Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin.

(1) Background: Inflammatory responses induce the formation of both anti-tumor and pro-tumor neutrophils known as myeloid-derived suppressor cells (MDSCs). Intermittent intravesical infusion of Bacillus Calmette-Guérin (BCG) is an established cancer immunotherapy for non-muscle-invasive bladder cancer (NMIBC). However, the types of neutrophils induced via the inflammatory response to both tumor-bearing and BCG remain unclear. (2) Methods: We therefore analyzed neutrophil dynamics in the peripheral blood and urine of patients with NMIBC who received BCG therapy. Further, we analyzed the effects of BCG in a mouse intraperitoneal tumor model. (3) Results: BCG therapy induced the formation of CXCL10 and MHC class II-positive neutrophils in the urine of patients with NMIBC but did not induce MDSC formation. CXCL10- and MHC class II-expressing neutrophils were detected in peritoneal exudate cells formed after BCG administration. Partial neutrophil depletion using an anti-Ly6G antibody suppressed the upregulation of CXCL10 and MHC class II in neutrophils and reversed the anti-tumor activity of BCG in mouse models. (4) Conclusions: These results indicated that intracellular MHC class II- and CXCL10-expressing neutrophils indicate the state of anti-tumor activity induced via BCG. The status of neutrophils in mixed inflammation of immunosuppressive and anti-tumor responses may therefore be useful for evaluating immunological systemic conditions.

TMPRSS2 Activates Hemagglutinin-Esterase Glycoprotein of Influenza C Virus.

Influenza C virus (ICV) has only one kind of spike protein, the hemagglutinin-esterase (HE) glycoprotein. HE functions similarly to hemagglutinin (HA) and neuraminidase of the influenza A and B viruses (IAV and IBV, respectively). It has a monobasic site, which is cleaved by some host enzymes. The cleavage is essential to activating the virus, but the enzyme or enzymes in the respiratory tract have not been identified. This study investigated whether the host serine proteases, transmembrane protease serine S1 member 2 (TMPRSS2) and human airway trypsin-like protease (HAT), which reportedly cleave HA of IAV/IBV, are involved in HE cleavage. We established TMPRSS2- and HAT-expressing MDCK cells (MDCK-TMPRSS2 and MDCK-HAT). ICV showed multicycle replication with HE cleavage without trypsin in MDCK-TMPRSS2 cells as well as IAV did. The HE cleavage and multicycle replication did not appear in MDCK-HAT cells infected with ICV without trypsin, while HA cleavage and multistep growth of IAV appeared in the cells. Amino acid sequences of the HE cleavage site in 352 ICV strains were completely preserved. Camostat and nafamostat suppressed the growth of ICV and IAV in human nasal surface epithelial (HNE) cells. Therefore, this study revealed that, at least, TMPRSS2 is involved in HE cleavage and suggested that nafamostat could be a candidate for therapeutic drugs for ICV infection. IMPORTANCE Influenza C virus (ICV) is a pathogen that causes acute respiratory illness, mostly in children, but there are no anti-ICV drugs. ICV has only one kind of spike protein, the hemagglutinin-esterase (HE) glycoprotein on the virion surface, which possesses receptor-binding, receptor-destroying, and membrane fusion activities. The HE cleavage is essential for the virus to be activated, but the enzyme or enzymes in the respiratory tract have not been identified. This study revealed that transmembrane protease serine S1 member 2 (TMPRSS2), and not human airway trypsin-like protease (HAT), is involved in HE cleavage. This is a novel study on the host enzymes involved in HE cleavage, and the result suggests that the host enzymes, such as TMPRSS2, may be a target for therapeutic drugs of ICV infection.

The proton ATPase inhibitor bafilomycin A1 reduces the release of rhinovirus C and cytokines from primary cultures of human nasal epithelial cells.

