Transcriptomic analysis of Porphyromonas gingivalis-infected head and neck cancer cells: Identification of PLAU as a candidate prognostic biomarker.

Periodontal disease is a risk factor for head and neck squamous cell carcinoma (HNSCC), and Porphyromonas gingivalis, a major periodontal pathogen, has been identified as a specific and potentially independent microbial factor that increases the risk of cancer mortality. Gene expression in HNSCC due to P. gingivalis infection and how changes in gene expression affect the prognosis of HNSCC patients are not clarified. When P. gingivalis was cultured with HNSCC cells, it efficiently adhered to these cells and enhanced their invasive ability. A transcriptome analysis of P. gingivalis -infected HNSCC cells showed that genes related to migration, including CCL20, CITED2, CTGF, C8orf44-SGK3, DUSP10, EGR3, FUZ, HBEGF, IL1B, IL24, JUN, PLAU, PTGS2, P2RY1, SEMA7A, SGK1 and SIX2, were highly up- or down-regulated. The expression of up-regulated genes was examined using the expression data of HNSCC patients obtained from The Cancer Genome Atlas (TCGA) database, and the expression of 5 genes, including PLAU, was found to be higher in cancer tissue than in solid normal tissue. An analysis of protein-protein interactions revealed that these 5 genes formed a dense network. A Cox regression analysis showed that high PLAU expression levels were associated with a poor prognosis in patients with TCGA-HNSCC. Furthermore, the prognostic impact correlated with tumour size and the presence or absence of lymph node metastasis. Collectively, these results suggest the potential of PLAU as a molecular prognostic marker in HNSCC patients. Further in vivo and in vitro studies are needed to verify the findings of this study.

Potential Role of the Intratumoral Microbiota in Prognosis of Head and Neck Cancer.

The tumor microbiome, a relatively new research field, affects tumor progression through several mechanisms. The Cancer Microbiome Atlas (TCMA) database was recently published. In the present study, we used TCMA and The Cancer Genome Atlas and examined microbiome profiling in head and neck squamous cell carcinoma (HNSCC), the role of the intratumoral microbiota in the prognosis of HNSCC patients, and differentially expressed genes in tumor cells in relation to specific bacterial infections. We investigated 18 microbes at the genus level that differed between solid normal tissue (n = 22) and primary tumors (n = 154). The tissue microbiome profiles of Actinomyces, Fusobacterium, and Rothia at the genus level differed between the solid normal tissue and primary tumors of HNSCC patients. When the prognosis of groups with rates over and under the median for each microbe at the genus level was examined, rates for Leptotrichia which were over the median correlated with significantly higher overall survival rates. We then extracted 35 differentially expressed genes between the over- and under-the-median-for-Leptotrichia groups based on the criteria of >1.5 fold and p < 0.05 in the Mann-Whitney U-test. A pathway analysis showed that these Leptotrichia-related genes were associated with the pathways of Alzheimer disease, neurodegeneration-multiple diseases, prion disease, MAPK signaling, and PI3K-Akt signaling, while protein-protein interaction analysis revealed that these genes formed a dense network. In conclusion, probiotics and specific antimicrobial therapy targeting Leptotrichia may have an impact on the prognosis of HNSCC.

Roles of Porphyromonas gulae proteases in bacterial and host cell biology.

Porphyromonas gulae, an animal-derived periodontal pathogen, expresses several virulence factors, including fimbria, lipopolysaccharide (LPS) and proteases. We previously reported that its invasive efficiency was dependent on fimbriae types. In addition, P. gulae LPS increased inflammatory responses via toll-like receptors. The present study was conducted to investigate the involvement of P. gulae proteases in bacterial and host cell biology. Porphyromonas gulae strains showed an ability to agglutinate mouse erythrocytes and also demonstrated co-aggregation with Actinomyces viscosus, while the protease inhibitors antipain, PMSF, TLCK and leupeptin diminished P. gulae proteolytic activity, resulting in inhibition of haemagglutination and co-aggregation with A. viscosus. In addition, specific proteinase inhibitors were found to reduce bacterial cell growth. Porphyromonas gulae inhibited Ca9-22 cell proliferation in a multiplicity of infection- and time-dependent manner. Additionally, P. gulae-induced decreases in cell contact and adhesion-related proteins were accompanied by a marked change in cell morphology from well spread to rounded. In contrast, inhibition of protease activity prevented degradation of proteins, such as E-cadherin, ß-catenin and focal adhesion kinase, and also blocked inhibition of cell proliferation. Together, these results indicate suppression of the amount of human proteins, such as γ-globulin, fibrinogen and fibronectin, by P. gulae proteases, suggesting that a novel protease complex contributes to bacterial virulence.

Porphyromonas gulae lipopolysaccharide elicits inflammatory responses through toll-like receptor 2 and 4 in human gingivalis epithelial cells.

