell cytotoxicity. On the other hand, the asymptomatic human immunodeficiency virus (HIV) carriers have potentially lowered EBV-specific cytotoxic T cell function, which is shown by exposure of the lymphocytes to an immunosuppressive substance. The EBV-specific T cell cytotoxicity may therefore tell the timing to cope with such opportunistic viral infections.
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Adult T-cell leukemia/lymphoma (ATLL) is a well-known human T-cell lymphotropic virus type-1-related disease. We studied Epstein-Barr virus (EBV) in the tumor cells of ATLL, to investigate the etiological significance of double infection with these viruses. We used polymerase chain reaction and EBV-encoded small RNA-1 in situ hybridization to investigate the presence of EBV and immunohistochemistry to detect EBV-related oncoproteins, such as EBV-determined nuclear antigen-2 and latent membrane protein. Polymerase chain reaction performed on DNA of frozen specimens from 96 cases of ATLL revealed that the tumor tissue from 21 cases contained EBV DNA. EBV-encoded small RNA-1 in situ hybridization performed on the paraffin sections of the polymerase chain reaction-positive cases indicated EBV in the nuclei of ATLL tumor cells in 16 cases, nine of which were in the pleomorphic nuclei. Latent membrane protein was also detected in the cytoplasm of ATLL tumor cells in 15 cases, and EBV nuclear antigen-2 was observed in the nuclei of ATLL tumor cells in 11 cases. We conclude that EBV was present within tumor cells in about 17% of cases with ATLL and expressed EBV oncoprotein in the tumor cells. It is hypothesized that EBV and human T-cell lymphotropic virus-1 may infect the same T cells in early life and may play a role in the oncogenesis of ATLL.
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Two human Burkitt's lymphoma cell lines (HBL-4 and HBL-5) were established individually from two patients with small noncleaved cell lymphoma (Burkitt's type). The HBL-4 cell line is Epstein-Barr virus (EBV)-negative, and the HBL-5 cell line is EBV-positive. Cytogenetically, both cell lines had the same chromosomal translocation, t(8;14)(q24;q32) as those observed in the primary malignant cells from individual patients. Morphologic, immunophenotypic, cytogenetic, and molecular studies confirmed that both cell lines were derived from the primary lymphoma cells in vivo. HBL-4 cells lacked CD23(H107), CD11a(LFA-1), and latent membrane protein (LMP) but expressed CD54(ICAM-1) at low levels, whereas HBL-5 cells showed the high level of expression of CD54 and faint expression of LMP but lacked CD11a. In addition, the EBV-positive lymphoblastoid cell line (LCL) expressed CD11a, CD23, CD54, and LMP at high levels. Therefore, an HBL-5 phenotype with expression of CD54 and LMP tends toward an LCL phenotype, and the augmentation of CD54 on the HBL-5 cells in comparison with primary lymphoma cells is likely to be upregulated by LMP, probably resulting from the EBV infection. There was little difference in the BrdUrd uptake in vivo and in vitro, doubling time, tumorigenicity, and dynamics of tumor growth in athymic nude mice between both cell lines. These findings indicate that the potentiality of cell growth and tumorigenicity of these two cell lines are unlikely to be related with EBV.
Epstein-Barr virus (EBV), a ubiquitous human B-lymphotropic virus, is associated with certain lymphoproliferative diseases of T-cell lineage. To understand the mechanism by which EBV infects T cells, we have tested the susceptibility of various human T-cell lines to the virus. We report here that the HTLV-I-harbouring T-lymphoblastoid line MT-2 carries a high level of CD21/EBV receptors on their surface, adsorbs fluorescein isothiocyanate-labeled EBV, and synthesizes virus latent antigens (EBNA-1 and LMP) following EBV infection. Pretreatment of MT-2 cells with anti-CD21 monoclonal antibody OKB7 inhibited the virus binding as well as the synthesis of virus latent antigen. These data suggest that human T-cells can be infected with EBV via functionally active virus receptors.
