Kohtaro Yamamoto

Common cytological and cytogenetic features of Epstein-Barr virus (EBV)-positive natural killer (NK) cells and cell lines derived from patients with nasal T/NK-cell lymphomas, chronic active EBV infection and hydroa vacciniforme-like eruptions

Summary. In this study, we describe the cytological and cytogenetic features of six Epstein‐Barr virus (EBV)‐infected natural killer (NK) cell clones. Three cell clones, SNK‐1, ‐3 and ‐6, were derived from patients with nasal T/NK‐cell lymphomas; two cell clones, SNK‐5 and ‐10, were isolated from patients with chronic active EBV infection (CAEBV); and the other cell clone, SNK‐11, was from a patient with hydroa vacciniforme (HV)‐like eruptions. An analysis of the number of EBV‐terminal repeats showed that the SNK cell clones had monoclonal EBV genomes identical to the original EBV‐infected cells of the respective patients, and SNK cells had the type II latency of EBV infection, suggesting that not only the cell clones isolated from nasal T/NK‐cell lymphomas but also those isolated from CAEBV and HV‐like eruptions had been transformed by EBV to a certain degree. Cytogenetic analysis detected deletions in chromosome 6q in five out of the six SNK cell clones, while 6q was not deleted in four control cell lines of T‐cell lineage. This suggested that a 6q deletion is a characteristic feature of EBV‐positive NK cells, which proliferated in the diseased individuals. The results showed that EBV‐positive NK cells in malignant and non‐malignant lymphoproliferative diseases shared common cytological and cytogenetic features.

Telomerase inhibition enhances apoptosis in human acute leukemia cells : possibility of antitelomerase therapy

Telomerase is a ribonucleoprotein enzyme that maintains protective structures at the ends of eukaryotic chromosomes. We examined the impact of telomerase inhibition by the dominant-negative human catalytic subunit of telomerase (DN-hTERT) on the biological features of acute leukemia. We introduced vectors encoding dominant- negative (DN)-hTERT, wild-type (WT)-hTERT, or a control vector expressing only a drug-resistant marker into a telomerase-positive human acute lymphoblastic leukemia cell line, HAL-01. Expression of DN-hTERT dramatically inhibited telomerase activity, leading to apoptotic cell death. Mutant telomerase expression also enhanced daunorubicin-induced apoptosis. Nude mice (n=5 per group) received subcutanous implants of HAL-01 cells expressing the control vector or DN-hTERT or WT-hTERT. Implantation of HAL-01 cells expressing control vector (n=5) rapidly produced tumors, whereas implantation of those expressing DN-hTERT (n=5) did not. Thus, telomerase inhibition both growth of HAL-01 cells in vitro and tumorigenic capacity in vivo. Furthermore, the G-quadruplex-interactive telomerase-specific inhibitor, telomestatin, shortened the telomere length and induced apoptosis in freshly isolated primary acute leukemia cells. These results suggest that antitelomerase therapy may be useful in some acute leukemias in combination with antileukemic agents such as daunorubicin.

The biological activity of hydrogen peroxide: I. Induction of chromosome-type aberrations susceptible to inhibition by scavengers of hydroxyl radicals in human embryonic fibroblasts

The cytogenetic effect of hydrogen peroxide (H2O2) was investigated in human embryonic fibroblasts. Chromosome-type aberrations were found together with chromatid-type aberrations in metaphase cells harvested 24 h after a single 10-min treatment with 10(-5)-10(-3) M H2O2 in 0.9% NaCl solution. The chromosome-type aberrations were observed to be predominantly dicentrics and deletions. Both types of aberration showed a dose-response relationship to the dose of H2O2 over the range of 10(-5)-1.5 X 10(-4) M H2O2. The intercellular distribution of dicentrics showed a Poisson distribution. Centric and acentric rings and abnormal monocentrics were a minor fraction of the chromosome-type aberrations. The chromatid-type aberrations observed, such as breaks, exchanges and gaps, showed no dose-response relationship. The frequency of isochromatid breaks was higher than that of chromatid breaks and approximately 70% of the isochromatid breaks were found in the centromeric or pericentromeric region. The intercellular distribution of chromatid exchanges showed an over-dispersed distribution. The generation of aberrations by H2O2 was effectively suppressed by catalase and several scavengers of hydroxyl radicals (.OH) such as ethanol, dimethyl sulfoxide (DMSO) and mannitol. This result suggest that .OH plays an essential role in the generation of the chromosome aberrations by H2O2.

Prevalence of a human retrovirus in native Japanese: Evidence for a possible ancient origin

The origin of a human retrovirus (ATLV or HTLV-I) is, at present, unknown although carriers of the virus have been found in Japan, the Caribbean basin and Africa. By means of a sero-epidemiological study, the Ainu people of Hokkaido, located in the northernmost island of Japan, were shown to have antibody to the virus in high frequency. Since the Ainu are regarded as descendants of the pre-agriculture native population of northern Japan, this finding appears to indicate that the retrovirus was already present in the aboriginal Japanese of prehistoric times.

Seroepidemiological study of antibodies to adult T-cell leukemia virus-associated antigen (ATLA) in free-ranging Japanese monkeys (Macaca fuscata).

