Seiko Iwata

Identification of Epstein-Barr Virus (EBV)-Infected Lymphocyte Subtypes by Flow Cytometric In Situ Hybridization in EBV-Associated Lymphoproliferative Diseases

To diagnose Epstein-Barr virus (EBV)-associated diseases and to explore the pathogenesis of EBV infection, not only must the EBV load be measured, but EBV-infected cells must also be identified. We established a novel flow cytometric in situ hybridization assay to detect EBV(+) suspension cells using a peptide nucleic acid probe specific for EBV-encoded small RNA (EBER). By enhancing fluorescence and photostability, we successfully stained EBER and surface antigens on the same cells. In 3 patients with hydroa vacciniforme-like lymphoproliferative disease, we demonstrated that 1.7%-25.9% of peripheral lymphocytes were infected with EBV and specifically identified these lymphocytes as CD3(+)CD4(-)CD8(-) gammadelta T cell receptor-positive T cells. The results indicate that this novel and noninvasive assay is a direct and reliable method of characterizing EBV-infected lymphocytes that can be used not only to diagnose EBV infection but also to clarify the pathogenesis of EBV-associated diseases.

Plasma Viral MicroRNA Profiles Reveal Potential Biomarkers for Chronic Active Epstein–Barr Virus Infection

BACKGROUND Chronic active Epstein-Barr virus (CAEBV) infection has high mortality and morbidity, and biomarkers for disease severity and prognosis are required. MicroRNAs (miRNAs) are small noncoding RNAs, and EBV encodes multiple miRNAs. Because plasma contains sufficiently stable miRNAs, circulating EBV-associated miRNA profiles were investigated as novel biomarkers in CAEBV infection. METHODS Plasma miRNA expression was assessed for 12 miRNAs encoded within 2 EBV open reading frames (BART and BHRF). Expression levels were investigated in 19 patients with CAEBV infection, 14 patients with infectious mononucleosis, and 11 healthy controls. Relative expression levels of plasma miRNAs were determined by TaqMan probe-based quantitative assay. RESULTS Plasma miR-BART1-5p, 2-5p, 5, and 22 levels in patients with CAEBV infection were significantly greater than those in patients with infectious mononucleosis and in controls. Plasma miR-BART2-5p, 4, 7, 13, 15, and 22 levels were significantly elevated in patients with CAEBV infection with systemic symptoms, compared with levels in patients with no systemic symptoms. The levels of miR-BART2-5p, 13, and 15 showed clinical cutoff values associated with specific clinical conditions, in contrast to plasma EBV loads. CONCLUSIONS Levels of specific plasma EBV miRNAs were elevated differentially in patients with CAEBV infection. Several EBV miRNAs, particularly miR-BART2-5p, 13, and 15, are potentially biomarkers of disease severity or prognosis.

Quantitative analysis of Epstein-Barr virus (EBV)-related gene expression in patients with chronic active EBV infection.

Chronic active Epstein-Barr virus (CAEBV) infection is a systemic Epstein-Barr virus (EBV)-positive lymphoproliferative disorder characterized by persistent or recurrent infectious mononucleosis-like symptoms in patients with no known immunodeficiency. The detailed pathogenesis of the disease is unknown and no standard treatment regimen has been developed. EBV gene expression was analysed in peripheral blood samples collected from 24 patients with CAEBV infection. The expression levels of six latent and two lytic EBV genes were quantified by real-time RT-PCR. EBV-encoded small RNA 1 and BamHI-A rightward transcripts were abundantly detected in all patients, and latent membrane protein (LMP) 2 was observed in most patients. EBV nuclear antigen (EBNA) 1 and LMP1 were detected less frequently and were expressed at lower levels. EBNA2 and the two lytic genes were not detected in any of the patients. The pattern of latent gene expression was determined to be latency type II. EBNA1 was detected more frequently and at higher levels in the clinically active patients. Quantifying EBV gene expression is useful in clarifying the pathogenesis of CAEBV infection and may provide information regarding a patients disease prognosis, as well as possible therapeutic interventions.

