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As a result of the novel coronavirus disease 2019 pandemic, strengthening control measures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become an urgent global issue. In addition to antiviral therapy and vaccination strategies, applying available virucidal substances for SARS-CoV-2 inactivation is also a target of research to prevent the spread of infection. Here, we evaluated the SARS-CoV-2 inactivation activity of a copper iodide (CuI) nanoparticle dispersion, which provides Cu+ ions having high virucidal activity, and its mode of actions. In addition, the utility of CuI-doped film and fabric for SARS-CoV-2 inactivation was evaluated. The CuI dispersion exhibited time-dependent rapid virucidal activity. Analyses of the modes of action of CuI performed by Western blotting and real-time reverse transcription-PCR targeting viral proteins and the genome revealed that CuI treatment induced the destruction of these viral components. In this setting, the indirect action of CuI-derived reactive oxygen species contributed to the destruction of viral protein. Moreover, the CuI-doped film and fabric demonstrated rapid inactivation of the SARS-CoV-2 solution in which the viral titer was high. These findings indicated the utility of the CuI-doped film and fabric as anti-SARS-CoV-2 materials for the protection of high-touch environmental surfaces and surgical masks/protective clothes. Throughout this study, we demonstrated the effectiveness of CuI nanoparticles for inactivating SARS-CoV-2 and revealed a part of its virucidal mechanism of action. IMPORTANCE The COVID-19 pandemic has caused an unprecedented number of infections and deaths. As the spread of the disease is rapid and the risk of infection is severe, hand and environmental hygiene may contribute to suppressing contact transmission of SARS-CoV-2. Here, we evaluated the SARS-CoV-2 inactivation activity of CuI nanoparticles, which provide the Cu+ ion as an antiviral agent, and we provided advanced findings of the virucidal mechanisms of action of Cu+. Our results showed that the CuI dispersion, as well as CuI-doped film and fabric, rapidly inactivated SARS-CoV-2 with a high viral titer. We also demonstrated the CuI’s virucidal mechanisms of action, specifically the destruction of viral proteins and the genome by CuI treatment. Protein destruction largely depended on CuI-derived reactive oxygen species. This study provides novel information about the utility and mechanisms of action of promising virucidal material against SARS-CoV-2.

We report formation of low resistivity n-type doped silicon region at 300°C by the method of two-step ion implantation. Phosphorus ions with a dose of 2×1014 cm−2 at 70 keV followed by hydrogen ions with 1×1016 cm−2 at 8 keV were implanted at RT to the both surfaces of single crystalline silicon substrates. Post heating at 300°C for 90 min decreased the sheet resistivity to 417 Ω/sq. The activation ratio was estimated as 75% under the assumption of electron mobility of 50 cm2/Vs in the doped region. Moreover, the minority carrier effective lifetime (τ eff ) was also increased to 253 μs. This indicates a low density of surface recombination defect states of 6.4×1010 cm−2. In contrast, a high sheet resistivity of 8×104 Ω/sq and a low τ eff of 64 μs resulted from a single phosphorus ion implantation with 2×1014 cm−2 at 70 keV followed by 300°C heating for 90 min. The hydrogen ion implantation probably decreased the silicon bonding energy to easily move phosphorus atoms in the silicon lattice sites at 300°C and passivated the doped region by terminating silicon dangling bonds.

Background Monovalent copper compounds have high antiviral and antibacterial activity. Unlike divalent copper compounds, monovalent copper compounds can generate hydroxyl radical which causes oxidative damage to microorganisms. However, the antibacterial activity of monovalent copper compounds against antibiotic-resistant bacteria is not clear. The objective of this study is to evaluate the sustained antibacterial activity of alcohol disinfectant containing monovalent copper compound against antibiotic-resistant bacteria and to compare its antibacterial activity with those of conventional disinfectants. Methods Monovalent copper compound dispersed in 70% ethanol (Ethanol/monovalent copper) was applied on stainless plate by swabbing with cotton fabric. Ethanol (70%), sodium hypochlorite (5000 ppm), and benzalkonium chloride (0.2%) were also applied for comparison. After each disinfectant was dried, each bacterial suspension of antibiotic-resistant bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Acinetobacter baumannii (MDRA), was dropped onto the stainless plate sample. Viable bacteria were evaluated at 1 and 3 hours after each bacterial suspension was applied. Results As compared to the untreated stainless plate, the stainless plates treated with Ethanol/monovalent copper, ethanol, sodium hypochlorite, and benzarconium chloride showed log reductions in the average number of CFUs, 1.08, -0.10, -0.56, and 0.83 log10 at 1 hour contact, >4.01, 0.08, -0.86, and 1.63 log10 at 3 hour contact for MRSA, and >4.86, -0.16, -0.08, and 3.61 log10 at 1 hour contact, >5.14, -0.04, -0.07, and 4.28 log10 at 3 hour contact for MDRA, respectively. Conclusions This study demonstrated that ethanol/monovalent copper have sustained antibacterial activity against MRSA and MDRA even after drying. The antibacterial activity is also higher than a quaternary ammonium salt, benzalkonium chloride which is widely used in healthcare settings. These study findings suggest that monovalent copper compounds serve as a continuously active disinfectant against antibiotic-resistant bacteria in the healthcare environment.

The H+ or Ar+ precursor implantation at room temperature (RT) was proposed to activate boron atoms subsequently implanted in single crystalline silicon by post heating at a low temperature. The two-step ion implantation of 1.5×1016 cm−2 H+ at 8 keV followed by 1.0×1015 cm−2 B+ at 40 keV for both surfaces of silicon and 1.0×1014 cm−2 Ar+ at 70 keV followed by 1.0×1015 cm−2 B+ at 40 keV resulted in low sheet resistivities of 520 and 890 Ω/sq, respectively by post heating at 300°C for 30 min. Their activation ratios were estimated as 22 and 13%. The optimization of the projected ranges of 79 nm for 6×1013 cm−2 Ar+ at 70 keV and of 46 nm for B+ at 10 keV further decreased the sheet resistivity to 392 Ω/sq and increased the activation ratio to 29.5%. The activation of B+ implanted region at 300°C probably resulted from a decrease in the activation energy induced by a decrease in the silicon bonding energy promoted by the first H+ or Ar+ precursor implantation.

