Hiroyuki Tsutsumi

Detection of enteric viruses in rectal swabs from children with acute gastroenteritis attending the pediatric outpatient clinics in Sapporo, Japan

BACKGROUND Gastroenteritis is a world-wide disorder. Numerous studies to identify causative viral agents have been reported for hospitalized patients but there are only a few for outpatients with mild symptoms who are usually managed in the outpatient clinics. OBJECTIVES Our aim was to clarify the epidemiological and clinical characteristics of acute gastroenteritis in children who visited the outpatient clinics with various complaints suggestive of gastroenteritis. STUDY DESIGN From December 2003 to December 2005, 877 rectal swabs were collected from patients attending outpatient clinics in Sapporo, Japan. Viral genomes of major five enteric viruses (rotavirus, norovirus, adenovirus, astrovirus and sapovirus) and bocavirus were investigated by RT-PCR or PCR. RESULTS At least one viral agent was found in 326 (37.2%) cases of the 877 studied. Rotaviruses were the most prevalent and were detected in 143 (16.3%) followed by norovirus in 116 (13.2%), adenovirus in 42 (4.8%), astrovirus in 40 (4.6%) and sapovirus in 15 (1.7%) cases. Bocavirus was detected in only 4 (0.5%) cases. Frequent diarrhea and frequent vomiting were prominent in rotavirus and norovirus infection, respectively. CONCLUSIONS The prevalence of each enteric virus in outpatients resembled that previously estimated in hospitalized patients, although the detection rate of rotavirus was slightly low. The contribution of bocavirus appears to be small.

Regulation of Tight Junctions in Upper Airway Epithelium

The mucosal barrier of the upper respiratory tract including the nasal cavity, which is the first site of exposure to inhaled antigens, plays an important role in host defense in terms of innate immunity and is regulated in large part by tight junctions of epithelial cells. Tight junction molecules are expressed in both M cells and dendritic cells as well as epithelial cells of upper airway. Various antigens are sampled, transported, and released to lymphocytes through the cells in nasal mucosa while they maintain the integrity of the barrier. Expression of tight junction molecules and the barrier function in normal human nasal epithelial cells (HNECs) are affected by various stimuli including growth factor, TLR ligand, and cytokine. In addition, epithelial-derived thymic stromal lymphopoietin (TSLP), which is a master switch for allergic inflammatory diseases including allergic rhinitis, enhances the barrier function together with an increase of tight junction molecules in HNECs. Furthermore, respiratory syncytial virus infection in HNECs in vitro induces expression of tight junction molecules and the barrier function together with proinflammatory cytokine release. This paper summarizes the recent progress in our understanding of the regulation of tight junctions in the upper airway epithelium under normal, allergic, and RSV-infected conditions.

Respiratory syncytial virus-induced cytokine production by neonatal macrophages

The induction of immunoregulatory cytokines IL‐1β, IL‐6, IL‐12, tumour necrosis factor‐alpha (TNF‐α) and interferon‐gamma (IFN‐γ) was studied with neonatal (cord blood) monocyte‐derived macrophages (MDM) after in vitro infection with respiratory syncytial virus (RSV). The expression of mRNAs for these cytokines in RSV‐infected MDM was examined by reverse transcriptase‐polymerase chain reaction (RT‐PCR). The activities of these cytokines were assayed by ELISA. Significant increase of expression of mRNA for IL‐6, IL‐12, TNF‐α and IFN‐γ occurred within 2 h after infection and decreased within 6 h after infection. At 20 h after infection the MDM produced and secreted moderate levels of IL‐6 and TNF‐α; however, no IL‐12 and IFN‐γ activities were detected. Moderate IL‐1β mRNA was expressed before RSV infection, and its expression increased at 2 h after infection. However, no detectable IL‐1β was secreted in culture fluids. These observations suggest that RSV‐infected neonatal macrophages produce and secrete IL‐6 and TNF‐α quickly during the eclipse phase of RSV infection and therefore may play a prominent role in the initiation of the immune response to RSV.