Heat Shock Protein 90 Inhibitors Repress Latent Membrane Protein 1 (LMP1) Expression and Proliferation of Epstein-Barr Virus-Positive Natural Killer Cell Lymphoma

Epstein-Barr virus (EBV) LMP1 is a major oncoprotein expressed in latent infection. It functions as a TNFR family member and constitutively activates cellular signals, such as NFκB, MAPK, JAK/STAT and AKT. We here screened small molecule inhibitors and isolated HSP90 inhibitors, Radicicol and 17-AAG, as candidates that suppress LMP1 expression and cell proliferation not only in EBV-positive SNK6 Natural Killer (NK) cell lymphoma cells, but also in B and T cells. Tumor formation in immuno-defficient NOD/Shi-scid/IL-2Rγnull (NOG) mice was also retarded. These results suggest that HSP90 inhibitors can be alternative treatments for patients with EBV-positive malignancies.

Immunologic and Virologic Analyses in Pediatric Liver Transplant Recipients with Chronic High Epstein-Barr Virus Loads

BACKGROUND Long-term Epstein-Barr virus (EBV) monitoring for potentially life-threatening posttransplant lymphoproliferative disorder (PTLD) has identified asymptomatic patients who maintain high EBV loads over long periods. METHODS Thirty-one pediatric liver transplant recipients were designated as 11 chronic high EBV load carriers (EBV DNA level >5000 copies/mL of whole blood for >6 months) and 20 control recipients. Serial quantification of EBV DNA, measurement of interleukin 10 (IL-10) concentrations, EBV-specific tetramer staining, and relative quantification of EBV gene expression in peripheral blood mononuclear cells were performed. RESULTS Most of the chronic high EBV load carriers were seronegative at transplant, the median time to resolution of a chronic high EBV load was 23 months, and no recipient developed late-onset PTLD. EBV DNA was detected predominantly in CD19(+) cells. The plasma concentration of IL-10 and the EBV-specific CD8(+) cell frequency did not differ significantly between the chronic high EBV load carriers and the control recipients. Analysis of gene expression showed that EBV-encoded small RNA 1, BamHI A rightward transcripts, and latent membrane protein 2 were positive in peripheral blood mononuclear cells from chronic high EBV load carriers. CONCLUSIONS EBV-infected cells in the blood of chronic high EBV load carriers expressed a highly restricted set of latency genes, suggesting that the EBV-infected cells escaped from a T cell response.

Antitumor activities of valproic acid on Epstein-Barr virus-associated T and natural killer lymphoma cells.

Epstein–Barr virus (EBV), which infects B cells, T cells, and natural killer (NK) cells, is associated with multiple lymphoid malignancies. Recently, histone deacetylase (HDAC) inhibitors have been reported to have anticancer effects against various tumor cells. In the present study, we evaluated the killing effect of valproic acid (VPA), which acts as an HDAC inhibitor, on EBV‐positive and ‐negative T and NK lymphoma cells. Treatment of multiple T and NK cell lines (SNT13, SNT16, Jurkat, SNK6, KAI3 and KHYG1) with 0.1‐5 mM of VPA inhibited HDAC, increased acetylated histone levels and reduced cell viability. No significant differences were seen between EBV‐positive and ‐negative cell lines. Although VPA induced apoptosis in some T and NK cell lines (SNT16, Jurkat and KHYG1) and cell cycle arrest, it did not induce lytic infection in EBV‐positive T or NK cell lines. Because the killing effect of VPA was modest (1 mM VPA reduced cell viability by between 22% and 56%), we tested the effects of the combination of 1 mM of VPA and 0.01 μM of the proteasome inhibitor bortezomib. The combined treated of cells with VPA and bortezomib had an additive killing effect. Finally, we administered VPA to peripheral blood mononuclear cells from three patients with EBV‐associated T or NK lymphoproliferative diseases. In these studies, VPA had a greater killing effect against EBV‐infected cells than uninfected cells, and the effect was increased when VPA was combined with bortezomib. These results indicate that VPA has antitumor effects on T and NK lymphoma cells and that VPA and bortezomib may have synergistic effects, irrespective of the presence of EBV. (Cancer Sci 2012; 103: 375–381)

Bortezomib induces apoptosis in T lymphoma cells and natural killer lymphoma cells independent of Epstein-Barr virus infection

