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We used the results of secondary prevention analyses for patients with a history of stroke or TIA in the large-scale RE-LY, ROCKET-AF (J-ROCKET-AF), and ARISTOTLE clinical trials to investigate the choice of new oral anticoagulants (NOACs) to prevent recurrent stroke. In light of these results, we concluded that dabigatran 150 mg BID should be the first-choice treatment for comparatively young patients with no apparent renal dysfunction, and apixaban for other patients, and that their efficacy and safety can be broadly guaranteed.

The role of Epstein-Barr virus (EBV) reactivation in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease was examined.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.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.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.

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.

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.

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.

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.

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.

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.

Endemicity of Burkitt's lymphoma (BL) coincides with profusion of a plant Euphorbia tirucalli in tropical Africa. E. tirucalli contains 4-deoxyphorbol ester that enhances Epstein-Barr virus (EBV) infection of B lymphocytes. In this study, we found that 4-deoxyphorbol ester reduced EBV-specific cytotoxic T-cell function. Furthermore, the B lymphocytes dually exposed to EBV and 4-deoxyphorbol ester were resistant to EBV-specific T cell cytotoxicity, through down-regulation of latent membrane protein 1 (LMP1), the major target to EBV-specific cytotoxic T-cells. These immunologic findings strengthen the notion that E. tirucalli may be an important environmental risk factor for the genesis of African BL.

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.

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.

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.

The tumor promoter teleocidin activates latent Epstein-Barr virus (EBV) genomes and enhances EBV-induced growth transformation of human B cells, with an activity comparable to that of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). It has been suggested that the TPA induction of EBV genomes is mediated by protein kinase C (PKC), an enzyme closely linked to signal transduction. We examined newly isolated, highly selective PKC inhibitors, UCN-01 (7-hydroxyl-staurosporine) and calphostin C, for the possible suppression of teleocidin enhancement of cord blood B lymphocyte growth transformation by EBV. 0.2 nM teleocidin B4 enhanced EBV-induced 3H-thymidine uptake 6 times, outgrowth in limiting dilution culture 5 times, and colony formation in semisolid agar 3 times. All these events were suppressed by exposure to 10-100 nM UCN-01 or to calphostin C. Our findings suggest that the tumor promoter enhancement of EBV growth transformation is probably mediated by PKC.

Five cases of lethal midline granuloma were identified histologically and phenotypically as peripheral T-cell lymphomas. Epstein-Barr virus (EBV) DNA was detected in the nasal tumour biopsy specimens by Southern blotting and in-vitro hybridisation with simultaneous detection of EBV-determined nuclear antigen (EBNA) and T-cell surface markers by two-colour immunofluorescence. Further immunofluorescence and northern blotting revealed that EBNA2 gene and also latent membrane protein gene were expressed in the nasal tumour cells. The patients had high titres of antibodies to EBV. These findings suggest that lethal midline granuloma is causally associated with EBV.

A B-cell population of human cord blood lymphocytes was exposed to the B95-8 strain of Epstein-Barr virus (EBV), and simultaneous observations of immunofluorescence, cellular morphology and autoradiography were carried out in each individual cell. It was evident that EBV-determined nuclear antigen (EBNA) synthesis occurred prior to blastogenic response and DNA synthesis. EBNA-positive cells could be observed as early as 12 h after infection and reached a maximum of 17% at 24 h, followed by a plateau for a subsequent 12 h. The positive cells were seen exclusively as morphologically normal lymphocytes until 18 h; at 24 h, blastogenesis became evident, without cell division. DNA synthesis was detected at 36 h in EBNA-positive blast cells, after which these cells increased rapidly. EBNA synthesis was similarly evident in the presence of cytosine arabinoside, but was significantly inhibited by a short-term exposure to cycloheximide immediately after infection. These findings suggest that the early events in EBV-induced transformation of human lymphocytes occur sequentially from EBNA synthesis, to blastogenesis, to DNA synthesis and that the crucial step of such transformation is probably involved in protein synthesis occurring in the very early stage of EBV infection.