Porphyromonas gulae, a Gram-negative black-pigmented anaerobe, has been associated with periodontal disease in companion animals and its virulence has been attributed to various factors, including lipopolysaccharide (LPS), protease and fimbriae. Toll-like receptors (TLRs) recognise pathogen-associated molecular patterns, such as peptidoglycan, lipids, lipoproteins, nucleic acid and LPS. Following P. gulae infection, some inflammatory responses are dependent on both TLR2 and TLR4. In addition, a recent clinical study revealed that acute and persistent inflammatory responses enhance the expressions of TLR2 and TLR4 in the oral cavity. In this study, we investigated the interaction between P. gulae LPS and human gingivalis epithelial cells (Ca9-22 cells). P. gulae LPS was found to increase TLR2 and TLR4 mRNA expressions and protein productions, and enhanced inflammatory responses, such as COX2 , TNF-ɑ, IL-6 and IL-8. Stimulated Ca9-22 cells exhibited phosphorylation of ERK1/2 and p38, and their inhibitors diminished inflammatory responses, while knockdown of the TLR2 and/or TLR4 genes with small interfering RNA (siRNA) prevented inflammatory responses. Moreover, p38 and ERK1/2 phosphorylation was decreased in TLR2 and TLR4 gene knockdown cells. These findings suggest that P. gulae LPS activates p38 and ERK1/2 via TLR2 and TLR4, leading to inflammatory responses in human gingival epithelial cells.

Application of surface plasmon resonance imaging to monitoring G protein-coupled receptor signaling and its modulation in a heterologous expression system.

BACKGROUND: G protein-coupled receptors (GPCRs) are ubiquitous surface proteins mediating various biological responses and thus, important targets for therapeutic drugs. GPCRs individually produce their own signaling as well as modulate the signaling of other GPCRs. Real-time observation of GPCR signaling and modulation in living cells is key to molecular study of biological responses and pharmaceutical development. However, fluorescence imaging, the technique widely used for this purpose, requires a fluorescent dye which may inhibit biological responses or a fluorescent-tagged target protein created through time-consuming genetic manipulation. In this study, we applied two-dimensional surface plasmon resonance (SPR) imaging to monitoring the translocation of protein kinase C (PKC), a major GPCR-coupled signaling molecule in the widely used HEK293 cell lines and examined whether the signaling of, and, modulation between heterologously expressed GPCRs can be measured without fluorescent labeling. RESULTS: We cultured HEK293 cells on the gold-plated slide glass and evoked SPR at the interface between the cell's plasma membrane and the gold surface with incident light. The translocation of activated native PKC to the plasma membrane is expected to alter the incident angle-SPR extent relation, and this could be detected as a change in the intensity of light reflection from the specimen illuminated at a fixed incident angle. Direct activation of PKC with 12-O-tetradecanoylphorbol-13-acetate increased the reflection intensity. This increase indeed reported PKC translocation because it was reduced by a pre-treatment with bisindolylmaleimide-1, a PKC inhibitor. We further applied this technique to a stable HEK293 cell line heterologously expressing the GPCRs type-1 metabotropic glutamate receptor (mGluR1) and adenosine A1 receptor (A1R). (RS)-3,5-dihydroxyphenylglycine, a mGluR1 agonist, increased the reflection intensity, and the PKC inhibitor reduced this increase. A pre-treatment with (R)-N(6)-phenylisopropyladenosine, an A1R-selective agonist suppressed mGluR1-mediated reflection increase. These results suggest that our technique can detect PKC translocation initiated by ligand binding to mGluR1 and its modulation by A1R. CONCLUSIONS: SPR imaging turned out to be utilizable for monitoring GPCR-mediated PKC translocation and its modulation by a different GPCR in a heterologous expression system. This technique provides a powerful yet easy-to-use tool for molecular study of biological responses and pharmaceutical development.

Green tea catechins inhibit Porphyromonas gulae LPS-induced inflammatory responses in human gingival epithelial cells.

OBJECTIVES: To determine the anti-inflammatory effects of green tea catechins in immortalized human gingival epithelial cells (Ca9-22) stimulated with Porphyromonas gulae lipopolysaccharide (LPS). METHODS: Ca9-22 cells were incubated with P. gulae LPS (10 µg/ml) with or without green tea catechins, epigallocatechin-3-gallate (EGCg), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epicatechin (EC) (each at 50 µM), for 6 or 24 h. Real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay were used to determine the induction of cyclooxygenase 2 (COX2), tumor necrosis factor alpha (TNF-ɑ), interleukin 6 (IL-6), and IL-8. Furthermore, the expression of toll-like receptors (TLRs) 2 and 4 was examined using real-time PCR and western blotting analysis, and phosphorylation of the p38 and ERK1/2 was examined using western blotting analysis. RESULTS: At the mRNA and protein levels, EGCg, EGC, ECG, and EC were found to significantly inhibit COX2, TNF-ɑ, IL-6, and IL-8. Furthermore, the levels of ERK1/2 and p38 phosphorylation induced by P. gulae LPS were decreased following the addition of each of the catechins, as well as TLR2 and 4 mRNA and protein. CONCLUSIONS: These findings indicate that green tea catechins are potent inhibitors of inflammatory responses induced by P. gulae LPS, and may also be useful for prevention and/or attenuation of periodontitis.