A plasma flood gun (PFG) has been developed for NISSIN Implanter “iG5” [1] and “iG6” for Gen5.5 and 6.0 FPD processing under high vacuum. This PFG contains a multi-cusp type arc chamber with a filament cathode. It generates the ions and electrons using a low feed Xenon (Xe) gas. In this paper, we investigated the neutralization of positive and negative charges on an insulator by the PFG under high vacuum conditions. The potential of the Al sheet on the insulator plate which was raised up to +1 or −1 kV in advance was neutralized to −0.005 kV in the 0.3 and 1.2 seconds by the PFG, respectively. We also reported the neutralization of the positive and negative charge pairs on the front and back surfaces of an insulator by the PFG.

Ion implantation of 1.5xl016-cm−2 H+ at 8 keV or 1.0xl014-cm−2 Ar+ at 70 keV was carried out at room temperature (RT) to crystalline silicon in advance of ion implantation of 1.0×10-15-cm−2B+ at RT. Decrease in the crystalline volume ratio and generation of crystalline defects were promoted by the first H+ or Ar+ implantation. Subsequently post heating at 300°C for 30 min decreased the sheet resistivity to 520 and 890 Ω/sq in the cases of the H+ and Ar+ implantation, respectively. The decreases in the sheet resistivity by 300°C post annealing is interpreted that formation of crystalline defects by the first H+ or Ar+ implantation decreased the activation energy for moving boron atoms from the interstitial to lattice sites and achieved carrier generation at the low temperature.

Activation and carrier generation are reported in the case of phosphorus implantation with a dose of 2.0 × 1015 cm−2 at 70 keV to crystalline silicon substrates under heating ranging from 200 to 500 °C. The analysis of the optical reflectivity spectra of implanted surfaces revealed that the effective amorphized thickness was low of 2.9 nm in the case of 200 °C-phosphorus implantation, while it was large of 140 nm for implantation at room temperature. The carrier density par unit area increased from 6.9 × 1013 to 4.8 × 1014 cm−2 and the photo-induced minority carrier effective lifetime increased from 2.2 × 10−6 to 1.6 × 10−4 s as the implantation temperature increased from 200 to 500 °C. Defect reduction with 1.3 MPa H2O vapor heating at 250 °C for 3 h increased the carrier density par unit area of the 200 °C-phosphorus-implanted sample to 2.7 × 1014 cm−2. The rectified characteristics were obtained by current–voltage measurement in the case of phosphorus implantation to p-type silicon substrate. Photovoltaic effect was also observed. These results show that the ion implantation under low temperature heating has a capability of p–n junction formation.

BACKGROUND Antineutrophil cytoplasmic autoantibody (ANCA) directed against myeloperoxidase (MPO), a diagnostic criterion in MPO-ANCA-associated vasculitis (MPO-AAV), does not always correlate with disease activity. Here, we detected autoantibodies against moesin, which was located on the surface of stimulated endothelial cells, in the serum of patients. METHODS The anti-moesin autoantibody titer was evaluated by ELISA. Seventeen kinds of cytokines/chemokines were measured by a Bio-Plex system. RESULTS Serum creatinine in the anti-moesin autoantibody-positive group was higher than that in the negative group. Additionally, interferon (IFN)-γ, macrophage chemotactic peptide-1 (MCP-1), interleukin (IL)-2, IL-7, IL-12p70, IL-13, granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor were significantly higher in the positive group. Furthermore, IL-7 and IL-12p70 levels correlated with the anti-moesin autoantibody titer. Based on these findings and the binding of anti-moesin IgG to neutrophils and monocytes, we detected the secretion of cytokines/chemokines such as IFN-γ, MCP-1 and GM-CSF from these cells. CONCLUSIONS The anti-moesin autoantibody existed in the serum of patients with MPO-AAV and was associated with the production of inflammatory cytokines/chemokines targeting neutrophils with a cytoplasmic profile, which suggests that the anti-moesin autoantibody has the possibility to be a novel autoantibody developing vasculitis via neutrophil and endothelial cell activation.

A plasma flood gun (PFG) has been developed for Nissin implanter "iG5 [1]" and "iG6" for Gen. 5.5 and 6.0 flat panel display (FPD) process. The PFG with a filament cathode can supply high electron flux in order to neutralize the surface charge of glass substrate irradiated by the high current ion beam [2]. We investigated the charge-up of the target glass substrates irradiated by the ion beam and the neutralization effect by the PFG using the Single Langmuir probes. The PFG effectively reduced charge-up voltage, and this neutralization effect appeared uniformly at the all part of the glass surface in the large size ribbon beam.

Because the pathogenesis of acute respiratory distress syndrome (ARDS) induced by influenza virus infection remains unknown, we can only improve on existing therapeutic interventions. To approach the subject, we investigated immunological etiology focused on cytokines and an acute lung damage factor in influenza-induced ARDS by using a PR-8 (A/H1N1)-infected mouse model. The infected mouse showed fulminant severe pneumonia with leukocyte infiltration, claudin alteration on tight junctions, and formation of hyaline membranes. In addition to interferon (IFN)-α, plenty of keratinocyte-derived chemokines (KC), macrophage inflammatory protein 2 (MIP-2), regulated on activation normal T-cell expressed and secreted (RANTES), and monocyte chemotactic protein 1 (MCP-1) were significantly released into bronchoalveolar lavage fluid (BALF) of the model. We focused on neutrophil myeloperoxidase (MPO) as a potent tissue damage factor and examined its contribution in influenza pneumonia by using mice genetically lacking in MPO. The absence of MPO reduced inflammatory damage with suppression of leakage of total BALF proteins associated with alteration of claudins in the lung. MPO(-/-) mice also suppressed viral load in the lung. The present study suggests that MPO-mediated OCl(-) generation affects claudin molecules and leads to protein leakage and viral spread as a damage factor in influenza-induced ARDS.