The interaction between EBV and type-C viruses was studied in our FVNC experimental system, in which EBV and type-C viral genomes are contained in each cell. The data indicate that the human lymphoid FVNC cells are sensitive to both EBV and type-C virus exposure, showing high frequencies of induction of both repressed viral genomes. The two different viral genomes may be associated with different chromosomes in individual cells.

In order to investigate the spread of adult T-cell leukemia (ATL) virus in the inhabitants of ATL-non-endemic Hokkaido, we surveyed the antibodies to ATL-associated antigen (ATLA) in 29516 healthy individuals by indirect immunofluorescence test using MT-1 for target cell. Of the healthy inhabitants, only 219 individuals (0.74%) were anti-ATLA positive as carriers. Prevalence of anti-ATLA was 0.63% (111 of 17590) in males and 0.91% (108 of 11926) in females. This difference by sex is significant. In several regions, the inhabitants were found to show relatively high incidence (greater than 2%) of the antibodies, and these regions have a tendency to cluster around the western side of Hidaka mountains or Akan area. The highest rate (5.2%) of anti-ATLA positive carriers was shown in a certain town of Hidaka area.

Leukemic lymphocytes with B-cell markers were obtained from the peripheral blood of 4 patients with chronic lymphocytic leukemia (CLL) or acute lymphocytic leukemia (ALL); a significant number of T cells was not involved. The lymphocytes were inoculated with the transforming B95-8 Epstein-Barr virus (EBV). Synthesis of EBV-determined nuclear antigen (EBNA) occurred in three CLL preparations: 15.1--25.4% of cells expressed maximum EBNA 24--48 h after viral exposure. A characteristic immunofluorescence pattern similar to a coarsely clumped chromatin structure was observed. In contrast to EBV infection of normal lymphocytes, EBNA synthesis in CLL cells (morphologically similar to normal lymphocytes) was not followed by either blastogenesis or DNA synthesis. EBNA-positive cells, which usually degenerate within 1--2 weeks, could be maintained for more than 30 days on feeder human embryo fibroblasts pretreated with mitomycin C. DNA synthesis and mitosis occurred, but unlimited cell growth did not. 41.9% of ALL cells were EBNA-positive at 36 h prior to DNA synthesis, and they subsequently grew rapidly for up to 9 days. The cell proliferation, however, was temporary, and death resulted in about 2 weeks. No significant synthesis of EBV-related early antigens and viral capsid antigen was noted in any of the infected cultures throughout the incubation period. These results indicate that certain intracellular restrictions may have exceeded the potent transforming capacity of EBV in these target leukemic cells.

Hybrid clones and subclones between human lymphoblastoid FV5 cells and MCB2 fibroblasts were examined for the presence of Epstein-Barr virus (EBV) DNA, the expression of the virus-determined nuclear antigen (EBNA), and the presence of human chromosomes, in the course of serial passage in vitro. Among 14 hybrid clones examined, three were positive for EBNA and EBV DNA and had human chromosome 14, but the remaining 11 clones were totally negative for both EBV markers and did not reveal the presence of this particular chromosome. Furthermore, ten subclones isolated from one of the three positive clones contained only chromosome 14 as the human chromosome. In all of these subclones EBNA, EBV DNA and chromosome 14 were segregated concordantly. These results seem to indicate an apparent linkage relationship between human chromosome 14 and the EBNA antigen gene in human lymphoblastoid cells.

Sera and peripheral blood lymphocytes in 40 clinical cases of adult T-cell leukemia (ATL) and 66 cases of mature T-cell malignancies in Hokkaido district, the most northern part of Japan, were examined for the infection with ATL virus (ATLV). All of the 40 ATL patients (100%) had antibodies to ATLV-associated antigen (ATLA) and 13 (19.7%) of the 66 patients with mature T-cell malignancies other than ATL were also positive for anti-ATLA. When the peripheral ATL lymphocytes were assessed after short time cultivation, both ATLA antigen and C type virus particles were detectable. In contrast to the high prevalence of ATLV antibodies in these patients, the positivity in 30,000 control individuals in this district was 0.73%. It was calculated that 8 patients of a 40,000 seropositive population are diagnosed annually as ATL in this particular northern part of Japan.