Prognostic association of starvation-induced gene expression in head and neck cancer.

Autophagy-related genes (ARGs) have been implicated in the initiation and progression of malignant tumor promotion. To investigate the dynamics of expression of genes, including ARGs, head and neck squamous cell carcinoma (HNSCC) cells were placed under serum-free conditions to induce growth retardation and autophagy, and these starved cells were subjected to transcriptome analysis. Among the 21 starvation-induced genes (SIGs) located in the autophagy, cell proliferation, and survival signaling pathways, we identified SIGs that showed prominent up-regulation or down-regulation in vitro. These included AGR2, BST2, CALR, CD22, DDIT3, FOXA2, HSPA5, PIWIL4, PYCR1, SGK3, and TRIB3. The Cancer Genome Atlas (TCGA) database of HNSCC patients was used to examine the expression of up-regulated genes, and CALR, HSPA5, and TRIB3 were found to be highly expressed relative to solid normal tissue in cancer and the survival rate was reduced in patients with high expression. Protein-protein interaction analysis demonstrated the formation of a dense network of these genes. Cox regression analysis revealed that high expression of CALR, HSPA5, and TRIB3 was associated with poor prognosis in patients with TCGA-HNSCC. Therefore, these SIGs up-regulated under serum starvation may be molecular prognostic markers in HNSCC patients.

Efficacy of FimA antibody and clindamycin in silkworm larvae stimulated with Porphyromonas gulae.

Objective: Porphyromonas gulae, a major periodontal pathogen in animals, possesses fimbriae that have been classified into three genotypes (A, B, C) based on the diversity of fimA genes encoding fimbrillin protein (FimA). P. gulae strains with type C fimbriae were previously shown to be more virulent than other types. In this study, we further examined the host toxicity mediated by P. gulae fimbriae by constructing recombinant FimA (rFimA) expression vectors for each genotype and raised antibodies to the purified proteins. Methods and Results: All larvae died within 204 h following infection with P. gulae type C at the low-dose infection, whereas type A and B did not. Among fimA types, the survival rates of the larvae injected with rFimA type C were remarkably decreased, while the survival rates of the larvae injected with rFimA type A and type B were greater than 50%. Clindamycin treatment inhibited the growth of type C strains in a dose-dependent manner, resulting in an increased rate of silkworm survival. Finally, type C rFimA-specific antiserum prolonged the survival of silkworm larvae stimulated by infection with P. gulae type C strain or injection of rFimA type C protein. Conclusion: These results suggested that type C fimbriae have high potential for enhancement of bacterial pathogenesis, and that both clindamycin and anti-type C rFimA-specific antibodies are potent inhibitors of type C fimbriae-induced toxicity. This is the first report to establish a silkworm infection model using P. gulae for toxicity assessment.

Inhibitory effect of a mouth rinse formulated with chlorhexidine gluconate, ethanol, and green tea extract against major oral bacterial species.

Mouth rinses are a useful supplementary tool for the prevention of oral infectious diseases. Although the antimicrobial effects of mouth rinses have been investigated, there are few studies focusing on the comparison of the effects among various oral bacterial species. In the present study, the inhibitory effect of a commercial mouth rinse, "ConCoolF," and each of its major components, chlorhexidine gluconate, ethanol, and green tea extract, on multiple species of oral bacteria were investigated. Inhibition of bacterial growth was observed in all cariogenic streptococcal species with different genera, serotypes, and strains isolated from different countries when either the complete mouth rinse or chlorhexidine gluconate were used. However, no growth inhibition was observed when the bacteria were exposed to ethanol or green tea extract. Interestingly, growth inhibition was greatly reduced in non-cariogenic streptococci compared with cariogenic streptococci. In addition, both the mouth rinse and chlorhexidine gluconate inhibited the biofilms formed by both Streptococcus mutans (S. mutans) and Porphyromonas gingivalis (P. gingivalis), among which the inhibitory effect against S. mutans was higher than that against P. gingivalis. These results suggest that a mouth rinse containing chlorhexidine gluconate, ethanol, and green tea extract, or chlorhexidine gluconate alone, exhibits antimicrobial activity against several oral bacteria species, having greater activity against pathogenic bacteria.