We investigated the antiviral activity of nanosized copper(I) iodide (CuI) particles having an average size of 160 nm. CuI particles showed aqueous stability and generated hydroxyl radicals, which were probably derived from monovalent copper (Cu + ). We confirmed that CuI particles showed antiviral activity against an influenza A virus of swine origin (pandemic [H1N1] 2009) by plaque titration assay. The virus titer decreased in a dose-dependent manner upon incubation with CuI particles, with the 50% effective concentration being approximately 17 μg/ml after exposure for 60 min. SDS-PAGE analysis confirmed the inactivation of the virus due to the degradation of viral proteins such as hemagglutinin and neuraminidase by CuI. Electron spin resonance (ESR) spectroscopy revealed that CuI generates hydroxyl radicals in aqueous solution, and radical production was found to be blocked by the radical scavenger N -acetylcysteine. Taken together, these findings indicate that CuI particles exert antiviral activity by generating hydroxyl radicals. Thus, CuI may be a useful material for protecting against viral attacks and may be suitable for applications such as filters, face masks, protective clothing, and kitchen cloths.

As is the case for tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), degranulated substances (DS) released from polymorphonuclear leukocytes (PMN) and H2O2 cause endothelial cell apoptosis through the phosphorylation of members of the mitogen-activated protein kinase (MAPK) family. Stimulation of human umbilical vein endothelial cells (HUVEC) with IL-1β or TNF-α/cycloheximide (CHX) was found to enhance the phosphorylation of p38 and Jun-N-terminal kinase (JNK) in a time-dependent fashion, but did not affect the time-dependent phosphorylation of extracellular signal-regulated kinase. In addition, IL-1β and TNF-α/CHX induced the phosphorylation of activating transcription factor-2 (ATF-2), but not c-Jun. Moreover, the p38 in HUVEC was phosphorylated by DS released from PMN and also by H2O2, but not by •O2 − induced by myeloperoxidase (MPO) of PMN. On the other hand, caspase 8 in HUVEC was activated by DS, but not by H2O2 and/or •O2 −. In addition, caspases 3 and 7 were cleaved by the treatment of DS and turned into active forms. DS was concentrated, analyzed by electrophoresis, and revealed to contain precursor and subunits of MPO (90, 60, and 14 kDa) and another peptide with a molecular weight of about 28 kDa. Because SB203580 that was an inhibitor of p38 MAPK did not repress phosphorylation of ATF-2 in HUVEC, it was suggested that JNK was more important than p38 in a series of signaling courses. These results suggest the possibility that not only TNF-α/CHX and IL-1β but also DS released from PMN and the cell-permeable reactive oxygen species H2O2 induce blood vessel injury through endothelial apoptosis.

Objectives. The MPO-specific antineutrophil cytoplasmic antibodies (MPO-ANCA) are associated with renal failure. Epitopes of MPO-ANCA and immunoglobulin G (IgG) subclass and cytokine levels in the recovery phase were analysed by the administration of 15-deoxyspergualin (DSG) to SCG/Kj mice, which show spontaneous crescentic GN (CrGN). Methods. We treated SCG/Kj mice by using DSG and MPO deletion mutants to investigate epitopes of MPO-ANCA associated with renal failure in SCG/Kj mice. After DSG treatment for 30 days, we observed histological changes in a crescentic formation and infiltration of neutrophils and lymphocytes into kidney, cytokines/chemokines and MPO-ANCA epitopes by deletion mutants. Results. MPO-ANCA were reduced by the administration of DSG, and epitopes of MPO-ANCA, mainly H-6, decreased. Moreover, the IgG2b subclass of the H-6 epitope of MPO-ANCA was greatly decreased by DSG treatment. These observations correlated with a decrease in renal failure and proteinuria, infiltration of neutrophils and lymphocytes into glomeruli, and crescent formation. The CD4/CD8 ratio of splenocytes ranged from 1.68 (0.24) in the non-treated group to 0.90 (0.12) at 100mg/mouse/day in the DSG-treated group. In addition, elevated levels of IL-12p40, IL-10 and IL-13 in the active phase of CrGN clearly decreased with DSG treatment but not with TNF-a. In contrast, the IL-1a level increased, and IL-17 and IL-12p70 slightly increased with DSG treatment. Conclusion. These results strongly suggest that DSG treatment of SCG/Kj mice leads to the reduction of risk antibodies in IgG2b and normalization of B-cell clones accompanied by recovery of the cytokine and chemokine balance.

Bronchoalveolar lavage (BAL) was performed in 5 patients with acute respiratory distress syndrome (ARDS; ARDS group hereafter) and 2 patients with respiratory failure (non-ARDS group hereafter) within 3 hours of intubation before ARDS treatment other than mechanical ventilation. ARDS was diagnosed based on the criteria defined by the American-European Consensus Conference. BAL fluid (BALF) and sera samples were obtained on the same occasion. The presence of 17 cytokines/chemokines in these specimens was simultaneously determined using a Bio-Plex® Suspension Array System. The levels of these cytokines/chemokines in BALF and sera samples were compared between the ARDS group and the non-ARDS group. The levels of chemokine granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein-1 (MCP-1) (monocyte chemoattractant factor), and macrophage inflammatory protein-1β (MIP-1β) in the BALF samples were significantly higher in the ARDS group than in the non-ARDS group. The level of pro-inflammatory cytokine interleukin (IL)-6 in BALF was also higher in the ARDS group than in the non-ARDS group. In contrast, the levels of IL-1β and IL-8 in BALF were higher in the non-ARDS group than in the ARDS group. The levels of these cytokines in BALF were higher than the corresponding sera levels in both groups. These results indicate that severe initial alveolar injury significantly activates chemokines in patients with ARDS caused by direct lung injury (such as pneumonia), unlike the increased levels of the inflammatory cytokines IL-1β and IL-8 in BALF of patients with early ARDS.