Curcumin Prevents Replication of Respiratory Syncytial Virus and the Epithelial Responses to It in Human Nasal Epithelial Cells

The human nasal epithelium is the first line of defense during respiratory virus infection. Respiratory syncytial virus (RSV) is the major cause of bronchitis, asthma and severe lower respiratory tract disease in infants and young children. We previously reported in human nasal epithelial cells (HNECs), the replication and budding of RSV and the epithelial responses, including release of proinflammatory cytokines and enhancement of the tight junctions, are in part regulated via an NF-κB pathway. In this study, we investigated the effects of the NF-κB in HNECs infected with RSV. Curcumin prevented the replication and budding of RSV and the epithelial responses to it without cytotoxicity. Furthermore, the upregulation of the epithelial barrier function caused by infection with RSV was enhanced by curcumin. Curcumin also has wide pharmacokinetic effects as an inhibitor of NF-κB, eIF-2α dephosphorylation, proteasome and COX2. RSV-infected HNECs were treated with the eIF-2α dephosphorylation blocker salubrinal and the proteasome inhibitor MG132, and inhibitors of COX1 and COX2. Treatment with salubrinal, MG132 and COX2 inhibitor, like curcumin, prevented the replication of RSV and the epithelial responses, and treatment with salubrinal and MG132 enhanced the upregulation of tight junction molecules induced by infection with RSV. These results suggest that curcumin can prevent the replication of RSV and the epithelial responses to it without cytotoxicity and may act as therapy for severe lower respiratory tract disease in infants and young children caused by RSV infection.

The middle ear mucosal immune system in otitis media with effusion.

The mucous membrane of the middle ear cavity in otitis media with effusion has a number of immunobiological mechanisms capable of defending the organ. A humoral immune mechanism, with all the attributes of a local mucosal immune system, appears to be present and capable of preventing viral and bacterial access to middle ear tissue. Putative cells of a cell-mediated immune response also appear to be present, and the distribution of T cells and B cells and T-helper and T-suppressor cells is described. The exact role of these cells in either cell-mediated immunity or delayed hypersensitivity remains to be defined in otitis media. Lymphocyte--macrophage interaction is briefly described and may represent an important aspect of immune modulation in the middle ear in otitis media with effusion. Finally, the effect of middle ear supernatants on natural killer cell activity is discussed. Serous effusions appear to augment natural killer cell activity of peripheral blood lymphocytes.

Clinical characteristics and computed tomography findings in children with 2009 pandemic influenza A (H1N1) viral pneumonia

Abstract In this article we review the clinical characteristics and computed tomography (CT) findings in children with 2009 pandemic H1N1 influenza viral pneumonia. The medical charts of 88 children with pandemic H1N1 influenza virus infection, admitted to our hospital in Japan from 10 August to 28 December 2009, were reviewed; we compared the clinical features of these children with those of 61 children admitted with seasonal influenza A during the previous 3 seasons. Of 88 patients, 53 (60%) had radiographic findings consistent with pneumonia and 34 patients underwent a chest computed tomography (CT) scan. Pneumonia was a more frequent complication in children with pandemic H1N1 influenza compared with those with seasonal influenza (60% vs 11%; p < 0.001). The predominant CT findings were unilateral or bilateral multifocal consolidation (15/34; 44%) associated with ground-glass opacities in the peribronchovascular region. The second most common CT finding was unilateral diffuse consolidation or atelectasis in 1 or more lung zones (12/34; 35%). The chest CT findings of unilateral or bilateral multifocal consolidation often associated with ground-glass opacities were commonly seen in children with pandemic H1N1 influenza viral pneumonia. Atelectasis was seen in patients who required oxygen administration.

Marked induction of matrix metalloproteinase‐10 by respiratory syncytial virus infection in human nasal epithelial cells

Respiratory syncytial virus (RSV) is an important pathogen of bronchiolitis, asthma, and severe lower respiratory tract disease in infants and young children. Matrix metalloproteinases (MMPs) play key roles in viral infection, inflammation and remodeling of the airway. However, the roles and regulation of MMPs in human nasal epithelial cells (HNECs) after RSV infection remain unclear. To investigate the regulation of MMP induced after RSV infection in HNECs, an RSV‐infected model of HNECs in vitro was used. It was found that mRNA of MMP‐10 was markedly increased in HNECs after RSV infection, together with induction of mRNAs of MMP‐1, ‐7, ‐9, and ‐19. The amount of MMP‐10 released from HNECs was also increased in a time‐dependent manner after RSV infection as was that of chemokine RANTES. The upregulation of MMP‐10 in HNECs after RSV infection was prevented by inhibitors of NF‐κB and pan‐PKC with inhibition of RSV replication, whereas it was prevented by inhibitors of JAK/STAT, MAPK, and EGF receptors without inhibition of RSV replication. In lung tissue of an infant with severe RSV infection in which a few RSV antibody‐positive macrophages were observed, MMP‐10 was expressed at the apical side of the bronchial epithelial cells and alveolar epithelial cells. In conclusion, MMP‐10 induced by RSV infection in HNECs is regulated via distinct signal transduction pathways with or without relation to RSV replication. MMP‐10 may play an important role in the pathogenesis of RSV diseases and it has the potential to be a novel marker and therapeutic target for RSV infection. J. Med. Virol. 85:2141–2150, 2013.