Epstein‐Barr virus (EBV), which infects not only B cells, but also T cells and natural killer (NK) cells, is associated with multiple lymphoid malignancies. Recently, the proteasome inhibitor bortezomib was reported to induce apoptosis of EBV‐transformed B cells. We evaluated the killing effect of this proteasome inhibitor on EBV‐associated T lymphoma cells and NK lymphoma cells. First, we found that bortezomib treatment decreased the viability of multiple T and NK cell lines. No significant difference was observed between EBV‐positive and EBV‐negative cell lines. The decreased viability in response to bortezomib treatment was abrogated by a pan‐caspase inhibitor. The induction of apoptosis was confirmed by flow cytometric assessment of annexin V staining. Additionally, cleavage of caspases and polyadenosine diphosphate‐ribose polymerase, increased expression of phosphorylated IκB, and decreased expression of inhibitor of apoptotic proteins were detected by immunoblotting in bortezomib‐treated cell lines. We found that bortezomib induced lytic infection in EBV‐positive T cell lines, although the existence of EBV did not modulate the killing effect of bortezomib. Finally, we administered bortezomib to peripheral blood mononuclear cells from five patients with EBV‐associated lymphoproliferative diseases. Bortezomib had a greater killing effect on EBV‐infected cells. These results indicate that bortezomib killed T or NK lymphoma cells by inducing apoptosis, regardless of the presence or absence of EBV.

mTOR Inhibitors Induce Cell-Cycle Arrest and Inhibit Tumor Growth in Epstein–Barr Virus–Associated T and Natural Killer Cell Lymphoma Cells

Purpose: Epstein–Barr virus (EBV) infects B cells, as well as T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoid malignancies. In various tumor cells, mTOR performs an essential function together with Akt with regard to cell growth. We investigated the effects of mTOR inhibitors on EBV-associated T- and NK-cell lymphomas. Experimental Design: We investigated the Akt/mTOR activation pathway in EBV-positive and -negative T- and NK-cell lines (SNT13, SNT16, Jurkat, SNK6, KAI3, and KHYG1). We evaluated the antitumor effects of mTOR inhibitors (rapamycin and its analogue, CCI-779) against these cell lines in culture and in a murine xenograft model that was established by subcutaneous injection of SNK6 cells into NOG mice. Results: All EBV-positive and -negative T- and NK-cell lines tested displayed activation of the Akt/mTOR pathway, and treatment with mTOR inhibitors suppressed mTOR activation. The inhibitors induced G1 cell-cycle arrest and inhibited cell proliferation in T- and NK-cell lines. Overall, T cell lines were more sensitive to rapamycin, but there were no significant differences between EBV-positive and -negative cell lines. Treatment with rapamycin did not affect lytic or latent EBV gene expression. Intraperitoneal treatment with CCI-779 significantly inhibited the growth of established tumors in NOG mice and reduced the EBV load in peripheral blood. Conclusion: These results suggest that inhibition of mTOR signaling is a promising new strategy for improving treatment of EBV-associated T- and NK-cell lymphoma. Clin Cancer Res; 20(21); 5412–22. ©2014 AACR.

Multiplex Real-Time PCR Assay for Simultaneous Quantification of BK Polyomavirus, JC Polyomavirus, and Adenovirus DNA

ABSTRACT In recent years, virus-induced nephropathy caused mainly by BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), and adenovirus has emerged as a problem in renal transplant patients. In the present study, we developed a multiplex real-time PCR assay to quantify the viral load of BKPyV, JCPyV, and adenovirus simultaneously. The dynamic range covered at least 6 orders of magnitude. This system was specific and reproducible, even in the presence of large amounts of DNA of other viruses. To validate this assay, urine samples from 124 renal transplant patients and serum samples from 18 hemorrhagic cystitis patients after hematopoietic stem cell transplantation were examined. In the urine samples from renal transplant patients, BKPyV was detected in 28 patients (22.6%), JCPyV was detected in 51 patients (41.1%), and adenovirus was detected in 2 patients (1.6%). The maximum amounts of each virus detected were 2.7 × 109, 8.7 × 108, and 1.2 × 102 copies/ml, respectively. Decoy cells were observed in 31 patients. The quantities of both BKPyV and JCPyV DNA were greater in samples with decoy cells. Two patients whose BKPyV viral loads exceeded 108 copies/ml had elevated serum creatinine levels and were diagnosed with BKPyV nephropathy based on graft biopsies. In serum samples from hemorrhagic cystitis patients, BKPyV, JCPyV, and adenovirus was detected in six, two, and three patients, respectively. Strong correlations were observed between the viral DNA copy numbers determined in the multiplex assays and those determined in single assays. Since this new assay is rapid, sensitive, and specific, it can be used for quantitative analyses of viruses in urine and serum samples after transplantation.

Rare occurrence of JAK3 mutations in natural killer cell neoplasms in Japan

Extranodal natural killer (NK)/T cell lymphoma, nasal type (ENKTL), which is prevalent in East Asia and Latin America, is a refractory neoplasm with resistance to chemotherapies [1]. Its pathogenes...