Adult T-cell leukemia (ATL) in man is a newly described disease entity (7). A new human retrovirus named ATL-virus (ATLV) has been detected in cells derived from ATL patients, and specific antibodies to ATL associated antigen (ATLA) synthesized by ATLV-producer cells have been detected in sera from ATL patients (1). It was suggested that ATLA is ATLV-associated antigen(s) (9, 10). One of the striking characteristics of ATL is geographical clustering restricted to southwestern japan (8) and a high incidence of ATLA antibodies has been documented in adults in ATL endemic areas by seroepidemiological studies (2). We have detected the presence of antibodies to the antigens associated with Epstein-Barr virus (EBV), which is thought to be a human oncogenic herpes virus, in plasmas of free-ranging japanese monkeys, suggesting that they are susceptible to infection with EBV or EBV-like viral agents (Ishida and Yamamoto, in preparation). This result prompted us to examine whether or not these japanese monkeys are infected with another human tumor virus, ATLV. The present study shows the natural occurrence of anti-ATLA antibodies among a free-ranging japanese monkey (Macaca fuscata) population. The age dependency, sexual difference and familial-clustering of ATLA-antibody seropositive incidence were also revealed by an ethological study on the japanese monkey population. Fifty-one plasma specimens were obtained from a free-ranging japanese monkey population (5) consisting of 126 individuals in Nagano Prefecture (Honshu) where ATL in humans is rare (8). Since this japanese monkey population has been followed ethologically for the last thirty years, their age, sex, and mother-infant relations have been studied and recorded (Tokita, unpublished data). The age of the subjects (20 males and 31 females) ranged between 2 and 25 years old (mean age: 7.0). The ATLA antibodies and their titers were detected and determined using ATLA-bearing MT-I cells by means of the indirect immunofluorescence test described previously (I). The acetone-fixed MT-I cells were first treated with test

Quantification of human herpesvirus 6 in healthy volunteers and patients with lymphoproliferative disorders by PCR-ELISA

To determine whether actual numbers of human herpesvirus 6 (HHV-6) genome in hematologic neoplasias are associated with disease condition, we developed a quantitative PCR-ELISA for detection of HHV-6. The amount of viral DNA was determined using externally amplified known amounts of the plasmid DNA containing the viral target sequences. First, we determined a viral burden in peripheral blood leukocytes obtained from 23 healthy volunteers and four specimens of lymph nodes with reactive hyperplasia. Using 1 microg of DNA, the prevalence of HHV-6 was 43.4% (10/23), ranging from 0 to 100 HHV-6 genomes in blood obtained from healthy volunteers. The amounts of HHV-6 genomes were < 10 in four non-neoplastic lymph node specimens. We next examined the amount of viral DNA in 21 blood specimens and 19 lymph node specimens obtained from patients with lymphoproliferative diseases (LPD) at the time of diagnosis. The number of HHV-6 genomes in most of the B-cell lymphoma was < 5 in both blood and lymph node specimens, however, two lymph node specimens obtained from immunoblastic lymphadenopathy (IBL) and T-cell lymphoma had very high levels of HHV-6 viral DNA (3705 and 810, respectively). We also found that HHV-6 genomes in peripheral blood were more than 1000 in two patients with chronic lymphocytic leukemia. For all LPD patients combined, there were significantly higher levels of viral DNA (200.6 +/- 654.8 HHV-6 genomes per 1 microg purified DNA) compared to those in healthy volunteers (10.0 +/- 21.0 HHV-6 genomes per 1 microg purified DNA) (P < 0.05). This study demonstrates that a high level of HHV-6 viral DNA is occasionally associated with LPD patients. Although it is still uncertain whether HHV-6 is related to the pathogenesis in LPD or not, our results suggest that measurement of HHV-6 genomes using PCR-ELISA may be useful not only to understand the mechanism of HHV-6 infection in hemopoietic neoplasia but also to manage the care of immnocompromised patients such as bone marrow transplant patients.

Evaluation of epstein-barr virus DNA load in gastric mucosa with chronic atrophic gastritis using a real-time quantitative PCR assay.

AbstractPurpose. It is hypothesized that Epstein-Barr virus (EBV) has already infected the noncarcinomatous gastric mucosa before carcinogenesis of EBV-associated gastric carcinoma. However, the frequency and distribution of EBV infection in the gastric mucosa of chronic atrophic gastritis (CAG) are still unclear. To clarify these points, we evaluated the EBV DNA load in gastric mucosa with CAG. Methods. We tested samples from 35 CAG cases. Paired biopsy specimens from five sites of the stomach were obtained according to the Updated Sydney System. One of each pair of specimens was subjected to a real-time quantitative polymerase chain reaction (Q-PCR) assay to detect EBV. Q-PCR was performed using the LightCycler System (Roche, Mannheim, Germany). The other was subjected to hematoxylin and eosin (H&E) and Giemsa staining. The histological degree of CAG was graded according to the Updated Sydney System. To evaluate the surface distribution of gastric mucosal atrophic changes of CAG, we modified the endoscopic classification of Kimura and Takemoto. Result. EBV DNA was detected in 65.7% (23 of 35 cases) of the gastric biopsy specimens of the cases examined. EBV DNA was detected most frequently (92.3%; 12 of 13 cases) in the cases with endoscopically moderate CAG (p<0.01). There was a significant association between EBV detection and the presence of inflammatory cell infiltration and atrophy in the stomach with endoscopically moderate CAG. Conclusion. EBV mainly infects the gastric mucosa of patients with moderate CAG.

