Yuka Torii

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.

Increased levels of cytokines and high-mobility group box 1 are associated with the development of severe pneumonia, but not acute encephalopathy, in 2009 H1N1 influenza-infected children

BACKGROUND The 2009 A(H1N1) influenza virus has caused a large outbreak, and resulted in major complications of severe pneumonia and acute encephalopathy in the pediatric population in Japan. METHODS This study examined six patients with acute encephalopathy, 34 patients with severe pneumonia, five patients with both pneumonia and encephalopathy, and 46 patients without severe complications. The concentrations of 27 cytokines were examined in the cerebrospinal fluid of patients with encephalopathy, and the levels of these cytokines, Cytochrome c, high-mobility group box 1 (HMGB1) were measured in the serum of all patients. RESULTS Patients with severe pneumonia had higher serum concentrations of 16 cytokines, including Th1 cytokines, Th2 cytokines, chemokines, and growth factors, than patients with uncomplicated influenza. The distribution of 27 cytokines in the CSF did not parallel the serum levels in 11 patients with acute encephalopathy. HMGB1 concentrations in the serum were significantly higher in pneumonia patients with or without encephalopathy than in uncomplicated influenza patients, and were significantly associated with the upregulation of 10 cytokines. CONCLUSIONS Elevated levels of Th2 cytokines appear to be unique to influenza caused by 2009 H1N1 influenza virus and HMGB1 could play an important role in the pathogenesis of severe pneumonia. There appear to be different pathologic processes for encephalopathy and pneumonia.

Serum concentrations of complement anaphylatoxins and proinflammatory mediators in patients with 2009 H1N1 influenza

Anaphylatoxins (C5a, C4a, and C3a) are fragments of activated complement and are leading mediators of the inflammatory response for controlling viral infection. However, an excessive response may increase the severity of infectious diseases. Serum concentrations of proinflammatory mediators, including cytokines, high‐mobility group box 1 and anaphylatoxins, were measured in pediatric 2009 H1N1 influenza patients in order to investigate the pathology of this new influenza. The concentrations of all three anaphylatoxins were significantly enhanced by 2009 H1N1 influenza infection. However, there were no significant differences in anaphylatoxin concentrations between 2009 H1N1 influenza patients with and without severe complications during the early stages of the disease. C3a concentrations dropped significantly during the recovery phase, whereas there were no significant differences between the acute and recovery phases in C5a and C4a concentrations. There was a correlation between C5a and IL‐2. C4a was associated with IL‐1ra, eotaxin, MCP‐1, PDGFbb, and VEGF. C3a was correlated with IL‐2 and IFN‐γ. Taken together, these findings indicate that complement activation occurs in patients infected with 2009 H1N1 influenza virus and demonstrate that anaphylatoxins are involved in increased production of proinflammatory mediators in this new influenza.

Identification of Viruses in Cases of Pediatric Acute Encephalitis and Encephalopathy Using Next-Generation Sequencing

Acute encephalitis/encephalopathy is a severe neurological syndrome that is occasionally associated with viral infection. Comprehensive virus detection assays are desirable because viral pathogens have not been identified in many cases. We evaluated the utility of next-generation sequencing (NGS) for detecting viruses in clinical samples of encephalitis/encephalopathy patients. We first determined the sensitivity and quantitative performance of NGS by comparing the NGS-determined number of sequences of human herpesvirus-6 (HHV-6) in clinical serum samples with the HHV-6 load measured using real-time PCR. HHV-6 was measured as it occasionally causes neurologic disorders in children. The sensitivity of NGS for detection of HHV-6 sequences was equivalent to that of real-time PCR, and the number of HHV-6 reads was significantly correlated with HHV-6 load. Next, we investigated the ability of NGS to detect viral sequences in 18 pediatric patients with acute encephalitis/encephalopathy of unknown etiology. A large number of Coxsackievirus A9 and mumps viral sequences were detected in the cerebrospinal fluid of 2 and 1 patients, respectively. In addition, Torque teno virus and Pepper mild mottle viral sequences were detected in the sera of one patient each. These data indicate that NGS is useful for detection of causative viruses in patients with pediatric encephalitis/encephalopathy.

Immunogenicity of inactivated 2009 H1N1 influenza vaccine in pediatric liver transplant recipients

To assess the safety and immunogenicity of 2009 H1N1 influenza vaccination, 13 pediatric liver transplant recipients and 31 immunocompetent controls received inactivated influenza vaccine without adjuvant according to Japanese guidelines. Serious adverse events and acute allograft rejections were not observed in participants. Seroprotection rates (hemagglutinin-inhibition (HI) antibody titer ≥ 1:40) were 53.8% among recipients and 58.1% among controls (p=0.797). Seroconversion rates (4-fold or more HI antibody rise) were 46.2% for the recipient group and 51.6% for the control group (p=0.741). Geometric mean titers were elevated after vaccination in both groups. In comparison with the seasonal influenza vaccination, the seroconversion rate for 2009 H1N1 appeared to be higher than that for seasonal influenza antigens, and the seroprotection rate for 2009 H1N1 clearly increased after vaccination. These findings suggest that pediatric liver transplant patients may respond safely to inactivated 2009 H1N1 influenza vaccines in a manner similar to immunocompetent children.

Clinicoepidemiologic status of mother-to-child infections: a nationwide survey in Japan.