Rhinovirus species C (RV-C) causes more severe asthma attacks than other rhinovirus species. However, the modulation of RV-C replication by drugs has not been well studied. Primary human nasal epithelial (HNE) cells cultured on filter membranes with air-liquid interface methods were infected with RV-C03, and the levels of RV-C03 RNA collected from the airway surface liquid (ASL) of HNE cells were measured with a SYBR Green assay. Pretreatment of HNE cells with the specific vacuolar H+-ATPase inhibitor bafilomycin A1 reduced the RV-C03 RNA levels in the ASL; inflammatory cytokines, including interleukin (IL)-1ß, IL-6 and IL-8, in the supernatant; the mRNA expression of the RV-C receptor cadherin-related family member 3 (CDHR3) in the cells; and the number of acidic endosomes where RV-B RNA enters the cytoplasm. The levels of RV-C03 RNA in the ASL obtained from HNE cells with the CDHR3 rs6967,330 G/A genotype tended to be higher than those obtained from HNE cells with the G/G genotype. Pretreatment with the Na+/H+ exchanger inhibitor ethyl-isopropyl amiloride or either of the macrolides clarithromycin or EM900 also reduced RV-C03 RNA levels in the ASL and the number of acidic endosomes in HNE cells. In addition, significant levels of RV-A16, RV-B14 and RV-C25 RNA were detected in the ASL, and bafilomycin A1 also decreased the RV-C25 RNA levels. These findings suggest that bafilomycin A1 may reduce the release of RV-Cs and inflammatory cytokines from human airway epithelial cells. RV-Cs may be sensitive to drugs, including bafilomycin A1, that increase endosomal pH, and CDHR3 may mediate virus entry through receptor-mediated endocytosis in human airway epithelial cells.

The clinically used serine protease inhibitor nafamostat reduces influenza virus replication and cytokine production in human airway epithelial cells and viral replication in mice.

The effects of the clinically used protease inhibitor nafamostat on influenza virus replication have not been well studied. Primary human tracheal (HTE) and nasal (HNE) epithelial cells were pretreated with nafamostat and infected with the 2009 pandemic [A/Sendai-H/108/2009/(H1N1) pdm09] or seasonal [A/New York/55/2004(H3N2)] influenza virus. Pretreatment with nafamostat reduced the titers of the pandemic and seasonal influenza viruses and the secretion of inflammatory cytokines, including interleukin-6 and tumor necrosis factor-α, in the supernatants of the cells infected with the pandemic influenza virus. HTE and HNE cells exhibited mRNA and/or protein expression of transmembrane protease serine 2 (TMPRSS2), TMPRSS4, and TMPRSS11D. Pretreatment with nafamostat reduced cleavage of the precursor protein HA0 of the pandemic influenza virus into subunit HA1 in HTE cells and reduced the number of acidic endosomes in HTE and HNE cells where influenza virus RNA enters the cytoplasm. Additionally, nafamostat (30 mg/kg/day, intraperitoneal administration) reduced the levels of the pandemic influenza virus [A/Hyogo/YS/2011 (H1N1) pdm09] in mouse lung washes. These findings suggest that nafamostat may inhibit influenza virus replication in human airway epithelial cells and mouse lungs and reduce infection-induced airway inflammation by modulating cytokine production.

Antigenic changes among the predominantly circulating C/Sao Paulo lineage strains of influenza C virus in Yamagata, Japan, between 2015 and 2018.