Analysis of Nucleotide Sequences of Human Parvovirus B19 Genome Reveals Two Different Modes of Evolution, a Gradual Alteration and a Sudden Replacement: a Retrospective Study in Sapporo, Japan, from 1980 to 2008

ABSTRACT There have been no long-term systematic analyses of the molecular epidemiology of human parvovirus B19 (B19V). We investigated the variations of nucleotide sequences of B19V strains collected in Sapporo, Japan, from 1980 to 2008. In that period, six outbreaks of erythema infectiosum occurred regularly at 5-year intervals. The B19V strains collected successively, regardless of the outbreak, were analyzed for nucleotide variation in the subgenomic NS1-VP1u junction. The isolated strains can be classified into 10 subgroups. Two patterns of change of endemic strains were observed. One was a dynamic replacement of strains that occurred almost every 10 years, and the other was a gradual change consisting of an accumulation of point mutations.

Phylogenetic and computational structural analysis of VP7 gene of group a human rotavirus G1P[8] strains obtained in Sapporo, Japan from 1987 to 2000

Many studies indicate that G1P[8] genotypes are the most prevalent rotavirus strains worldwide. Although two vaccines have been licensed and their value proven in many countries, continuous surveillance for genetic evolution of circulating rotavirus strains before and after the introduction of the vaccines is desirable. G and P typing were carried out on all field strains isolated during 1987–2000 in Sapporo, Japan. Phylogenetic analysis for the VP7 gene of rotavirus G1P[8] strains was performed. Amino acid substitutions were mapped on the predicted three‐dimensional VP7 protein image. G1P[8] genotype predominated. One hundred thirteen strains with G1P[8] genotype were analyzed. Phylogenetic studies of the VP7 gene classified these strains into three lineages. The mean estimated substitution rate was 7.25 × 10−4 nucleotide substitutions per site per year. One predominant lineage contained the mutant strains which had VP7 amino acid substitutions at residue 91 and 212 that is in the neutralization domains. They were estimated to locate in or near intersubunit boundary of VP7 trimer. It is suggested that the most prevalent G1P[8] lineage strains in Sapporo obtained some survival advantages by changing the neutralization domains of VP7. J. Med. Virol. 84:832–838, 2012.

Fosfomycin Suppresses Chemokine Induction in Airway Epithelial Cells Infected with Respiratory Syncytial Virus

ABSTRACT Respiratory syncytial virus (RSV) infects airway epithelial cells, causing bronchiolitis and pneumonia. Inflammation is mediated by various cytokines secreted from RSV-infected airway epithelial cells, and it promotes the pathogenesis of RSV-related diseases. Fosfomycin (FOF) is approved as a treatment for various bacterial infectious diseases, including respiratory infectious diseases, in Japan. FOF is suggested to exhibit immunomodulatory effects on lipopolysaccharide-stimulated monocytes and T lymphocytes, in addition to its antimicrobial activity. We investigated the effect of FOF on the cytokine production of an airway epithelial cell line, A549, infected with RSV. RSV-induced cytokines, such as regulated on activation, normal T-cell expressed and secreted (RANTES), interleukin-8 (IL-8), and IL-6, in infected A549 cells. We found that FOF decreased the levels of RSV-induced RANTES and IL-8 but not the level of RSV-induced IL-6. The RANTES promoter was activated by RSV infection. Site-directed mutagenesis analysis of the RANTES promoter showed that NF-κB-binding motifs had a critical role in RSV-induced RANTES promoter activity. A luciferase reporter gene assay and a DNA-binding assay indicated that FOF suppressed the NF-κB activity induced by RSV infection. These results demonstrate that FOF treatment suppresses the RSV-induced transcription of the chemokines RANTES and IL-8 in airway epithelial cells.