Kazumichi Kuroda

Peroxiredoxin family proteins are key initiators of post-ischemic inflammation in the brain

Post-ischemic inflammation is an essential step in the progression of brain ischemia-reperfusion injury. However, the mechanism that activates infiltrating macrophages in the ischemic brain remains to be clarified. Here we demonstrate that peroxiredoxin (Prx) family proteins released extracellularly from necrotic brain cells induce expression of inflammatory cytokines including interleukin-23 in macrophages through activation of Toll-like receptor 2 (TLR2) and TLR4, thereby promoting neural cell death, even though intracellular Prxs have been shown to be neuroprotective. The extracellular release of Prxs in the ischemic core occurred 12 h after stroke onset, and neutralization of extracellular Prxs with antibodies suppressed inflammatory cytokine expression and infarct volume growth. In contrast, high mobility group box 1 (HMGB1), a well-known damage-associated molecular pattern molecule, was released before Prx and had a limited role in post-ischemic macrophage activation. We thus propose that extracellular Prxs are previously unknown danger signals in the ischemic brain and that its blocking agents are potent neuroprotective tools.

Persistent infection of hepatitis E virus transmitted by blood transfusion in a patient with T-cell lymphoma.

Aim:  With advent of reverse‐transcription (RT)/polymerase chain reaction (PCR) for detection of the hepatitis E viral genome, we carried out retrospective examinations.

Expression of Mas-related gene X2 on mast cells is upregulated in the skin of patients with severe chronic urticaria.

BACKGROUND Wheal reactions to intradermally injected neuropeptides, such as substance P (SP) and vasoactive intestinal peptide, are significantly larger and longer lasting in patients with chronic urticaria (CU) than in nonatopic control (NC) subjects. Mas-related gene X2 (MrgX2) has been identified as a receptor for basic neuropeptides, such as SP and vasoactive intestinal peptide. Mast cell (MC) responsiveness to eosinophil mediators contributes to the late-phase reaction of allergy. OBJECTIVE We sought to compare the frequency of MrgX2 expression in skin MCs from patients with CU and NC subjects and to identify the receptor for basic eosinophil granule proteins on human skin MCs. METHODS MrgX2 expression was investigated by using immunofluorescence in skin tissues from NC subjects and patients with severe CU and on skin-derived cultured MCs. MrgX2 expression in human MCs was reduced by using a lentiviral small hairpin RNA silencing technique. Ca(2+) influx was measured in CHO cells transfected with MrgX2 in response to eosinophil granule proteins. Histamine and prostaglandin D2 levels were measured by using enzyme immunoassays. RESULTS The number of MrgX2(+) skin MCs and the percentage of MrgX2(+) MCs in all MCs in patients with CU were significantly greater than those in NC subjects. Eosinophil infiltration in urticarial lesions was observed in 7 of 9 patients with CU. SP, major basic protein, and eosinophil peroxidase, but not eosinophil-derived neurotoxin, induced histamine release from human skin MCs through MrgX2. CONCLUSION MrgX2 might be a new target molecule for the treatment of wheal reactions in patients with severe CU.

Unmutated Immunoglobulin M Can Protect Mice from Death by Influenza Virus Infection

To elucidate the role of class switch recombination (CSR) and somatic hypermutation (SHM) in virus infection, we have investigated the influence of the primary and secondary infections of influenza virus on mice deficient of activation-induced cytidine deaminase (AID), which is absolutely required for CSR and SHM. In the primary infection, AID deficiency caused no significant difference in mortality but did cause difference in morbidity. In the secondary infection with a lethal dose of influenza virus, both AID−/− and AID+/− mice survived completely. However, AID−/− mice could not completely block replication of the virus and their body weights decreased severely whereas AID+/− mice showed almost complete prevention from the reinfection. Depletion of CD8+ T cells by administration of an anti-CD8 monoclonal antibody caused slightly severer body weight loss but did not alter the survival rate of AID−/− mice in secondary infection. These results indicate that unmutated immunoglobulin (Ig)M alone is capable of protecting mice from death upon primary and secondary infections. Because the titers of virus-neutralizing antibodies were comparable between AID−/− and AID+/− mice at the time of the secondary infection, a defect of AID−/− mice in protection of morbidity might be due to the absence of either other Ig classes such as IgG, high affinity antibodies with SHM, or both.