Influenza C virus is a pathogen that causes acute respiratory illness in children and results in the hospitalization of infants. The antigenicity of the hemagglutinin esterase (HE) glycoprotein is highly stable, and it is not yet known whether antigenic changes contribute to the worldwide transmission and the occurrence of outbreaks of influenza C virus. Here, we performed antigenic analysis of 84 influenza C viruses isolated in Yamagata, Japan, during a 4-year period from 2015 to 2018 and analyzed sequence data for strains of the virus from Japan and many other parts of the world. Antigenic and phylogenetic analyses revealed that 83 strains belonged to the C/Sao Paulo lineage, and two sublineage strains, the Aichi99 sublineage and Victoria2012 sublineage, cocirculated between 2016 and 2018. Aichi99 sublineage strains exhibiting decreased reactivity with the monoclonal antibody YA3 became predominant after 2016, and these strains possessed the K190N mutation. Residue 190 is located in the 190-loop on the top side of the HE protein within a region that is known to show variation that does not impair the biological activity of the protein. The Aichi99 sublineage strains possessing the K190N mutation were detected after 2012 in Europe, Australia, the USA, and Asia as well as Japan. These observations suggest that antigenic variants with K190N mutations have circulated extensively around the world and caused outbreaks in Japan between 2016 and 2018. Our study indicated that the 190-loop is an important antigenic region, and the results suggested that changes in the 190-loop have contributed to the extensive transmission of the virus.

Inhibitory effects of glycopyrronium, formoterol, and budesonide on coronavirus HCoV-229E replication and cytokine production by primary cultures of human nasal and tracheal epithelial cells.

BACKGROUND: Coronavirus 229E (HCoV-229E), one of the causes of the common cold, exacerbates chronic obstructive pulmonary disease (COPD) and bronchial asthma. Long-acting muscarinic antagonists and ß2-agonists and inhaled corticosteroids inhibit the exacerbation of COPD and bronchial asthma caused by infection with viruses, including HCoV-229E. However, the effects of these drugs on HCoV-229E replication and infection-induced inflammation in the human airway are unknown. METHODS: Primary human nasal (HNE) and tracheal (HTE) epithelial cell cultures were infected with HCoV-229E. RESULTS: Pretreatment of HNE and HTE cells with glycopyrronium or formoterol decreased viral RNA levels and/or titers, the expression of the HCoV-229E receptor CD13, the number and fluorescence intensity of acidic endosomes where HCoV-229E RNA enters the cytoplasm, and the infection-induced production of cytokines, including IL-6, IL-8, and IFN-ß. Treatment of the cells with the CD13 inhibitor 2'2'-dipyridyl decreased viral titers. Pretreatment of the cells with a combination of three drugs (glycopyrronium, formoterol, and budesonide) exerted additive inhibitory effects on viral titers and cytokine production. Pretreatment of HNE cells with glycopyrronium or formoterol reduced the susceptibility to infection, and pretreatment with the three drugs inhibited activation of nuclear factor-kappa B p50 and p65 proteins. Pretreatment with formoterol increased cAMP levels and treatment with cAMP decreased viral titers, CD13 expression, and the fluorescence intensity of acidic endosomes. CONCLUSIONS: These findings suggest that glycopyrronium, formoterol, and a combination of glycopyrronium, formoterol, and budesonide inhibit HCoV-229E replication partly by inhibiting receptor expression and/or endosomal function and that these drugs modulate infection-induced inflammation in the airway.

Neutralizing Epitopes and Residues Mediating the Potential Antigenic Drift of the Hemagglutinin-Esterase Protein of Influenza C Virus.

We mapped the hemagglutinin-esterase (HE) antigenic epitopes of the influenza C virus on the three-dimensional (3D) structure of the HE glycoprotein using 246 escape mutants that were selected by a panel of nine anti-HE monoclonal antibodies (MAbs), including seven of the C/Ann Arbor/1/50 virus and two of the C/Yamagata/15/2004 virus. The frequency of variant selection in the presence of anti-HE MAbs was very low, with frequencies ranging from 10-4.62 to 10-7.58 for the C/Ann Arbor/1/50 virus and from 10-7.11 to 10-9.25 for the C/Yamagata/15/2004 virus. Sequencing of mutant HE genes revealed 25 amino acid substitutions at 16 positions in three antigenic sites: A-1, A-2, and A-3, and a newly designated Y-1 site. In the 3D structure, the A-1 site was widely located around the receptor-binding site, the A-2 site was near the receptor-destroying enzyme site, and the Y-1 site was located in the loop on the topside of HE. The hemagglutination inhibition reactions of the MAbs with influenza C viruses, circulating between 1947 and 2016, were consistent with the antigenic-site amino acid changes. We also found some amino acid variations in the antigenic site of recently circulating strains with antigenic changes, suggesting that viruses that have the potential to alter antigenicity continue to circulate in humans.

