Jun-ichi Kawada

Systemic Cytokine Responses in Patients with Influenza-Associated Encephalopathy

Influenza-associated encephalopathy, a severe neurologic complication of influenza, is being reported more frequently in Japan. We investigated the transcription of cytokine genes in peripheral blood leukocytes and compared patients with influenza and with encephalopathy or febrile convulsions and patients with influenza but without neurologic complications. A quantitative polymerase chain reaction (PCR) revealed that transcription of the interleukin (IL)-6, IL-10, and tumor necrosis factor-alpha genes was up-regulated to a greater extent in patients with encephalopathy than in those without neurologic complications. Plasma IL-6 levels also were higher in patients with encephalopathy, although the difference was marginal. Viral RNA in throat swabs was quantified using a real-time quantitative PCR. The virus load was similar among patients with encephalopathy or febrile convulsions or without neurologic complications. Furthermore, virus load was not correlated with either the transcription of cytokine genes or plasma cytokine concentrations. These results suggest that influenza-associated encephalopathy might be a consequence of systemic immune responses.

Differences between T cell-type and natural killer cell-type chronic active epstein-barr virus infection

Infections of T cells and natural killer (NK) cells play a central role in the pathogenesis of chronic active Epstein-Barr virus (CAEBV) infection. To characterize the virologic and cytokine profiles of T cell-type and NK cell-type infection, 39 patients with CAEBV infection were analyzed. Patients with T cell-type infection had higher titers of immunoglobulin G against early and late EBV antigens, suggesting lytic cycle infection. However, the pattern of EBV gene expression was latency type II; BZLF1, which is a hallmark of lytic cycle infection, could not be detected in any patients, regardless of infection type. Patients with CAEBV infection had high concentrations of proinflammatory, T helper cell type 1, and anti-inflammatory cytokines. The cytokine profile in patients with NK cell-type infection was similar to that in patients with T cell-type infection, but the concentration of IL-13 was high in patients with NK cell-type infection. These findings should help to clarify the pathogenesis of CAEBV infection and facilitate the development of more-effective treatments.

Bortezomib Induces Apoptosis of Epstein-Barr Virus (EBV)-Transformed B Cells and Prolongs Survival of Mice Inoculated with EBV-Transformed B Cells

ABSTRACT Bortezomib, an inhibitor of the 26S proteasome, is currently approved for treatment of multiple myeloma and is being studied for therapy of non-Hodgkins lymphoma. We found that Epstein-Barr virus (EBV)-positive B cells with type III latency were more susceptible to killing by bortezomib than those with type I latency. Bortezomib induced apoptosis of EBV lymphoblastoid cell lines (LCLs) by inducing cleavage of caspases 8 and 9; apoptosis was inhibited by pretreatment with a pan-caspase inhibitor. Bortezomib reduced the levels of the p50 and p65 components of the canonical NF-κB pathway and reduced the level of p52 in the noncanonical NF-κB pathway, which is induced by EBV LMP1. Bortezomib inhibited expression of cIAP-1, cIAP-2, and XIAP, which are regulated by NF-κB and function as inhibitors of apoptosis. Bortezomib did not inhibit expression of several other antiapoptotic proteins, including Bcl-2 and Bcl-XL. Finally, bortezomib significantly prolonged the survival of severe combined immunodeficiency mice inoculated with LCLs. These findings suggest that bortezomib may represent a novel strategy for the treatment of certain EBV-associated lymphomas.

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.

Tubacin Kills Epstein-Barr Virus (EBV)-Burkitt Lymphoma Cells by Inducing Reactive Oxygen Species and EBV Lymphoblastoid Cells by Inducing Apoptosis