AIM Non-parenchymal liver cells (NPLC) play an important role in the regulation of immune responses and the inflammatory process. In this study, we hypothesized that F4/80(+)Mac-1(high+) cells were involved in the regulative feedback-modulated regulation of inflammatory responses during concanavalin A (Con A)-induced hepatitis. METHODS Hepatitis was induced in BALB/c mice by the intravenous injection of Con A. Liver injury was assessed using serum aminotransferase and pathology. The function of NPLC was assessed by FACS analysis. Accessory cell function of adherent Con A NPLC was performed with an ovalbumin specific T-helper 1 (Th1) clone proliferation assay. The culture supernatant nitric oxide (NO) content was quantified by the Griess reaction. Inducible NO synthase (iNOS) expression was demonstrated by immunohistochemistry and Western blot analysis. RESULTS The number of hepatic F4/80(+)Mac-1(high+)cells increased in a time-dependent manner after Con A administration, which consequently suppressed Th1 cell proliferation by a mechanism likely to involve NO. The iNOS expression of NPLC was elevated at 24 h post-Con A injection. In nude mice, F4/80(+)Mac-1(high+)cells did not increase in the Con A-treated liver; the NPLC did not suppress Th1 clone proliferation. CONCLUSION These findings suggest that the in vivo activation of F4/80(+)Mac-1(high+)cells by Con A administration suppresses Th1 cell proliferation by increasing NO, and subsequently reducing liver injury.

The elevation of serum anti-neutrophil cytoplasmic autoantibodies (ANCA) is significantly associated with the progression of some patients with systemic vasculitis. Especially, myeloperoxidase-specific ANCA (MPO-ANCA) play a pivotal role in the progression of systemic vasculitis including crescentic glomerulonephritis. Here we demonstrated that MPO-ANCA-activated neutrophils allow the local environment to differentiate Th(17) cells through IL-6, IL-17A, and IL-23 production. We found a variety of elevated serum cytokines, especially IL-17A, in ANCA-mediated systemic vasculitis mice. Furthermore, activated peritoneal neutrophils in vitro also produced IL-17A and IL-23 in response to MPO-ANCA. Co-stimulation of fungal mannoprotein and complements significantly enhanced the MPO-ANCA-mediated IL-17A expression, but F(ab)'(2) fragments of MPO-ANCA diminished the cytokine response. These results suggest that the activated neutrophils produce IL-17A and IL-23 in response to MPO-ANCA via their Fc-region and classical complement pathway, which initiate the first steps of chronic autoimmune inflammation by allowing the local environment to develop Th(17)-mediated autoimmunity.

We recently found that the inhibitory effect of (-)-epigallocatechin gallate (EGCG) on epidermal growth factor (EGF) binding to the epidermal growth factor receptor (EGFR) is associated with alterations in lipid organization in the plasma membrane of colon cancer cells. Since changes in lipid organizations are thought to play a role in the trafficking of several membrane proteins, in this study we examined the effects of EGCG on cellular localization of the EGFR in SW480 cells. Treatment of the cells for 30 min with as little as 1 microg/ml of EGCG caused a decrease in cell surface-associated EGFRs and this was associated with internalization of EGFRs into endosomal vesicles. Similar effects were seen with a green fluorescent protein (GFP)-EGFR fusion protein. As expected, the EGFR protein was phosphorylated at tyrosine residues, ubiquitinated and partially degraded when the cells were treated with EGF, but treatment with EGCG caused none of these effects. The loss of EGFRs from the cell surface induced by treating the cells with EGF for 30 min persisted for at least 2 h. However, the loss of EGFRs from the cell surface induced by temporary exposure to EGCG was partially restored within 1-2 h. These studies provide the first evidence that EGCG can induce internalization of EGFRs into endosomes, which can recycle back to the cell surface. This sequestrating of inactivated EGFRs into endosomes may explain, at least in part, the ability of EGCG to inhibit activation of the EGFR and thereby exert anticancer effects.

Fluorescent nanocrystal quantum dots (QDs) have been applied to a wide range of biological studies by taking advantage of their fluorescence properties. Here we show that QDs conjugated with antibody against neutrophil peroxidase, myeloperoxidase (MPO). We designed a novel method to conjugate QDs to antibody without losing any antibody function including their antigen recognizing and Fc-receptor binding activities. When we applied anti-MPO antibody (Ab) with conventional organic probes in the case of immunostaining of living cells, the antibodies lost their fluorescence because of MPO enzymic activity to produce reactive oxygen species. Our QD-conjugated anti-MPO (alpha-MPO-QDs) can detect MPO on the surface of activated neutrophils. In addition, anti-MPO-QDs did not react to the inactivated neutrophils. In conclusion, we demonstrated that antibody visualized the expression of MPO on the neutrophil surface after stimulation with proinflammatory cytokines. Taken together, these techniques have the possibility that QDs can reveal the activation of neutrophils by immunostaining and flow cytometric analysis as a powerful tool for diagnosis of the neutrophil activation in vitro.

Background. Anti-neutrophil cytoplasmic antibody directed against myeloperoxidase (MPO-ANCA) has been implicated in pauci-immune crescentic glomerulonephritis. It stimulates primed neutrophils to adhere to glomerular endothelial cells (GECs), thereby releasing reactive oxygen and other toxic substances and ultimately damaging the GECs. Though, a pathogenic role for MPO-ANCA is not fully understood, we hypothesized that MPO-ANCA modulates GEC functions by the increases in expression of adhesion molecules. Methods. A polyclonal rabbit anti-recombinant mouse MPO antibody (anti-rmMPO IgG) was evaluated in mouse GEC (mGEC) for its effect on adhesion molecule expression. The primary culture of mGEC was incubated with anti-rmMPO IgG or isotype control and the expression of intercellular adhesion molecules-1 (ICAM-1) was evaluated by real-time reverse transcription–polymerase chain reaction (RT–PCR) analysis and ICAM-1 cell ELISA. Results. The real-time RT–PCR analysis showed that a treatment with 100mg/ml anti-rmMPO IgG increased the expression of mRNAs for ICAM-1, vascular cell adhesion molecule-1 and E-selectin by approximately 12.5, 7.5 and 10.5-fold, respectively. ICAM-1 cell ELISA also substantiated increased expression of ICAM-1. This enhancement of ICAM-1 expression was mediated by the antigen specificity of anti-rmMPO IgG. In addition, there were several proteins in mGEC specifically immunoprecipitated with anti-rmMPO IgG. Conclusions. These results showed that anti-MPO antibody activates not only neutrophils, but also GEC, indicating that anti-rmMPO IgG-induced direct activation of GEC contributes to neutrophil adhesion to GEC, thereby increasing glomerular neutrophil infiltration in initiation and progression of pauci-immune glomerulonephritis.