Anti-influenza virus activity of Chaenomeles sinensis.

AIM OF THE STUDY This investigation evaluated anti-influenza virus activity of 50% ethanol extract of the fruit of Chaenomeles sinensis K(OEHNE), which is widely used as a traditional Chinese medicine to treat throat diseases. MATERIAL AND METHODS Type A and B influenza viruses were treated with the extract at various concentrations for 1h at room temperature; then the plaque titers of the treated viruses were determined. The neutralizing component in the extract was partially purified using HP20 column chromatography. RESULTS Treatment with the extract at concentrations greater than 5mg/ml reduced the plaque titers of the both viruses to less than 10% of those of untreated viruses. The treatment inhibited viral hemagglutination activity, too. When the 50mg/ml extract was added to the culture medium after inoculation of the virus, viral NS2 protein synthesis was selectively inhibited and progeny virus was not detected in the infected cell medium. Partial purification showed that the neutralizing component consisted of high molecular weight polyphenols. CONCLUSION High molecular weight polyphenols in the fruits of C. sinensis neutralizes influenza virus by inhibiting hemagglutination activity and by suppressing NS2 protein synthesis.

Dual Wavelength Imaging Allows Analysis of Membrane Fusion of Influenza Virus inside Cells

ABSTRACT Influenza virus hemagglutinin (HA) is a determinant of virus infectivity. Therefore, it is important to determine whether HA of a new influenza virus, which can potentially cause pandemics, is functional against human cells. The novel imaging technique reported here allows rapid analysis of HA function by visualizing viral fusion inside cells. This imaging was designed to detect fusion changing the spectrum of the fluorescence-labeled virus. Using this imaging, we detected the fusion between a virus and a very small endosome that could not be detected previously, indicating that the imaging allows highly sensitive detection of viral fusion.

Bacterial Neuraminidase Rescues Influenza Virus Replication from Inhibition by a Neuraminidase Inhibitor

Influenza virus neuraminidase (NA) cleaves terminal sialic acid residues on oligosaccharide chains that are receptors for virus binding, thus playing an important role in the release of virions from infected cells to promote the spread of cell-to-cell infection. In addition, NA plays a role at the initial stage of viral infection in the respiratory tract by degrading hemagglutination inhibitors in body fluid which competitively inhibit receptor binding of the virus. Current first line anti-influenza drugs are viral NA-specific inhibitors, which do not inhibit bacterial neuraminidases. Since neuraminidase producing bacteria have been isolated from oral and upper respiratory commensal bacterial flora, we posited that bacterial neuraminidases could decrease the antiviral effectiveness of NA inhibitor drugs in respiratory organs when viral NA is inhibited. Using in vitro models of infection, we aimed to clarify the effects of bacterial neuraminidases on influenza virus infection in the presence of the NA inhibitor drug zanamivir. We found that zanamivir reduced progeny virus yield to less than 2% of that in its absence, however the yield was restored almost entirely by the exogenous addition of bacterial neuraminidase from Streptococcus pneumoniae. Furthermore, cell-to-cell infection was severely inhibited by zanamivir but restored by the addition of bacterial neuraminidase. Next we examined the effects of bacterial neuraminidase on hemagglutination inhibition and infectivity neutralization activities of human saliva in the presence of zanamivir. We found that the drug enhanced both inhibitory activities of saliva, while the addition of bacterial neuraminidase diminished this enhancement. Altogether, our results showed that bacterial neuraminidases functioned as the predominant NA when viral NA was inhibited to promote the spread of infection and to inactivate the neutralization activity of saliva. We propose that neuraminidase from bacterial flora in patients may reduce the efficacy of NA inhibitor drugs during influenza virus infection. (295 words).