Tubacin is a small molecule inhibitor of histone deacetylase 6 and blocks aggresome activity. We found that Epstein-Barr virus (EBV)-positive Burkitt lymphoma (BL) cells were generally killed by lower doses of tubacin than EBV-transformed lymphoblastoid cells (LCLs) or EBV-negative BL cells. Tubacin induced apoptosis of LCLs, which was inhibited by pretreatment with a pancaspase inhibitor but not by butylated hydroxyanisole, which inhibits reactive oxygen species. In contrast, tubacin killed EBV-positive BL cells in a caspase-3-independent pathway that involved reactive oxygen species and was blocked by butylated hydroxyanisole. Previously, we showed that bortezomib, a proteasome inhibitor, induces apoptosis of EBV LCLs and that LCLs are killed by lower doses of bortezomib than EBV-positive BL cells. Here we found that the combination of bortezomib and tubacin acted in synergy to kill EBV-positive BL cells and LCLs. Tubacin or the combination of bortezomib and tubacin did not induce EBV lytic replication. These findings suggest that the combination of a proteasome inhibitor and an HDAC6 inhibitor may represent a useful strategy for the treatment of certain EBV-associated B cell lymphomas.

Hemiconvulsion–hemiplegia syndrome and primary human herpesvirus 7 infection

We report a case of hemiconvulsion-hemiplegia (HH) syndrome. An 18-month-old female infant had a hemiconvulsion followed by left hemiplegia. Magnetic resonance imaging immediately after the onset of hemiplegia showed high intensity in the right hemisphere in diffusion-weighted images (DWI), while T1- and T2-weighted images were normal. Single photon emission computed tomography showed hypoperfusion of the right hemisphere in the acute phase. Virological analyses proved primary human herpesvirus 7 (HHV-7) infection. DWI are useful for the early evaluation of HH syndrome. Vascular disorders due to HHV-7 infection may have been related to the development of HH syndrome in this patient.

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.

Multiplex real‐time PCR for the simultaneous detection of herpes simplex virus, human herpesvirus 6, and human herpesvirus 7

A simultaneous detection system to quantify HSV, HHV‐6, and HHV‐7 DNA via multiplex real‐time PCR using different fluorochromes was developed. The minimum quantitative level established via this multiplex assay was four copies per reaction for HSV type 1, four copies for HHV‐6, and three copies for HHV‐7, respectively. The dynamic range encompassed at least six orders of magnitude. The system was specific and reproducible even in the presence of large amounts of other viral DNA. We then applied this multiplex real‐time PCR assay to 105 CSF specimens obtained from subjects less than 15 years old in whom a diagnosis of viral encephalitis/encephalopathy was suspected on clinical grounds. The detection rate for each viral DNA was 6.7% for HSV, 9.5% for HHV‐6, and 1.9% for HHV‐7. These results indicate that our system is reliable and may be useful for the rapid diagnosis of viral encephalitis/encephalopathy.

Evaluation of Systemic Inflammatory Responses in Neonates with Herpes Simplex Virus Infection

Neonatal herpes simplex virus (HSV) infection is a severe disease with high mortality and morbidity. To investigate the pathogenesis of neonatal HSV infection, we examined inflammatory responses and markers of apoptosis in patients with neonatal HSV infection. Concentrations of inflammatory cytokines and markers of apoptosis were significantly higher in patients with disseminated HSV infection and were correlated with HSV load. It appears that the immunopathological damage that results from host responses to viral infection leads to organ dysfunction in patients with neonatal HSV infection.

Comparison of Real-Time and Nested PCR Assays for Detection of Herpes Simplex Virus DNA

We performed a real‐time PCR assay to detect herpes simplex virus (HSV) DNA, and compared it prospectively with a nested PCR assay in 164 clinical samples (109 cerebrospinal fluid and 55 sera) from patients suspected of having neonatal HSV infection or HSV encephalitis. In 25 of 164 samples, HSV DNA was detected by the nested PCR assay. All samples positive for HSV DNA in the nested PCR assay were also positive in the real‐time PCR assay, and all but two samples negative for HSV DNA in the nested assay were negative in the real‐time assay. The real‐time PCR assay thus had a sensitivity of 100% and a specificity of 99%, when compared with the nested assay. Sequential assays in a case of disseminated HSV showed that a decrease in HSV DNA paralleled clinical improvement. Quantification of HSV DNA by real‐time PCR was useful for diagnosing and monitoring patients with HSV encephalitis and neonatal HSV infection.