Myeloperoxidase-specific anti-neutrophil cytoplasmic antibody (MPO-ANCA) is associated with rapidly progressive glomerulonephritis (RPGN) and glomerular crescent formation. Pathogenic factors in RPGN were analyzed by using SCG/Kj mice, which spontaneously develop MPO-ANCA-associated RPGN. The serum concentration of soluble IL-6R was determined by using ELISA and those of another 23 cytokines and chemokines by Bio-Plex analysis. Sections of frozen kidney tissue were examined by fluorescence microscopy and the CD3(+) B220(+) T cell subset in the spleen determined by a flow cytometry. Concentrations of IL-6 and monocyte chemotactic protein-1 were significantly correlated with the percentages of crescent formation. Anti-IL-6R antibody, which has been effective in patients with rheumatoid arthritis, was administered to SCG/Kj mice to elucidate the role of IL-6 in the development of RPGN. MPO-ANCA titers decreased after administration of anti-IL-6R antibody, but not titers of mizoribine, which is effective in Kawasaki disease model mice. These results suggest that IL-6-mediated signaling is involved in the production of MPO-ANCA.

Severe respiratory disease arising from influenza virus infection has a high fatality rate. Neutrophil myeloperoxidase (MPO) has been implicated in the pathogenesis of severe influenza-induced pneumonia because extracellularly released MPO mediates the production of hypochlorous acid, a potent tissue injury factor. To search for candidate anti-influenza compounds, we screened leucomycin A3 (LM-A3), spiramycin (SPM), an erythromycin derivative (EM900, in which anti-bacterial activity has been eliminated), and clarithromycin (CAM), by analyzing their ability to inhibit MPO release in neutrophils from mice and humans. When each candidate was injected into mice infected with a lethal dose of A/H1N1 influenza virus (PR-8), LM-A3 produced the highest survival rate (80.9%). We found that LM-A3 induced beneficial effects on lung pathology and viral proliferation involved in the regulatory activity of MPO release, pro-inflammatory cytokines and interferon-α production in the lung. SPM and EM900 also induced positive survival effects in the infected mice, whereas CAM did not. We further found that these compounds inhibit virus proliferation in human pneumonia epithelial A549 cells in vitro. LM-A3 showed effective action against influenza A virus infection with high anti-viral activity in human host cells, indicating the possibility that LM-A3 is a prospective lead compound for the development of a drug for human influenza. The positive survival effect induced by EM900 suggests that pharmacological architectures between anti-bacterial and anti-influenza virus activities can be dissociated in macrolide derivatives. These observations provide valuable evidence for the potential development of novel macrolide derivatives that have strong anti-viral but no anti-bacterial activity.

Myeloperoxidase (MPO)-specific antineutrophil cytoplasmic antibody (ANCA) is associated with rapidly progressive glomerulonephritis (RPGN) in microscopic polyangiitis (MPA). MPO-ANCA activates neutrophils by binding to cell surface MPO expressed on tumor necrosis factor-α (TNF-α)-primed neutrophils and induces neutrophil degranulation and production of reactive oxygen species, consequently resulting in glomerular endothelial damage. Recently, anti-MPO antibody has been found to activate glomerular endothelial cells, leading to an upregulation of adhesion molecules. MPO-ANCA, however, is not always correlated with disease activity in MPA. Accordingly, the molecule(s) responsible for the anti-MPO antibody have been explored on mouse glomerular endothelial cells. The molecule was identified as moesin, which is a heparin-binding protein and belongs to the ezrin/radixin/moesin family of proteins distributed in the plasma membrane in the cellular cortex. Interestingly, anti-moesin is observed in sera of SCG/Kj mice, which spontaneously develop MPO-ANCA-associated RPGN, and of patients with MPO-AAV. The activation of glomerular endothelial cells by the anti-MPO antibody appears to be associated with signaling through moesin.

Anti-neutrophil cytoplasmic autoantibodies (ANCA) are useful diagnostic markers in systemic vasculitic disorders with small-vessel involvement, but depending on the particular test used, the myeloperoxidase (MPO)-ANCA results are variable. In the present study, we performed a comparative analysis between our originally developed nMPO-ANCA assay that targets the native MPO antigen and other commercially available assays using sera of patients with clinical features of ANCA-associated vasculitis (AAV).Sera of 24 patients strongly suspected of having AAV were examined for the presence of MPO-ANCAs by our nMPO-ANCA assay and by other commercial-based MPO-ANCA assays. These results were correlated to indirect immunofluorescence microscopy staining patterns and patient clinical parameters.Eighteen out of 24 patients (75 %) were positive for nMPO-ANCA, compared with 13 out of 24 patients (54 %) by one of the most frequently used commercial-based MPO-ANCA enzyme-linked immunosorbent assays (ELISAs) in Japan. Interestingly, the patients who tested positive with our nMPO-ANCA assay alone showed clinical features of AAV marked by continuous fever, polyarthritis, and mild nephritis. The titers of nMPO-ANCA decreased in association with clinical improvement after treatment.Our data suggest that a positive nMPO-ANCA result, which identifies antibodies to human native MPO antigen, correlates with AAV disease activity. Moreover, the nMPO-ANCA test has clinical utility in detecting AAV-affected patients who have tested negative using commercially available assays.