The effect of calcium on germination of coat-modified Bacillus cereus T spores was investigated. Coat-modified spores produced either by chemical extraction (SDS-DTT-treated spores) or by mutagenesis (10LD mutant spores) were unable to germinate in response to inosine. While SDS-DTT-treated spores could germinate slowly in the presence of L-alanine, 10LD mutant spores could not germinate at all. The lost or reduced germinability of coat-modified spores was restored when exogenous Ca2+ was supplemented to the germination media. The calcium requirement of coat-modified spores for germination was fairly specific. The simultaneous presence of germinant with Ca2+ was also required for germination of coat-modified spores. The optimal recovery of germinability was observed in the presence of 1.0 mM of calcium acetate. The calcium requirement itself was remarkably diminished under the condition in which L-alanine and a certain purine nucleoside analog, adenosine or inosine, coexisted. The lost or diminished germinability observed in SDS-DTT-treated spores or 10LD mutant spores may be attributed to the loss of calcium associated with the spore integuments.
Eight cell lines were established from patients with adult T-cell leukemia, and from normal adults, by cocultivation with human T-cell leukemia virus type I(HTLV-I)-producer cell lines in the presence of interleukin-2. All of these cell lines harbored HTLV-I and showed T-cell markers CD2, CD3 and CD4, but not B-cell markers. Unexpectedly, all eight cell lines expressed a myeloid marker CD13 and three of the eight lines also expressed another myeloid marker CD33. Dual staining showed the simultaneous expression of CD3 and CD13 on the same cells. Thus, evidence was obtained for the expression of myeloid antigens on HTLV-I-harboring T cells.
To evaluate the relations between salivary gland tumors and Epstein-Barr virus (EBV), the levels of EBV-related antibodies were examined, and detection of EBV nuclear antigen (EBNA) and EBVDNA in tumor tissue was attempted by the anti-complement immunofluorescence technique and polymerase chain reaction, respectively. The mean VCA-IgG antibody level was increased to 925 (80-2560) in Warthin's tumor, 496 (40-2560) in mucoepidermoid tumor, and 206 (40-640) in pleomorphic adenoma. The positive rate of EA-IgG was high in Warthin's and mucoepidermoid tumors. VCA-IgA antibody was positive in 2 of the 7 cases of Warthin's tumor. EA-IgA antibody was negative in all cases. EBVDNA was detected in 7 of the 7 cases of Warthin's tumor, 3 of the 5 cases of mucoepidermoid tumor, and 2 of the 26 cases of pleomorphic adenoma. A relationship between Warthin's tumor and EBV was suggested by the 100% detection rate of the viral DNA.
Seven Japanese patients with Burkitt's lymphoma (BL), residing in Hokkaido, were studied during the period, 1979-1991. Immunological analyses of their lymphoma cells showed all to express surface and/or cytoplasmic immunoglobulins. Chromosomal analysis was performed in five cases. Four of the five lymphomas had the chromosomal translocation, t(8;14), and one had t(2;8). Three patients had extremely high IgG antibody titers to Epstein-Barr virus (EBV) viral capsid antigen (VCA) and to EBV early antigens (EA). Two patients had positive antibodies to EA, and two others had normal antibody patterns comparable to those of EBV-seropositive age- and sex-matched healthy controls. Four of the seven lymphomas (57.1%) were positive for EBV-determined nuclear antigen (EBNA) by anticomplement immunofluorescence and/or EBV DNA using DNA-DNA reassociation kinetics, and/or Southern blot analysis. The frequency of EBV positivity in BL patients residing in Hokkaido was higher than that of cases previously reported in Japan. Three of the four EBV genome-positive BL patients responded well to chemotherapy, radiotherapy and/or surgical treatment, with no significant relapse being observed during the study period. In contrast, EBV genome-negative patients had poor prognoses despite having similar levels of clinical staging at the time of diagnosis.
Dual exposure to Epstein-Barr virus and purified 4-deoxyphorbol ester derived from the plant Euphorbia tirucalli induced a high frequency of chromosomal rearrangements in human B lymphocytes in vitro. Rearrangements most commonly affected chromosome 8, the chromosome most often showing structural changes in Burkitt's lymphoma (BL) cells. E tirucalli is indigenous in parts of Africa where BL is endemic and may be an important risk factor for the disease.
An Epstein-Barr virus (EBV)-producer line, designated Akata, was established from a Japanese patient with Burkitt's lymphoma. The Akata line possessed the Burkitt's-type chromosome translocation, t(8q- ; 14q+), and was derived from the tumor cell. Akata cells produced a large quantity of transforming virus upon treatment of cells with anti-immunoglobulin antibodies (Takada, 1984). Southern blot analysis of viral DNA indicated that the Akata EBV is nondefective and more representative of wild-type viruses. Akata cells should be useful as a source of EBV.