Effect of Phosphorylation of CM2 Protein on Influenza C Virus Replication.

CM2 is the second membrane protein of the influenza C virus and has been demonstrated to play a role in the uncoating and genome packaging processes in influenza C virus replication. Although the effects of N-linked glycosylation, disulfide-linked oligomerization, and palmitoylation of CM2 on virus replication have been analyzed, the effect of the phosphorylation of CM2 on virus replication remains to be determined. In this study, a phosphorylation site(s) at residue 78 and/or 103 of CM2 was replaced with an alanine residue(s), and the effects of the loss of phosphorylation on influenza C virus replication were analyzed. No significant differences were observed in the packaging of the reporter gene between influenza C virus-like particles (VLPs) produced from 293T cells expressing wild-type CM2 and those from the cells expressing the CM2 mutants lacking the phosphorylation site(s). Reporter gene expression in HMV-II cells infected with VLPs containing the CM2 mutants was inhibited in comparison with that in cells infected with wild-type VLPs. The virus production of the recombinant influenza C virus possessing CM2 mutants containing a serine-to-alanine change at residue 78 was significantly lower than that of wild-type recombinant influenza C virus. Furthermore, the virus growth of the recombinant viruses possessing CM2 with a serine-to-aspartic acid change at position 78, to mimic constitutive phosphorylation, was virtually identical to that of the wild-type virus. These results suggest that phosphorylation of CM2 plays a role in efficient virus replication, probably through the addition of a negative charge to the Ser78 phosphorylation site.IMPORTANCE It is well-known that many host and viral proteins are posttranslationally modified by phosphorylation, which plays a role in the functions of these proteins. In influenza A and B viruses, phosphorylation of viral proteins NP, M1, NS1, and the nuclear export protein (NEP), which are not integrated into the membranes, affects the functions of these proteins, thereby affecting virus replication. However, it was reported that phosphorylation of the influenza A virus M2 ion channel protein, which is integrated into the membrane, has no effect on virus replication in vitro or in vivo We previously demonstrated that the influenza C virus CM2 ion channel protein is modified by N-glycosylation, oligomerization, palmitoylation, and phosphorylation and have analyzed the effects of these modifications, except phosphorylation, on virus replication. This is the first report demonstrating that phosphorylation of the influenza C virus CM2 ion channel protein, unlike that of the influenza A virus M2 protein, plays a role in virus replication.

The serine protease inhibitor camostat inhibits influenza virus replication and cytokine production in primary cultures of human tracheal epithelial cells.

BACKGROUND: Serine proteases act through the proteolytic cleavage of the hemagglutinin (HA) of influenza viruses for the entry of influenza virus into cells, resulting in infection. However, the inhibitory effects of serine protease inhibitors on influenza virus infection of human airway epithelial cells, and on their production of inflammatory cytokines are unclear. METHODS: Primary cultures of human tracheal epithelial cells were treated with four types of serine protease inhibitors, including camostat, and infected with A/Sendai-H/108/2009/(H1N1) pdm09 or A/New York/55/2004(H3N2). RESULTS: Camostat reduced the amounts of influenza viruses in the supernatants and viral RNA in the cells. It reduced the cleavage of an influenza virus precursor protein, HA0, into the subunit HA1. Camostat also reduced the concentrations of the cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the supernatants. Gabexate and aprotinin reduced the viral titers and RNA levels in the cells, and aprotinin reduced the concentrations of TNF-α in the supernatants. The proteases transmembrane protease serine S1 member (TMPRSS) 2 and HAT (human trypsin-like protease: TMPRSS11D), which are known to cleave HA0 and to activate the virus, were detected at the cell membrane and in the cytoplasm. mRNA encoding TMPRSS2, TMPRSS4 and TMPRSS11D was detectable in the cells, and the expression levels were not affected by camostat. CONCLUSIONS: These findings suggest that human airway epithelial cells express these serine proteases and that serine protease inhibitors, especially camostat, may reduce influenza viral replication and the resultant production of inflammatory cytokines possibly through inhibition of activities of these proteases.