BackgroundIntravenous immunoglobulin (IVIg) treatment results in an effective response from patients with acute-phase Kawasaki disease (KD), but 16.5% of them remain nonresponsive to IVIg. To address this therapeutic challenge, we tried a new therapeutic drug, mizoribine (MZR), in a mouse model of KD, which we have established using injections ofCandida albicanswater-soluble fractions (CAWS).MethodsCAWS (4 mg/mouse) were injected intraperitoneally into C57BL/6N mice for 5 consecutive days. MZR or IgG was administered for 5 days. After 4 weeks, the mice were sacrificed and autopsied, the hearts were fixed in 10% neutral formalin, and plasma was taken to measure cytokines and chemokines using the Bio-Plex system.The incidence of panvasculitis in the coronary arteries and aortic root was 100% in the control group. The incidence of panvasculitis in the MZR group decreased to 50%. Moreover, the scope and severity of the inflammation of those sites were significantly reduced in the MZR group as well as the IgG group. On the other hand, increased cytokines and chemokines, such as IL-1α, TNF-α, KC, MIP-1α, GM-CSF, and IL-13, in the nontreatment group were significantly suppressed by treatment with MZR, but the MCP-1 level increased. In addition, IL-1α, TNF-α, IL-10, IL-13, and MIP-1α were suppressed by treatment in the IgG group.ResultsThe incidence of panvasculitis in the coronary arteries and aortic root was 100% in the control group. The incidence of panvasculitis in the MZR group decreased to 50%. Moreover, the scope and severity of the inflammation of those sites were significantly reduced in the MZR group as well as the IgG group. On the other hand, increased cytokines and chemokines, such as IL-1α TNF-α, KC, MIP-1α, GM-CSF, and IL-13, in the nontreatment group were significantly suppressed by treatment with MZR, but the MCP-1 level increased. In addition, IL-1α, TNF-α, IL-10, IL-13, and MIP-1α were suppressed by treatment in the IgG group.ConclusionMZR treatment suppressed not only the incidence, range, and degree of vasculitis, but also inflammatory cytokines and chemokines in the plasma of the KD vasculitis model mice, suggesting that MZR may be useful for treatment of KD.

Seven new macrolactins (named G~M) and known macrolactins A and F were isolated from a culture broth of Bacillus sp. PP19-H3. The strain had been isolated from the macroalga, Schizymenia dubyi. Macrolactin A, which was 24-membered lactone, had previously been reported to show antibacterial, cytotoxic and antiviral activities. The new macrolactins include 22-membered ring or dicyclic lactone in addition to geometric isomers of known macrolactins A and F. The antibacterial activities of all the macrolactins examined in this study were relatively weak.

Phylogenetic and taxonomic characterization was performed for 14 strains of bacteria that produce anticancer antibiotics (pelagiomicins) (represented by strain Ni-2088(T)) and one strain that produces UV-absorbing substances (strain F-104(T)), isolated from marine algae and seagrass collected from coastal areas of tropical Pacific islands and a subtropical island of Japan. All 15 isolates were Gram-negative, strictly aerobic, non-motile and non-spore-forming. Sequence analysis of the 16S rRNA gene showed that the isolates occupied positions in the phylogenetic radiation of the genus Microbulbifer, with similarities of 93.6-97.6 %. The cells possessed a clearly discernible rod-coccus cell cycle in association with the growth phase; cells were rods during the growth phase and all converted to coccoid-ovoid cells when proliferation ceased. The coccoid-ovoid cells were optically denser than the rod cells and were viable for extended periods. They were considered to constitute a resting form. The type strains of described species of Microbulbifer were also found to possess identical rod-coccus cell cycles. The G+C content of the DNA was 48.1-49.7 mol%. The major respiratory quinone system was ubiquinone-8. The major fatty acids were C(18 : 1)omega7c and C(16 : 0), and the hydroxy acids comprised C(10 : 0) 3-OH, C(12 : 0) 3-OH and iso-C(11 : 0) 3-OH. The polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine. The group of 14 pelagiomicin-producing strains and strain F-104(T) each constituted a single genomic species. Based on phylogenetic affiliation, phenotypic characteristics and genomic distinctness, the isolates represent two novel species in the genus Microbulbifer, for which the names Microbulbifer variabilis sp. nov. (type strain Ni-2088(T) =MBIC01082(T) =ATCC 700307(T)) and Microbulbifer epialgicus sp. nov. (type strain F-104(T) =MBIC03330(T) =DSM 18651(T)) are proposed.

The elevation of serum anti-neutrophil cytoplasmic autoantibodies (ANCA) is significantly associated with the progression of some patients with systemic vasculitis. Especially, myeloperoxidase-specific ANCA (MPO-ANCA) play a pivotal role in the progression of systemic vasculitis including crescentic glomerulonephritis. Here we demonstrated that MPO-ANCA-activated neutrophils allow the local environment to differentiate Th(17) cells through IL-6, IL-17A, and IL-23 production. We found a variety of elevated serum cytokines, especially IL-17A, in ANCA-mediated systemic vasculitis mice. Furthermore, activated peritoneal neutrophils in vitro also produced IL-17A and IL-23 in response to MPO-ANCA. Co-stimulation of fungal mannoprotein and complements significantly enhanced the MPO-ANCA-mediated IL-17A expression, but F(ab)'(2) fragments of MPO-ANCA diminished the cytokine response. These results suggest that the activated neutrophils produce IL-17A and IL-23 in response to MPO-ANCA via their Fc-region and classical complement pathway, which initiate the first steps of chronic autoimmune inflammation by allowing the local environment to develop Th(17)-mediated autoimmunity.

