Kensei Gotoh

EBV-associated T/NK–cell lymphoproliferative diseases in nonimmunocompromised hosts: prospective analysis of 108 cases

EBV-associated T/NK-cell lymphoproliferative disease (T/NK-LPD) is defined as a systemic illness characterized by clonal proliferation of EBV-infected T or NK cells. We prospectively enrolled 108 nonimmunocompromised patients with this disease (50 men and 58 women; median onset age, 8 years; age range, 1-50 years) evidenced by expansion of EBV(+) T/NK cells in the peripheral blood; these were of the T-cell type in 64 cases and of the NK-cell type in 44, and were clinically categorized into 4 groups: 80 cases of chronic active EBV disease, 15 of EBV-associated hemophagocytic lymphohistiocytosis, 9 of severe mosquito bite allergy, and 4 of hydroa vacciniforme. These clinical profiles were closely linked with the EBV(+) cell immunophenotypes. In a median follow-up period of 46 months, 47 patients (44%) died of severe organ complications. During the follow-up, 13 patients developed overt lymphoma or leukemia characterized by extranodal NK/T-cell lymphoma and aggressive NK-cell leukemia. Fifty-nine received hematopoietic stem cell transplantation, 66% of whom survived. Age at onset of disease (≥ 8 years) and liver dysfunction were risk factors for mortality, whereas patients who received transplantation had a better prognosis. These data depict clinical characteristics of systemic EBV(+) T/NK-LPD and provide insight into the diagnostic and therapeutic approaches for distinct disease.

Clinical and Virological Characteristics of 15 Patients with Chronic Active Epstein-Barr Virus Infection Treated with Hematopoietic Stem Cell Transplantation

BACKGROUND Chronic active Epstein-Barr virus (EBV) infection is characterized by recurrent infectious mononucleosis-like symptoms, and infected patients have high viral loads in their peripheral blood. Standard therapy for the disease has not yet been established. Recently, hematopoietic stem cell transplantation (HSCT) has been introduced and has the potential to become a standard treatment, although guidelines for HSCT to treat chronic active EBV infection have not yet been proposed. METHODS Fifteen patients were retrospectively analyzed, both clinically and virologically, to investigate the factors associated with prognosis of chronic active EBV infection treated with HSCT. RESULTS After HSCT, 7 patients died after survival periods that ranged from 1 to 16 months (mean duration of survival after HSCT, 5 months). Three patients were considered to have died of transplantation-related complications. The duration between infection onset and diagnosis was significantly longer in patients who died than in those who survived. Five of the 7 patients who died experienced > or =3 life-threatening complications. The plasma concentrations of interferon-gamma, interleukin-10, thrombomodulin, and soluble E-selectin did not differ significantly between the groups of patients. With regard to sequence variations in the EBV latent membrane protein 1 gene, no specific patterns were found in the patients who died. Importantly, the plasma EBV load at diagnosis was significantly higher in patients who died than in living patients. Moreover, plasma viral load was shown to be an important factor to monitor during follow-up for patients after HSCT. CONCLUSIONS The number of life-threatening complications and plasma viral load are indicative of the stage of disease progression and may be useful factors for predicting the outcome of HSCT.

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.

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.

Comparison of the Levels of Human Herpesvirus 6 (HHV-6) DNA and Cytokines in the Cerebrospinal Fluid and Serum of Children With HHV-6 Encephalopathy

Primary human herpesvirus‐6 (HHV‐6) infection is a common cause of acute sporadic encephalopathy in Japanese children. Occasionally, HHV‐6 is not detected in the cerebrospinal fluid (CSF) of patients with encephalopathy, for example, in those with focal viral encephalitis, such as herpes simplex viral encephalitis. This indicates that HHV‐6 encephalopathy is caused by an indirect mechanism, although this is not fully understood. HHV‐6 DNA, cytokines (interleukin (IL)‐1β, IL‐6, IL‐8, IL‐10, IL‐12 p70, tumor necrosis factor‐α, interferon‐γ), and matrix metalloproteinase‐9 were quantitated in both the CSF and serum of 13 patients with HHV‐6 encephalopathy during the acute phase of the disease. HHV‐6 DNA was detected in the CSF of seven patients with HHV‐6 encephalopathy. The viral DNA concentration was significantly higher in serum than in CSF (mean 1.64 × 104 vs. 5.70 × 101 copies/ml; P = 0.003). The lack or low level of viral DNA in the CSF samples suggests that direct invasion of the central nervous system by HHV‐6 is not the main cause of encephalopathy. Additionally, the IL‐10 concentration was significantly higher in serum than in CSF (P < 0.001), whereas there was no significant difference in IL‐6 levels between the CSF and serum samples. Interestingly, the IL‐8 concentration was significantly higher in CSF than in serum (P = 0.038). The distribution of these cytokines differed between CSF and serum. The high CSF concentration of IL‐8 could play an important role in the pathogenesis of encephalopathy. J. Med. Virol. 82:1410–1415, 2010.

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.

Effectiveness and safety of inactivated influenza vaccination in pediatric liver transplant recipients over three influenza seasons

Gotoh K, Ito Y, Suzuki E, Kaneko K, Kiuchi T, Ando H, Kimura H. Effectiveness and safety of inactivated influenza vaccination in pediatric liver transplant recipients over three influenza seasons.
Pediatr Transplantation 2011: 15:112–116.