The activities of two glycolipid synthetases, globoside synthase or UDP-N-acetylgalactosamine-trihexosylceramide beta-N-acetylgalactosaminyltransferase (beta-GalNAc transferase; EC 2.4.1.79) and Forssman synthase or UDP-N-acetylgalactosamine-globoside-alpha-N-acetylgalactosaminyltransfer ase (alpha-GalNAc transferase; EC 2.4.1.88), were assayed in various human lymphoblastic cell lines. The activity of beta-GalNAc transferase was much higher than that of alpha-GalNAc transferase except in Molt 3 and Molt 4 lines, which were derived from T-cells. In cultivated human peripheral lymphocytes concanavalin A (Con A), lipopolysaccharide (LPS), and Epstein-Barr virus (EBV) stimulated the activities of alpha- and beta-GalNAc transferases in addition to having their known stimulative effect on thymidine incorporation. Characteristic differences between alpha- and beta-GalNAc transferases were noted in the responses to the above mitogens, but activities of both enzymes were greatly increased by exposure of the lymphocytes to EBV. Treatment of lymphocytes with either dactinomycin (actinomycin D) or cycloheximide 24 hours after the addition of Con A, LPS, or EBV decreased the activities of the transferases. This observation suggests that stimulation of alpha- and beta-GalNAc transferases requires transcriptional and translational processes.

Cytotoxicity against lymphocytes freshly transformed by EBV was studied in an autologous system by trypan blue exclusion of EBNA-positive cells and 51Cr-release assay. When EBV-tranformed lymphocytes were incubated with EBV-positive serum and autologous unfractionated lymphocytes or K-cells from sero-positive healthy donors, the transformed cells were significantly damaged. The cytotoxicity against newly produced EBNA-positive lymphocytes was also remarkable when sero-positive healthy donor lymphocytes were exposed to EBV, immediately followed by incubation with positive serum. In contrast, EBV-transformed cells incubated with autologous lymphocytes or T-cells alone were not significantly affected. EBV-positive serum alone was also not cytotoxic, regardless of inactivation. These results strongly suggest that antibody-dependent cellular cytotoxicity (ADCC) plays an important role in immunological defence against EBV oncogenesis in healthy humans. However, ADCC was not seen when immunosuppressed patients were examined.

Family members of non-endemic Hokkaido patients with adult T-cell leukemia (ATL) were assessed for the prevalence of ATL virus (ATLV) infection. Immunofluorescence test showed that 53 (39.8%) of 133 healthy family members of 23 ATL patients were positive for antibodies of ATLV-associated antigen (ATLA). When seropositive T-cell malignancies other than ATL and healthy controls in Hokkaido were examined, 10 of 33 family members (30.3%) of 9 patients and 3 of 18 family members (16.7%) of 5 donors had ATLA antibodies, respectively. In sharp contrast, the overall seropositivity in this northern part of Japan was 0.73%. None were seropositive (0%), when 26 family members of 6 patients with acute myeloid leukemia sero-converted by blood transfusion during the course of the disease.

Immunologic cytotoxicity against lymphocytes transformed or infected by Epstein-Barr virus (EBV) was mainly studied in an autologous in vitro system by 51Cr release assay and EBV-determined nuclear antigen (EBNA)-specific trypan blue exclusion method. When the cells of newly established EBV-transformed or spontaneously transformed lines were incubated with unfractionated autologous healthy donor lymphocytes or T-cell-depleted lymphocytes in the presence of EBV-positive autologous or allogeneic serum, the transformed cells were killed with high frequency. Exposure to lymphocytes alone or to EBV-positive serum alone was not effective. The cytotoxic reaction was directed against cells positive for EBV-induced membrane antigens (MA) but not against MA-negative transformed cells. A very small fraction (1 of 200) of healthy donor lymp]hocytes exposed to EBV converted into EBNA-positive and MA-positive cells, and these were also killed by the remaining autologous lymphocytes in the presence of EBV-positive serum. These results indicated that the present cytotoxic reaction represents antibody-dependent cellular cytotoxicity (ADCC), and this particular mechanism probably plays an important role in the immunologic surveillance in the protection against EBV-induced oncogenesis in seropositive individuals. Such ADCC, however, does not seem to function effectively in patients with systemic lupus erythematosus.

