Shigeru Suga

Detection of adenovirus DNA in clinical samples by SYBR Green real-time polymerase chain reaction assay

Abstract Background : Adenoviruses are associated with a variety of diseases including upper respiratory tract infections, acute conjunctivitis, cystitis and gastroenteritis. Adenoviruses can also cause fatal disseminated infections in patients undergoing stem cell transplantation. Measurement of adenovirus load in clinical samples from localized adenovirus infections or disseminated adenovirus infections may provide important information for analyzing the pathogenesis of various adenovirus infections. The purpose of the present study was to develop and optimize a highly sensitive real‐time polymerase chain reaction (PCR) assay to detect a wide range of adenoviruses and to detect adenovirus DNA in clinical samples from immunocompetent children.

Binding of the V proteins to the nucleocapsid proteins of human parainfluenza type 2 virus

Abstract Interaction of the nucleocapsid (NP) and V proteins of human parainfluenza type 2 virus (HPIV-2) was investigated using a transient expression system. When the NP proteins were co-expressed with the V proteins, some of the NP proteins were translocated into the nuclei. These findings suggest that the NP protein interact with the V proteins. We examined the interaction of the NP proteins and the P, V proteins or deletion mutants of V protein using immunofluorescence and co-immunoprecipitation plus Western blotting analyses, and showed that the V proteins of HPIV-2 bind to the NP proteins and that the N-terminal domain of V protein interacts directly with the NP proteins. When the NP proteins were co-expressed with the V proteins or the N-terminal fragments (aa 1–46), the NP proteins were detected diffusely in the nuclei of the transfected cells, and were also detected in cytoplasmic inclusions. The NP and V proteins were co-localized in the nuclei or cytoplasm. Futhermore, the NP proteins were co-precipitated with the P, V, and V(1–164) proteins by a specific antibody. The P proteins interact more closely with the NP proteins than do the V proteins. These findings indicate that the V proteins have the ability to bind the NP proteins.

Serotypes, antimicrobial susceptibility, and molecular epidemiology of invasive and non-invasive Streptococcus pneumoniae isolates in paediatric patients after the introduction of 13-valent conjugate vaccine in a nationwide surveillance study conducted in Japan in 2012–2014

Pneumococcal infection in children is a major public health problem worldwide, including in Japan. The pneumococcal conjugate vaccine 7 (PCV7) was licensed for use in Japan in 2010 followed by PCV13 in 2013. This report includes the results of a nationwide surveillance of invasive pneumococcal disease (IPD) and non-IPD in paediatric patients from January 2012 to December 2014. We collected 343 isolates from 337 IPD patients and 286 isolates from 278 non-IPD patients. Of the IPD isolates, the most identified serotypes included 19A, 24F, and 15A. The prevalence of non-PCV13 serotype isolates increased significantly from 2012 to 2014 (51.6-71.4%, p=0.004). Serotypes 19A, 15A and 35B were highly non-susceptible to penicillin, and the rates of non-susceptible isolates from IPD patients to penicillin and cefotaxime significantly declined during the study period (p=0.029 and p=0.013, respectively). The non-susceptible rate to meropenem increased, particularly for serotype 15A. The IPD isolates comprised clonal complex (CC) 3111 (93.8% was serotype 19A) followed by CC2572 (81.5% was serotype 24F) and CC63 (97.1% was serotype 15A). CC3111, CC63 and CC156 (33.3% was serotype 23A, 28.6% was serotype 6B, and 14.3% was serotype 19A) were highly non-susceptible to penicillin. Of the non-IPD isolates, the most identified serotypes included 19A, 15A, and 3. In conclusion, the introduction of PCV7 and PCV13 resulted in increasing non-PCV13 serotypes and clones, including antimicrobial resistant serotypes 15A and CC63 (Sweden(15A)-25 clone).

Nationwide population-based surveillance of invasive pneumococcal disease in Japanese children: Effects of the seven-valent pneumococcal conjugate vaccine.

BACKGROUND In Japan, the seven-valent pneumococcal conjugate vaccine (PCV7) was introduced in 2010. PCV13 has replaced PCV7 since November 2013. METHODS The effectiveness of PCV7 in protecting against invasive pneumococcal disease (IPD) in children aged <5 years was evaluated in a nationwide active population-based surveillance of IPD in 2008-2013 in 10 prefectures in Japan. RESULTS 1181 cases were identified; 711 pneumococcal strains were analyzed for serotyping and antimicrobial resistance. Compared with the baseline IPD incidence (25.0 per 100,000), a 98% decline in IPD caused by PCV7 serotypes was found after the introduction of PCV7. This was partially offset by an increased incidence of IPD caused by PCV13 minus PCV7 and non-PCV13 serotypes, resulting in a 57% decline in overall IPD incidence. Absolute increases in the incidence rates of IPD caused by PCV13 minus PCV7 and non-PCV13 serotypes were 2.1 and 2.8 per 100,000 during the study period, respectively. The proportion of meropenem-nonsusceptible strains, especially with serotypes 19A and 15A, increased significantly after PCV7 introduction. CONCLUSIONS Our data confirmed a 98% decline in IPD incidence caused by PCV7 serotypes in children aged <5 years and serotype replacement after PCV7 introduction. This shows the importance of continuing surveillance of serotypes responsible for IPD and their antimicrobial resistance in Japan.