(−)-Epigallocatechin gallate (EGCG), a major biologically active constituent of green tea, inhibits activation of the epidermal growth factor (EGF) receptor (EGFR) and downstream signaling pathways in several types of human cancer cells, but the precise mechanism is not known. Because several plasma membrane-associated receptor tyrosine kinases (RTK) including EGFR are localized in detergent-insoluble ordered membrane domains, so-called “lipid rafts,” we examined whether the inhibitory effect of EGCG on activation of the EGFR is associated with changes in membrane lipid order in HT29 colon cancer cells. First, we did cold Triton X-100 solubility assays. Phosphorylated (activated) EGFR was found only in the Triton X-100–insoluble (lipid raft) fraction, whereas total cellular EGFR was present in the Triton X-100–soluble fraction. Pretreatment with EGCG inhibited the binding of Alexa Fluor 488–labeled EGF to the cells and also inhibited EGF-induced dimerization of the EGFR. To examine possible effects of EGCG on membrane lipid organization, we labeled the cells with the fluorescent lipid analogue 1, 1′-dihexadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, which preferentially incorporates into ordered membrane domains in cells and found that subsequent treatment with EGCG caused a marked reduction in the Triton X-100–resistant membrane fraction. Polyphenon E, a mixture of green tea catechins, had a similar effect but (−)-epicatechin (EC), the biologically inactive compound, did not significantly alter the Triton X-100 solubility properties of the membrane. Furthermore, we found that EGCG but not EC caused dramatic changes in the function of bilayer-incorporated gramicidin channels. Taken together, these findings suggest that EGCG inhibits the binding of EGF to the EGFR and the subsequent dimerization and activation of the EGFR by altering membrane organization. These effects may also explain the ability of EGCG to inhibit activation of other membrane-associated RTKs, and they may play a critical role in the anticancer effects of this and related compounds. [Cancer Res 2007;67(13):6493–501]

In systemic vasculitis, the serum level of myeloperoxidase (MPO)-specific anti-neutrophil cytoplasmic autoantibodies (MPO-ANCA) is significantly elevated with the progression of disease. We have established a model of murine systemic vasculitis by administration of MPO-ANCA and fungal mannoprotein to C57BL/6 mice. We examined the role of MPO and MPO-ANCA in the pathogenesis of glomerulonephritis and systemic vasculitis in this model using quantum dots (QDs). We demonstrated that QD-conjugated MPO-ANCA (ANCA-QD) visualized the translocation of MPO on the neutrophil membrane surface after stimulation with proinflammatory cytokines. We also observed that MPO translocation on neutrophils in both patients with rapid progressive glomerulonephritis and these model mice without any stimulation, suggesting that MPO translocation is certain to contribute to the development of glomerular lesion. In addition, blood flow on the kidney surface vessel was significantly decelerated in both SCG/Kj mice and this model, suggesting that ANCA induces the damage of blood vessel. These results indicate that MPO-ANCA and surface-translocated MPO on the activated neutrophils coordinately plays essential roles in the initial steps of the glomerulonephritis.

Objective— Atherogenesis begins as small subendothelial accumulations of foam cells that develop through unregulated uptake of modified and aggregated low-density lipoprotein (LDL). The reason why foam cells remain in the atherosclerotic plaque rather than migrating out of the area is unclear. We tested the hypothesis that elevated membrane cholesterol levels, which may result from interactions with aggregated LDL, affect macrophage migration. Methods and Results— Cholesterol loading by incubation with cholesterol-chelated methyl-β-cyclodextrin decreased migration of J774A.1 macrophages toward complement 5a (C5a) in transwell migration assays, even though cholesterol-loaded macrophages responded to a bath application of C5a. In a micropipette polarization assay, cholesterol-loaded cells polarized toward a C5a gradient. In a transwell migration assay, cholesterol-loaded cells extended lamellae through the filter pores but were unable to translocate their cell bodies. Cholesterol loading decreased both the cellular levels of GTP-bound active RhoA and the phosphorylation of myosin light chain. Expression of constitutively active RhoA largely prevented the inhibition of cell migration by cholesterol loading. Conclusions— These results suggest that increases in plasma membrane cholesterol content alter RhoA activation, resulting in inhibition of cell migration. These findings provide one possible explanation for the retention of foam cells in atherosclerotic lesions.

It is assumed that activated neutrophils contribute to the development of anti-neutrophil cytoplasmic auto-antibody (ANCA)-associated vasculitis due to the association of myelopeoxidase(MPO)-ANCA with MPO expressed on the surface of activated neutrophils. FITC-labeled antibody (Ab) used widely are not suitable for neutrophil examination because of the labile fluorescence emission of FITC. Therefore, it is necessary to develop specific fluorescent probes for MPO detection in neutrophils in vivo. Recently, fluorescent nanocrystal quantum dots (QDs) have been used for biotechnological and medical applications because of their greater and far longer fluorescence in. QDs have several advantages over organic fluorophores: high luminescence, far longer stability against photobleaching, and a range of fluorescence wavelengths from blue to infrared, depending on particle size. Thus, we examined the role of MPO and the Ab to MPO in the pathogenesis of glomerulonephritis associated with MPO-ANCA in experimental glomerulonephritis mice using QDs. We demonstrated the QD-conjugated anti-MPO Ab visualized the expression of MPO on the neutrophil surface after stimulation with proinflammatory cytokines. In addition, QD immuno-conjugates with anti-recombinant murine MPO (rmMPO) Ab revealed the trafficking of MPO-ANCA in vivo. Deceleration of blood flow in kidney vessels occurred in model mice, in which serum proteins including anti-rmMPO Ab were leaked out from collapsed glomeruli into the proximal tubule. Thus, sustained MPO expression on the neutrophil surface was significantly related to glomerulonephritis. These results indicate that the expressed MPO on the activated neutrophils with anti-MPO Ab may coordinately play essential roles in the initial steps for the development of glomerulonephritis.