We conducted a nationwide survey of the present status of 10 representative mother-to-child infections in Japan. Congenital syphilis, vertical human T-cell leukemia virus type 1 infection, congenital rubella and vertical HIV infection, for which effective preventative strategies have been established, were rare. Cytomegalovirus was the most common congenital pathogen in Japan, although most infants with congenital cytomegalovirus infection may remain undiagnosed.

Serum microRNAs as Potential Biomarkers of Juvenile Idiopathic Arthritis

MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression of targeted mRNAs, which are important in the pathogenesis of autoimmune diseases. MiRNAs may have the potential to serve as biomarkers of disease. We evaluated serum levels of selected miRNAs and their associations with disease activity in juvenile idiopathic arthritis (JIA). Sera and peripheral blood leukocytes were collected from patients with JIA (8 systemic onset, 16 polyarthritis) and healthy controls. Levels of miR-16, miR-132, miR-146a, miR-155, and miR-223 were quantified. Levels of miR-223 in sera were significantly higher in patients in the active phase of systemic onset JIA than in controls. MiRNAs of peripheral blood leukocytes did not exhibit any difference between patients with JIA and controls. In both systemic onset JIA and polyarthritis patients, levels of miR-223 and miR-16 correlated with erythrocyte sedimentation rate and matrix metalloproteinase-3, respectively. MiR-146a and miR-223 in polyarthritis showed correlations with matrix metalloproteinase-3. Expressions of miRNAs were altered in patients with JIA. Serum levels of miR-223 may be a potential disease biomarker. Investigation of miRNAs could be helpful in understanding the pathogenesis of JIA and could aid in the identification of additional disease biomarkers.

Successful treatment with rituximab of refractory idiopathic thrombocytopenic purpura in a patient with Kabuki syndrome

Kabuki syndrome (KS) is often associated with autoimmune abnormalities, such as idiopathic thrombocytopenic purpura (ITP), autoimmune hemolytic anemia, leukoplakia and thyroiditis, as well as congenital anomalies. We herein present a KS patient with refractory ITP who achieved durable and complete remission in response to a total of four once-monthly infusions of rituximab. KS patients are often more susceptible to infection, so splenectomy should be avoided. Therefore, rituximab therapy is an alternative option for KS patients with ITP who fail to respond to first-line therapy.

Epstein-Barr virus infection-induced inflammasome activation in human monocytes

Inflammasomes are cytoplasmic sensors that regulate the activity of caspase-1 and the secretion of interleukin-1β (IL-1β) or interleukin-18 (IL-18) in response to foreign molecules, including viral pathogens. They are considered to be an important link between the innate and adaptive immune responses. However, the mechanism by which inflammasome activation occurs during primary Epstein-Barr virus (EBV) infection remains unknown. Human B lymphocytes and epithelial cells are major targets of EBV, although it can also infect a variety of other cell types. In this study, we found that EBV could infect primary human monocytes and the monocyte cell line, THP-1, inducing inflammasome activation. We incubated cell-free EBV with THP-1 cells or primary human monocytes, then confirmed EBV infection using confocal microscopy and flow cytometry. Lytic and latent EBV genes were detected by real-time RT-PCR in EBV-infected monocytes. EBV infection of THP-1 cells and primary human monocytes induced caspase-dependent IL-1β production, while EBV infection of B-cell or T-cell lines did not induce IL-1β production. To identify the sensor molecule responsible for inflammasome activation during EBV infection, we examined the mRNA and the protein levels of NLR family pyrin domain-containing 3 (NLRP3), absent in melanoma 2 (AIM2), and interferon-inducible protein 16 (IFI16). Increased AIM2 levels were observed in EBV-infected THP-1 cells and primary human monocytes, whereas levels of IFI16 and NLRP3 did not show remarkable change. Furthermore, knockdown of AIM2 by small interfering RNA attenuated caspase-1 activation. Taken together, our results suggest that EBV infection of human monocytes induces caspase-1-dependent IL-1β production, and that AIM2, acting as an inflammasome, is involved in this response.

Viral load in children with congenital cytomegalovirus infection identified on newborn hearing screening

BACKGROUND Congenital cytomegalovirus (CMV) infection is the most common non-genetic cause of sensorineural hearing loss (SNHL) in children. However, congenital SNHL without other clinical abnormalities is rarely diagnosed as CMV-related in early infancy. OBJECTIVES The aim of this study was to identify and treat patients with congenital CMV-related SNHL or CMV-related clinical abnormalities other than SNHL. The association between CMV load and SNHL was also evaluated. STUDY DESIGN Newborns who had abnormal hearing screening results or other clinical abnormalities were screened for congenital CMV infection by PCR of saliva or urine specimens, and identified infected patients were treated with valganciclovir (VGCV) for 6 weeks. The CMV load of patients with or without SNHL was compared at regular intervals during as well as after VGCV treatment. RESULTS Of 127 infants with abnormal hearing screening results, and 31 infants with other clinical abnormalities, CMV infection was identified in 6 and 3 infants, respectively. After VGCV treatment, 1 case had improved hearing but the other 5 SNHL cases had little or no improvement. Among these 9 patients with or without SNHL at 1 year of age, there was no significant difference in CMV blood or urine load at diagnosis, but both were significantly higher in patients with SNHL during VGCV treatment. CONCLUSIONS Selective CMV screening of newborns having an abnormal hearing screening result would be a reasonable strategy for identification of symptomatic congenital CMV infection. Prolonged detection of CMV in blood could be a risk factor for SNHL.