Regulation of human immunodeficiency virus gp160-mediated cell fusion by antibodies against fusion regulatory protein 1.

We have isolated new MAbs directed against the human fusion regulatory protein 1 (FRP-1; CD98) molecule using human FRP-1-expressing L929 cells as antigens. The biological activities, and in particular the human immunodeficiency virus (HIV)-mediated fusion regulatory activity of seven anti-FRP-1/CD98 MAbs were analysed using the U937/gp160 cell line, which is a CD4+ U937 cell line expressing HIV gp160. Two MAbs induced multinucleated giant cell formation in U937/gp160 cells and the other five MAbs showed no fusion-inducing ability. However, four of these MAbs suppressed multinucleated giant cell formation of U937/gp160 cells induced by the activating anti-FRP-1 MAbs. Interestingly, five of the MAbs induced multinucleated giant cells in peripheral blood monocytes and one MAb showing fusion-inducing ability in U937/gp160 cells suppressed multinucleated giant cell formation of monocytes induced by anti-FRP-1 MAbs. Furthermore, four of the anti-FRP-1 MAbs suppressed cell fusion of Jurkat/gp160 cells, which are Jurkat cells expressing HIV gp160. Thus, FRP-1/CD98 is capable of either activating or inhibiting HIV-mediated cell fusion depending on whether an enhancing or inhibiting antibody is used, indicating that FRP-1/CD98 is a multipotential molecule. Thus, HIV-mediated cell fusion can be regulated by modification of the FRP-1 system. Furthermore, the present study demonstrates that the FRP-1 and FRP-2 systems are interdependent.

Protein tyrosine kinase activation provides an early and obligatory signal in anti-FRP-1/CD98/4F2 monoclonal antibody induced cell fusion mediated by HIV gp160.

Abstract The mechanism by which anti-fusion regulatory protein-1 (FRP-1) monoclonal antibody (mAb) induced cell fusion was investigated using U2ME-7 cells that are CD4+U937 cells transfected with the HIV gp160 gene. Protein kinase inhibitors (H-7, H-89, herbimycin A and genistein) suppressed cell fusion of Cd+U2ME-7 cells induced by anti-FRP-1 mAb. H-7 and H-89 also inhibited the cell aggregation, but herbimycin A and genistein did not. Intriguingly, only when herbimycin A was added either before or simultaneously with addition of anti-FRP-1 mAb, was cell fusion suppressed, suggesting that tyrosine kinase is related with the initial step of polykaryocyte formation. Anti-FRP-1 mAb induced the rapid tyrosine phosphorylation of multiple cellular proteins. These effects occurred within 1 min and returned to near baseline by 60 min. The rapid tyrosine phosphorylation was suppressed by herbimycin A and genistein. Although it remains to be determined which protein tyrosine kinase(s) is involved in this response, pp130 tyrosine phosphorylation appears to be a specific and early signal transmitted after the interaction of FRP-1 with a specific antibody. pp130 was present in the cytosol fraction and was distinct from pp125FAK, p130CAS, vinculin, and β1-integrin. Thus, our study may present evidence for a novel pathway of protein tyrosine kinases that phosphorylate specific, still unknown protein substrates during polykaryocyte formation.

Staphylococcus aureus directly activates eosinophils via platelet‐activating factor receptor