A chronic active Epstein-Barr virus (EBV) infection with no significant underlying diseases was evidenced in 7 Japanese children and adolescents. In these patients, massive EBV-positive polyclonal lymphoproliferation occurred with extremely high EBV antibody titers.

We have recently established a cell line, designated FVNC, by infection of non-EBV-productive human lymphoid NC-37 cells with the type-C virus FLV. The FVNC cell line has been maintained free of detectable EBV- and FLV-related immunofluorescent antigens for three years but both viral genomes exist in the cells in a repressed form. The FVNC cells were morphologically similar to the uninfected NC-37 cells but a D-group chromosome change was consistently seen. The colony-forming capacity of FVNC cells in semi-solid agar medium was about 1/10 of that of NC-37, although the former grew rather faster than the latter in fluid culture. FVNC cells were three times more sensitive to EBV superinfection than NC-37, as shown by immunofluorescence. In addition, an obvious induction of FLV antigenic markers occurred in FVNC cells on exposure to EBV. Nucleic acid hybridization experiments showed a striking decrease in the number of EBV genomes in FVNC cells. The number of genomes was too small to be measured, but EBNA was clearly present in all FVNC cells as well as in NC-37 cells. The implications of these findings were discussed from the point of view of a possible co-carcinogenesis in man by EBV and the type-C virus.

A 6-year-old girl was admitted for cervical lymphadenopathy. She was diagnosed histologically as having Burkitt's lymphoma with partial involvement of follicles. Immunological study of a biopsy specimen demonstrated that many cells were surface immunoglobulin (lambda light chain)-positive, and chromosomal analysis revealed a translocation of (8;14) (q24;q32) in all blastic cells. Her serum antibody titers to Epstein-Barr virus (EBV) capsid antigen and to early antigen were 1:320 and 1:160, respectively, and most of the cells from the lymphoma biopsy specimen were EBV-associated nuclear antigen (EBNA)-positive. In addition, EBV DNA/DNA reassociation kinetic analysis showed 22 EBV genome equivalents per cell. Complete remission was obtained by CHOP therapy (consisting of cyclophosphamide, adriamycin, vincristine, and prednisolone) combined with cervical irradiation. She is in complete remission now (17 months after the onset).

Lymphomas occurred in 3 of 16 Japanese patients with ataxia telangiectasia (AT) and Wiskott-Aldrich syndrome (WAS). The patients had a persistently reactivated Epstein-Barr virus (EBV) infection with a remarkable decrease in virus-specific cellular immunity. In these patients, the B lymphocytes were more sensitive to EBV-induced events and to cellular proto-oncogene activation than seen in the healthy counterparts. This immunologic and genetic background was considered to explain the massive lymphoproliferation in these primary immunodeficiency disorders.

Two continuously growing in vito cell lines, PB-1049 and LN-1049, were established from a patient with Ph1-positive chronic myelogenous leukemia in extramedullary blastic crisis. PB-1049 was established from Epstein-Barr virus (EBV)-infected peripheral lymphocytes of the patient and had a 46,XY,t(9;22) karyotype. This cell line was shown to be tumorigenic in nude mice, and cultured cells recovered from the tumor nodule showed a 46,XY,6p+,t(9;22) stem line karyotype. LN-1049 was derived from a lymph node culture of the same patient, and was found to be non-tumorigenic in nude mice and karyotypically normal. Immunological examinations on EBV-induced antigens, surface and cytoplasmic immunoglobulin, and monoclonal antibodies, and some enzymatic and electron microscopic studies revealed that both cell lines had attained differentiation into late-B cell stage.