Application of Copper Iodide Nanoparticle-Doped Film and Fabric To Inactivate SARS-CoV-2 via the Virucidal Activity of Cuprous Ions (Cu+).

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.

Leucomycin A3, a 16-membered macrolide antibiotic, inhibits influenza A virus infection and disease progression.

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.

A novel autoantibody against moesin in the serum of patients with MPO-ANCA-associated vasculitis.

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.

Effect of invariant natural killer T cells with IL-5 and activated IL-6 receptor in ventilator-associated lung injury in mice.

Mechanical ventilation (MV) is well known to potentially cause ventilator-associated lung injury (VALI). It has also been reported recently that activation of invariant natural killer T (iNKT) cells is involved in the onset/progression of airway inflammation. We analyzed the roles of inflammatory cells, including iNKT cells, and cytokines/chemokines in a mouse model of VALI. C57BL/6 and Vα14(+)NKT cell-deficient (Jα18KO) female mice were subjected to MV for 5 hours. The MV induced lung injury in the mice, with severe histological abnormalities, elevation in the percentages of neutrophils in the bronchoalveolar lavage fluid (BALF), and increase in the number of iNKT cells in the lung. Jα18KO mice subjected to MV for 5 hours also showed lung injury, with decrease of the PaO2/FiO2 ratio (P/F ratio) and elevation of the levels of total protein, IL-5, IL-6, IL-12p40, and keratinocyte-derived cytokine (KC) in the BALF. Intranasal administration of anti-IL-5 monoclonal antibody (mAb) or anti-IL-6 receptor (IL-6R) mAb into the Jα18KO mice prior to the start of MV resulted in significant improvement in the blood oxygenation. In addition, the anti-IL-5 mAb administration was associated with a decrease in the levels of IL-5, IL-9, and IL-6R in the BALF, and anti-IL-6R mAb administration suppressed the mRNA expressions of IL-5, IL-6, IL-6R, and KC. These results suggest that iNKT cells may play a role in attenuating the inflammatory caused by ventilation through IL-5 and IL-6R.

Proposal of anti-moesin as a novel biomarker for ANCA-associated vasculitis.

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.

Correlation of interleukin-6 and monocyte chemotactic protein-1 concentrations with crescent formation and myeloperoxidase-specific anti-neutrophil cytoplasmic antibody titer in SCG/Kj mice by treatment with anti-interleukin-6 receptor antibody or mizoribine.

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.

Clinical features of patients with anti-neutrophil cytoplasmic autoantibodies targeting native myeloperoxidase antigen.

OBJECTIVES: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Contribution of neutrophil-derived myeloperoxidase in the early phase of fulminant acute respiratory distress syndrome induced by influenza virus infection.

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.

Apoptotic signaling in endothelial cells with neutrophil activation.

As is the case for tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), degranulated substances (DS) released from polymorphonuclear leukocytes (PMN) and H(2)O(2) 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-1ß 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-1ß 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 H(2)O(2), but not by •O(2) (-) induced by myeloperoxidase (MPO) of PMN. On the other hand, caspase 8 in HUVEC was activated by DS, but not by H(2)O(2) and/or •O(2) (-). 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-1ß but also DS released from PMN and the cell-permeable reactive oxygen species H(2)O(2) induce blood vessel injury through endothelial apoptosis.

Novel antiviral characteristics of nanosized copper(I) iodide particles showing inactivation activity against 2009 pandemic H1N1 influenza virus.

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.

Mizoribine provides effective treatment of sequential histological change of arteritis and reduction of inflammatory cytokines and chemokines in an animal model of Kawasaki disease.

UNLABELLED: 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. BACKGROUND: Intravenous 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 of Candida albicans water-soluble fractions (CAWS). METHODS: CAWS (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. RESULTS: 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. CONCLUSION: MZR 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.

Direct activation of glomerular endothelial cells by anti-moesin activity of anti-myeloperoxidase antibody.

BACKGROUND: Glomerular neutrophil infiltration has been thought to be a key pathological event in the development of myeloperoxidase (MPO)-specific anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis involving glomerulonephritis. Accordingly, we sought to explore the molecules responsible for glomerular neutrophil accumulation. METHODS: Glomerular neutrophil infiltration and renal chemokine expression in mice treated with anti-MPO IgG were evaluated. Chemokine expression in vitro induced by anti-MPO IgG was measured in the primary mouse glomerular endothelial cells (mGEC). The target molecule reacted with anti-MPO IgG on the mGEC was determined by peptide mass fingerprint analysis. RESULTS: A significant glomerular neutrophil infiltration was observed in the mice administered with anti-MPO IgG. The expressions of CXC chemokines, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2), were significantly increased in the renal cortex, indicating that these chemokines contribute to the neutrophil infiltration. Based on the previous findings of upregulation of adhesion molecule expression in mGEC treated with anti-MPO IgG, we examined whether mGEC secrete these chemokines in response to anti-MPO IgG. Indeed, anti-MPO IgG induced secretion of KC and MIP-2, leading to neutrophil chemotaxis in vitro. Furthermore, complete depletion of MPO in mGEC and serum using MPO-deficient mice showed an upregulation of intercellular adhesion molecule-1, indicating cross-reactive molecule(s) were existing on mGEC. We identified the molecule as moesin by a proteomic approach. CONCLUSIONS: The endothelial CXC chemokines, KC and MIP-2, contribute to infiltration of neutrophils in MPO-ANCA-associated vasculitis involving glomerulonephritis. The activation of glomerular endothelial cells by anti-MPO IgG appeared to directly involve a signaling through moesin.

Reduction of MPO-ANCA epitopes in SCG/Kj mice by 15-deoxyspergualin treatment restricted by IgG2b associated with crescentic glomerulonephritis.

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 100 microg/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-alpha. In contrast, the IL-1alpha 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.

Microbulbifer variabilis sp. nov. and Microbulbifer epialgicus sp. nov., isolated from Pacific marine algae, possess a rod-coccus cell cycle in association with the growth phase.

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.

(-)-Epigallocatechin gallate causes internalization of the epidermal growth factor receptor in human colon cancer cells.

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.

Role of F4/80Mac-1 adherent non-parenchymal liver cells in concanavalin A-induced hepatic injury in mice.

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.

MPO-ANCA induces IL-17 production by activated neutrophils in vitro via classical complement pathway-dependent manner.

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.

The inhibitory effect of (-)-epigallocatechin gallate on activation of the epidermal growth factor receptor is associated with altered lipid order in HT29 colon cancer cells.

(-)-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.

Trafficking of QD-conjugated MPO-ANCA in murine systemic vasculitis and glomerulonephritis model mice.

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.

Elevated cholesterol levels in the plasma membranes of macrophages inhibit migration by disrupting RhoA regulation.

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-beta-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.