Colonization by SA is associated with exacerbation of AD. Eosinophilic inflammation is a cardinal pathological feature of AD, but little is known about possible direct interaction between SA and eosinophils. PAFR appears to be involved in phagocytosis of Gram‐positive bacteria by leukocytes. The objective of this study was to investigate whether SA directly induces eosinophil effector functions via PAFR in the context of AD pathogenesis. Peripheral blood eosinophils were cultured with heat‐killed SA, and EDN release, superoxide generation, and adhesion to fibronectin‐coated plates were measured. Cytokines, released in the supernatants, were quantified by multiplex bead immunoassays. FISH‐labeled SA was incubated with eosinophils and visualized by confocal laser‐scanning microscopy. PAFR‐blocking peptide and PAFR antagonists were tested for inhibitory effects on SA‐induced reactions. SA induced EDN release and superoxide generation by eosinophils in a dose‐dependent manner. IL‐5 significantly enhanced SA‐induced EDN release. IL‐5 and IL‐17A significantly enhanced SA‐induced superoxide generation. SA enhanced eosinophil adhesion to fibronectin, which was blocked by anti‐CD49d, and induced eosinophil secretion of various cytokines/chemokines (IL‐2R, IL‐9, TNFR, IL‐1β, IL‐17A, IP‐10, TNF‐α, PDGF‐bb, VEGF, and FGF‐basic). After incubation of eosinophils with SA, FISH‐labeled SA was visualized in the eosinophilsˈ cytoplasm, indicating phagocytosis. A PAFR‐blocking peptide and two PAFR antagonists completely inhibited those reactions. In conclusion, SA directly induced eosinophil activation via PAFR. Blockade of PAFR may be a novel, therapeutic approach for AD colonized by SA.

Moyamoya disease associated with bilateral renal artery stenosis

Recent research has suggested that an association exists between moyamoya disease and fibromuscular dysplasia which involves systemic vessels, including renal arteries. We report a 3 year old girl with moyamoya disease associated with bilateral renal artery stenosis. This case may support the common etiology of these two clinical conditions. To our knowledge this is the youngest case of moyamoya disease associated with bilateral renal artery stenosis.

Apoptosis of human peripheral blood mononuclear cells by wild-type measles virus infection is induced by interaction of hemagglutinin protein and cellular receptor, SLAM via caspase-dependent pathway.

Although MV infection causes lymphopenia and degradation of cell‐mediated immunity, the mechanisms are poorly known. MV interacts with cellular receptors which mediate virus binding and uptake and are on the surface of PBMC. In this study, apoptosis of MV‐infected PBMC in vitro was analyzed. Both PBMC treated with UV‐inactivated viruses and those infected with live MV underwent apoptosis. Apoptosis of wild‐type MV‐infected PBMC was blocked by anti‐SLAM and anti‐MV hemagglutinin antibodies, respectively. Furthermore, addition of soluble MV hemagglutinin recombinant protein induced apoptosis in PBMC. These data suggest that induction of apoptosis in MV‐infected PBMC is triggered by interaction between hemagglutinin protein of MV and receptor, without other viral components. To further determine the mechanisms of apoptosis, caspase activity was analyzed by Western blotting. Wild‐type virus Yonekawa strain‐induced apoptosis was blocked by pretreatment with pan‐caspase inhibitor (Z‐VAD‐fmk). Intriguingly, the laboratory‐adapted Nagahata strain‐induced apoptosis was not blocked by Z‐VAD‐fmk, indicating that there may be different apoptosis pathways which depend on the viral receptors, SLAM and CD46. Both extrinsic and intrinsic apoptotic pathways, including activation of caspase‐3, ‐8 and ‐9, are involved in Yonekawa strain‐induced apoptosis. Taken together, the findings of this study could open up a new avenue for understanding the molecular mechanisms of MV‐induced PBMC apoptosis and immunosuppression.

Human immunodeficiency virus type-1 envelope glycoprotein gp120 induces expression of fusion regulatory protein (FRP)-1/CD98 on CD4+ T cells: a possible regulatory mechanism of HIV-induced syncytium formation

Abstract Syncytium formation is one of major cytopathic effects of human immunodeficiency virus (HIV) infection, and requires the interaction of CD4 molecules on uninfected cells with HIV envelope glyoprotein gp120 expressed on HIV-infected cells. Recent evidence suggests chemokine receptors function as fusion cofactors. We have recently found that fusion regulatory protein (FRP)-1/ CD98 is involved in syncytium formation of HIV gp160-expressing U2ME-7 cells and TALL-1 cells persistently infected with HIV. However, resting lymphocytes were found to express no FRP-1 molecule. In this study, we demonstrated that recombinant gp120 (rpg120) has the ability to induce expression of FRP-1 on peripheral blood mononuclear cells (PBMC). Three-color flow cytometric analysis showed that rgp120-induced FRP-1 was expressed selectively on CD4+ T cells in a dose-dependent manner. FRP-1 expression level was maximum 3 days after addition of rgp120. Anti-CD4 and anti-gp120 antibodies blocked rgp120-induced FRP-1 expression. Co-cultivation of PBMC with HIV-1 gp160-expressing HeLa cells also resulted in the increased expression of FRP-1 on T cells. These results suggest that FRP-1 molecules are induced on CD4+ T cells via CD4-gp120 interaction and may play an important role in regulation of HIV-induced syncytium formation.