Epstein-Barr virus in the enlarged salivary tissues of patients with IgG4-related disease.

OBJECTIVE: Immunoglobulin G4-related disease (IgG4-RD) is a recently recognized disease entity characterized by high-serum IgG4 concentration and IgG4-producing plasma cell production with fibrotic or sclerotic changes in affected organs. We aimed to clarify the roles of Epstein-Barr virus (EBV) in patients with IgG4-RDs. STUDY DESIGN AND SETTING: A retrospective clinical study at the Yamagata University School of Medicine, Yamagata, Japan. METHODS: The patient group consisted of four males and four females with an average age of 62 years (range: 48-73). Expression of IgG4, latent member protein 1, EBV nuclear antigens-2, and EBV-encoded RNA in affected salivary glands from patients with IgG4-RD was examined by using immunohistochemistry and in situ hybridization. The copy number of EBV DNA in the salivary glands was also investigated by real-time polymerase chain reaction. RESULTS: All patients had hard masses in the salivary or lacrimal glands, or both, bilaterally. Serum concentrations of IgG4 were elevated in all cases (mean 589.1, range 129-1750), and IgG4-positive plasmacytes were observed in the involved salivary glands. Four patients developed potentially life-threatening systemic involvement after initial salivary gland swelling. EBV-associated molecules (EBNA and EBER) were overexpressed in the affected salivary glands. The copy number of EBV DNA was significantly higher in patients with potentially life-threatening systemic involvement than in patients without systemic involvement (P < 0.05). CONCLUSION: These results suggest that the copy number of EBV DNA could be useful as diagnostic findings in IgG4-RD to predict potentially life-threatening systemic involvement. LEVEL OF EVIDENCE: 4.

Antiproliferative activity of root extract from gentian plant (Gentiana triflora) on cultured and implanted tumor cells.

We describe a novel pharmacological activity of the gentian root, an ingredient of Chinese medicines. Root extract from Gentiana triflora triggered cell death of human Daudi cells in culture. In addition, daily administration of the extract to mice inhibited growth of implanted solid tumors. Extract treatment of cultured cells resulted in the appearance of shranken, fragmented, or condensed cell and nuclear morphologies, and in chromosomal DNA degradation. But, the extract-treated cells did not show DNA fragmentation, which exhibits a nucleosome ladder, suggesting that extract-triggered cell death is not mediated through a typical apoptotic pathway.

A binding site for Pur alpha and Pur beta is structurally unstable and is required for replication in vivo from the rat aldolase B origin.

The rat aldolase B promoter acts as a replication origin in vivo, as well as an autonomously replicating sequence (ARS). Here, we examined roles of a polypurine stretch (site PPu) in this origin, which is indispensable to the ARS activity. Purification of site PPu-binding protein revealed that site PPu binds Puralpha and Purbeta, i.e., single-stranded DNA-binding proteins whose roles in replication have been implicated, but less clear. Biochemical analyses showed that site PPu even in a longer DNA fragment is unstable in terms of double-helix, implying that Puralpha/beta may stabilize single-stranded state. Deletion of site PPu from the origin DNA, which was ectopically positioned in the mouse chromosome, significantly reduced replicator activity. Chromatin immunoprecipitation experiments showed that deletion of site PPu abolishes binding of the Puralpha/beta proteins to the origin. These observations suggest functional roles of site PPu and Puralpha/beta proteins in replication initiation.