Objective—During atherogenesis, macrophages migrate into the subendothelial space where they ingest deposited lipoproteins, accumulate lipids, and transform into foam cells. It is unclear why these macrophages do not remove their lipid loads from the region. This study was aimed at testing the hypothesis that macrophage behavior is altered when membrane cholesterol levels are elevated, as might be the case for cells in contact with lipoproteins within atherosclerotic lesions.Methods and Results—We examined the effects of elevating membrane cholesterol on macrophage behavior. J774 macrophages were treated with either acetylated low-density lipoprotein (ac-LDL) and ACAT inhibitor or cholesterol-chelated methyl-β-cyclodextrin (chol-MβCD) to increase membrane cholesterol levels. Our results show that elevating the membrane cholesterol of J774 macrophages induced dramatic ruffling, stimulated cell spreading, and affected F-actin organization. Cellular adhesion was required for these effects, and Rac-mediated signaling pathways were involved. Additionally, 3-dimensional transwell chemotaxis assays showed that migration of J774 macrophages was significantly inhibited when membrane cholesterol levels were raised.Conclusions—These findings indicate that increased membrane cholesterol causes dramatic effects on macrophage cellular functions related to the actin cytoskeleton. They should provide new insights into the early steps of atherogenesis.

CD69, known as an early activation marker antigen on T and B cells, is also expressed on platelets and activated neutrophils, suggesting certain roles in inflammatory diseases. In order to address the role of CD69 in the pathogenesis of arthritis, we established CD69-null mice. CD69-null mice displayed a markedly attenuated arthritic inflammatory response when injected with anti-type II collagen antibodies. Cell transfer experiments with neutrophils, but not T cells or spleen cells, from wild-type mice into CD69-null mice restored the induction of arthritis. These results indicate a critical role for CD69 in neutrophil function in arthritis induction during the effector phase. Thus, CD69 would be a possible therapeutic target for arthritis in human patients.

This study attempts to investigate the mechanism of the vascular shut down (VSD) effect during photodynamic therapy (PDT) with zinc coproporphyrin III tetrasodium salt as a photosensitizer. PDT is a treatment based on photochemical reactions and the resultant cytotoxic reactive oxygen species (ROS). Platelet thrombus formation leading to stasis observed in vivo during PDT is called the VSD effect. Leukocytes play an important role in the VSD effect in vivo, but the mechanism how activated monocytes generate ROS is not known in detail. To evaluate ROS generation by activated monocytes is especially important to clarify leukocyte-endothelium interactions in the VSD mechanism. The dichlorofluorescein fluorescence intensity of monocytes with four types of free radical scavenger was investigated by confocal laser scanning microscopy. The fluorescence intensity of monocytes that had been incubated with superoxide dismutase and incubated and added with L-histidine was decreased by about 20 and 30%, respectively. The result affirms the predominant role of singlet oxygen and superoxide anion radicals in monocyte activation in the VSD effect during PDT.

Seven new macrolactins (named G~M) and known macrolactins A and F were isolated from a culture broth of Bacillus sp. PP19-H3. The strain had been isolated from the macroalga, Schizymenia dubyi. Macrolactin A, which was 24-membered lactone, had previously been reported to show antibacterial, cytotoxic and antiviral activities. The new macrolactins include 22-membered ring or dicyclic lactone in addition to geometric isomers of known macrolactins A and F. The antibacterial activities of all the macrolactins examined in this study were relatively weak.

not Available.

A high minority carrier effective lifetime τeff of crystalline silicon was achieved by hydrogenated amorphous silicon (a-Si:H) films formed by a combination of plasma-enhanced chemical vapor deposition at 150 °C with high-pressure H2O vapor heat treatment. τeff was 1.6×10-4, 3.0×10-4, and 1.15×10-3 s for n-type silicon substrates coated with 3-, 10-, and 50-nm-thick a-Si:H films treated with 1.0×106 Pa H2O vapor heat treatment between 180 and 300 °C for 1 h. Light-induced passivation enhancement was demonstrated when 620-nm light was illuminated at the 50-nm-thick a-Si:H surface. τeff increased from 8.5×10-4 to 1.15×10-3 s probably caused by field effect passivation induced by hole trapping at the SiO x formed by H2O vapor heat treatment for 1 h. On the other hand, τeff was further increased to 1.2×10-3 s by 1.0×106 Pa H2O vapor heat treatment at 300 °C for 3 h for the sample formed with the 50-nm-thick a-Si:H film. However, no increase in τeff was observed by light illumination at the a-Si:H surface, probably because the SiO x clusters became stable and had no hole trapping property.

We report photoinduced carrier recombination induced by 13.56 MHz radio frequency argon plasma treatment for samples of silicon substrates coated with thermally grown SiO2 layers. The transmissivity of a 9.35 GHz microwave was measured and analyzed under illumination of 532 nm green light to the top or rear surface. Irradiation of argon plasma at 100 W for 5 min caused substantial carrier recombination. The recombination velocity of the plasma-irradiated surface was estimated to be 6000 cm/s. Investigation of 100 kHz capacitance response characteristics as a function of bias voltage for metal oxide p-type silicon structures revealed that irradiation of argon plasma increased the fixed charge density from 3.2×1011 to 3.0×1012 cm-2. Those changes were restored completely by heat treatment at 300 °C in air for 60 min.

We propose a measurement system for photoinduced minority carrier absorption of 9.35 GHz microwaves using periodically pulsed light illumination at 620 nm. The ratio of average carrier density when light illumination is ON to that when light illumination is OFF, P, was theoretically analyzed for different light pulse widths. The analysis of P resulted in a formula giving the minority carrier lifetime τeff of silicon under continuous light illumination. τeff for holes was experimentally determined using the formula, and its spatial distribution was obtained to be from 1.0 ×10-3 to 1.28 ×10-3 s for n-type silicon substrates with a thickness of 520 µm coated with 100-nm-thick thermally grown SiO2 layers. We also demonstrated that τeff depended on the means of light illumination for a defective sample. Two different τeff values were obtained, 7 ×10-5 and 1.73 ×10-4 s, in the cases of light illumination to the top surface and rear surface, respectively, when the SiO2 layer was etched up to 2 nm at the top surface.