Localization of influenza virus proteins to nuclear dot 10 structures in influenza virus-infected cells

We studied influenza virus M1 protein by generating HeLa and MDCK cell lines that express M1 genetically fused to green fluorescent protein (GFP). GFP-M1 was incorporated into virions produced by influenza virus infected MDCK cells expressing the fusion protein indicating that the fusion protein is at least partially functional. Following infection of either HeLa or MDCK cells with influenza A virus (but not influenza B virus), GFP-M1 redistributes from its cytosolic/nuclear location and accumulates in nuclear dots. Immunofluorescence revealed that the nuclear dots represent nuclear dot 10 (ND10) structures. The colocalization of authentic M1, as well as NS1 and NS2 protein, with ND10 was confirmed by immunofluorescence following in situ isolation of ND10. These findings demonstrate a previously unappreciated involvement of influenza virus with ND10, a structure involved in cellular responses to immune cytokines as well as the replication of a rapidly increasing list of viruses.

Immunofluorescence imaging of the influenza virus M1 protein is dependent on the fixation method

The distribution of the matrix (M1) protein of influenza virus in infected cells was examined using immunostaining. The fixation method influenced strongly the immunofluorescence pattern of the M1 protein. The M1 protein was distributed uniformly in both the cytoplasm and in nuclei when cells that had been infected with virus were fixed with paraformaldehyde. In cells that had been fixed with methanol, however, nuclear dots of the M1 protein were clearly visible. The dots were evident at 8h post-inoculation. Up to 6h post-inoculation, only a diffuse distribution of the M1 protein was observed. The dots were co-localized with promyelocytic leukemia (PML) protein, a major component of nuclear domain 10 (ND10), also called PML oncogenic domains (PODs) or PML-nuclear bodies (NBs). These results indicate that the nuclear dots of the M1 protein in cells that had been fixed with methanol are not artifacts of the fixation method. Furthermore, methanol fixation is preferred for localization of the influenza M1 protein in nuclei using immunostaining.

Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity

Background: It has been shown that heat shock protein 70 (Hsp70) plays a role in influenza A virus replication. Results: A correlation between viral replication/transcription activities and nuclear/cytoplasmic shuttling of Hsp70 was observed. Conclusion: Hsp70 modulates the influenza A virus polymerase activity. Significance: This study, for the first time, suggests that Hsp70 may actually assist in influenza A virus replication. The role of heat shock protein 70 (Hsp70) in virus replication has been discussed for many viruses. The known suppressive role of Hsp70 in influenza virus replication is based on studies conducted in cells with various Hsp70 expression levels. In this study, we determined the role of Hsp70 in influenza virus replication in HeLa and HEK293T cells, which express Hsp70 constitutively. Co-immunoprecipitation and immunofluorescence studies revealed that Hsp70 interacted with PB2 or PB1 monomers and PB2/PB1 heterodimer but not with the PB1/PA heterodimer or PB2/PB1/PA heterotrimer and translocated into the nucleus with PB2 monomers or PB2/PB1 heterodimers. Knocking down Hsp70 resulted in reduced virus transcription and replication activities. Reporter gene assay, immunofluorescence assay, and Western blot analysis of nuclear and cytoplasmic fractions from infected cells demonstrated that the increase in viral polymerase activity during the heat shock phase was accompanied with an increase in Hsp70 and viral polymerases levels in the nuclei, where influenza virus replication takes place, whereas a reduction in viral polymerase activity was accompanied with an increase in cytoplasmic relocation of Hsp70 along with viral polymerases. Moreover, significantly higher levels of viral genomic RNA (vRNA) were observed during the heat shock phase than during the recovery phase. Overall, for the first time, these findings suggest that Hsp70 may act as a chaperone for influenza virus polymerase, and the modulatory effect of Hsp70 appears to be a sequel of shuttling of Hsp70 between nuclear and cytoplasmic compartments.