Preferential Expansion of Vγ9-JγP/Vδ2-Jδ3 γδ T Cells in Nasal T-Cell Lymphoma and Chronic Active Epstein-Barr Virus Infection

We recently established an Epstein-Barr virus (EBV)-positive γδ T-cell line from a nasal T/natural killer (NK)-cell lymphoma (Nagata H, Konno A, Kimura N, Zhang Y, Kimura M, Demachi A, Sekine T, Yamamoto K, Shimizu N: Characterization of novel natural killer (NK)-cell and γδ T-cell lines established from primary lesions of nasal T/NK-cell lymphomas associated with the Epstein-Barr virus. Blood 2001, 97:708–713). Subsequently, we established two novel EBV-positive γδ T-cell lines from the peripheral blood of patients with chronic active EBV infection. Analysis of the terminal repeat of EBV showed that the three cell lines consisted of monoclonal populations, and flow cytometry showed that they had a common phenotype of γδ T cells: CD3 + CD4 − CD8 − CD16 − CD19 − CD56 + CD57 − HLA-DR + T-cell receptor (TCR) αβ − TCR γδ + . Analysis for the expression of TCR by flow cytometry showed that all three cell lines were Vγ9 + /Vδ2 + , but negative for VγI, Vδ1, or Vδ3 TCR. Southern blot analysis for TCR genes showed that the three cell lines had a common rearrangement of Vγ9-JγP and Jδ3 genes. Polymerase chain reaction and sequence analysis of the junction between Vδ and Jδ genes revealed that the Jδ3 genes were rearranged with the Vδ2 genes. In contrast, none of the EBV-negative γδ T-cell lines, Molt-14, Peer, or Loucy, which were analyzed for controls, had Vγ9 or Vδ2 TCR, or a rearrangement of Jδ3 genes. These results indicated that Vγ9-JγP/Vδ2-Jδ3 + γδ T cells were preferentially affected by EBV and expanded in patients with nasal γδ T-cell lymphoma and chronic active EBV infection. Jδ3 + γδ T cells are known to be a very minor population in γδ T cells of peripheral blood, whereas Vγ9-JγP/Vδ2-Jδ1 + cells are the major population. The close association of EBV with this particular γδ T-cell population may provide a key to the etiology of EBV-positive lymphoproliferative diseases.

Coexpression of CD40 and CD40 Ligand in Epstein-Barr Virus–Infected T and NK Cells and Their Role in Cell Survival

We investigated the role that CD40-CD40 ligand (CD40L) signaling plays in survival of Epstein-Barr virus (EBV)-infected T and NK cells. EBV-infected T and NK cell lines derived from patients with either chronic active EBV infection (CAEBV) or nasal T/NK cell lymphoma, as well as virus-infected peripheral T cells freshly isolated from a patient with CAEBV, were shown to express both CD40 and CD40L on their surface. Apoptosis of these cells was enhanced by blockade of CD40-CD40L signaling by a fusion protein of CD40 and immunoglobulin G (CD40Ig). Expression of CD40 was induced in human CD40L-positive Jurkat T cells after experimental EBV infection, and apoptosis of infected cells was enhanced by CD40Ig. These results suggest that CD40-CD40L signaling promotes survival of EBV-infected T and NK cells and, thus, plays an important role in the pathogenesis of T/NK lymphoproliferative disorders associated with the virus.

Presence of natural killer‐cell clones with variable proliferative capacity in chronic active Epstein–Barr virus infection

Chronic active Epstein–Barr virus infection (CAEBV) is a syndrome that takes diverse clinical courses and is often associated with lymphoproliferative disorders of T / natural killer (NK)‐cell lineage. We describe a patient with CAEBV associated with persistent pharyngeal ulcer, and with subsequent nasal T/NK‐cell lymphoma in her neck lymph nodes and nasopharynx. Immunophenotyping of lymphoid cells showed that the lineage of Epstein–Barr virus (EBV)‐positive cells in the patient was of NK‐cell origin. By means of high‐dose recombinant interleukin‐2, we established an EBV‐positive cell line of NK‐cell lineage from her peripheral blood. Southern blot analysis for the number of terminal repeat sequences of EBV detected three NK‐cell clones in the patient’s lymph node. One of these clones was identical to the established cell line but was not observed in the pharyngeal ulcer, while the other two clones were present in the pharyngeal ulcer. These results suggest that the patient had expansion of the three NK‐cell clones, one of which had proliferative capacity in vitro and was involved in the formation of the lymphoma. Moreover, the results suggest that the proliferative capacity of EBV‐positive cells can be variable even in a single patient, and this variability may explain the clinical diversity in CAEBV.