Author Correction: Inhibition of Porphyromonas gulae and periodontal disease in dogs by a combination of clindamycin and interferon alpha.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Inhibition of Porphyromonas gulae and periodontal disease in dogs by a combination of clindamycin and interferon alpha.

Porphyromonas gulae is a major periodontal pathogen in dogs, which can be transmitted to their owners. A major virulence factor of P. gulae consists of a 41-kDa filamentous appendage (FimA) on the cell surface, which is classified into three genotypes: A, B, and C. Thus far, inhibition of periodontal disease in dogs remains difficult. The present study assessed the inhibitory effects of a combination of clindamycin and interferon alpha (IFN-α) formulation against P. gulae and periodontal disease. Growth of P. gulae was significantly inhibited by clindamycin; this inhibition had a greater effect on type C P. gulae than on type A and B isolates. In contrast, the IFN-α formulation inhibited the expression of IL-1ß and COX-2 elicited by type A and B isolates, but not that elicited by type C isolates. Furthermore, periodontal recovery was promoted by the administration of both clindamycin and IFN-α formulation to dogs undergoing periodontal treatment; moreover, this combined treatment reduced the number of FimA genotypes in oral specimens from treated dogs. These results suggest that a combination of clindamycin and IFN-α formulation inhibit P. gulae virulence and thus may be effective for the prevention of periodontal disease induced by P. gulae.

Chitin-deacetylase activity induces appressorium differentiation in the rice blast fungus Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae differentiates a specialized infection structure called an appressorium to invade rice cells. In this report, we show that CBP1, which encodes a chitin-deacetylase, is involved in the induction phase of appressorium differentiation. We demonstrate that the enzymatic activity of Cbp1 is critical for appressorium formation. M. oryzae has six CDA homologues in addition to Cbp1, but none of these are indispensable for appressorium formation. We observed chitosan localization at the fungal cell wall using OGA488. This observation suggests that Cbp1-catalysed conversion of chitin into chitosan occurs at the cell wall of germ tubes during appressorium differentiation by M. oryzae. Taken together, our results provide evidence that the chitin deacetylase activity of Cbp1 is necessary for appressorium formation.

SCN5A(K817E), a novel Brugada syndrome-associated mutation that alters the activation gating of NaV1.5 channel.

BACKGROUND: Brugada syndrome (BrS) is an inherited lethal arrhythmic disorder characterized by syncope and sudden cardiac death from ventricular tachyarrhythmias. Here we identified a novel K817E mutation of SCN5A gene in a man with type 1 BrS electrocardiogram pattern using next-generation sequencing targeted for 73 cardiac disorder-related genes. SCN5A encodes the α-subunit of NaV1.5 voltage-gated Na(+) channel, and some of its mutations are linked to BrS. The proband had no mutation in any of the other arrhythmia-related genes sequenced. OBJECTIVE: We investigated whether the K817E mutation causes a functional change of NaV1.5 channel responsible for the BrS phenotype. METHODS: We compared the electrophysiological properties of the whole-cell currents mediated by wild-type and mutant channels heterologously expressed in human embryonic kidney 293 cells by using a voltage-clamp technique. RESULTS: The K817E mutation reduced the Na(+) current density by 39.0%-91.4% at membrane potentials from -55 to -5 mV. This reduction resulted from a ~24-mV positive shift in the voltage dependence of activation. The mutation also decelerated recovery from both fast and intermediate inactivation, whereas it had little effect on the cell surface expression, single-channel conductance, voltage-dependence of fast inactivation, entry into intermediate inactivation, use-dependent loss of channel availability, or closed-state inactivation. CONCLUSION: The K817E mutation of SCN5A gene leads to loss of function of NaV1.5 channel and may underlie the BrS phenotype of the proband.

Prognosis of hypercholesterolemic patients taking pravastatin for five years: the Chiba Lipid Intervention Program (CLIP) Study.

The Chiba Lipid Intervention Program (CLIP) Study was designed to clarify the prognosis of Japanese hypercholesterolemic patients taking pravastatin for 5 years. Hypercholesterolemic patients (n = 2,529) with a total cholesterol level > or = 220 mg/dl and without histories of ischemic coronary heart disease and/or cerebral infarction were administered pravastatin (10-20 mg/day). Among them, 2,131 took pravastatin fully (Pravastatin-continued group), and 398 discontinued the treatment (Discontinued group). The baseline total cholesterol level was 264.3 +/- 34.7 mg/dl (mean +/- standard deviation). The mean reduction rates of total cholesterol and low-density lipoprotein (LDL) cholesterol were 18.0% and 27.2%, respectively. Mild and moderate adverse events occurred in 86 cases (3.6%). Serious adverse events were not observed. Death rates of the pravastatin-continued group and of the discontinued group were 2.6 and 16.0/1,000 persons/year, respectively. Cardiac events (fatal and nonfatal myocardial infarction, cardiac death, angina pectoris) in all, occurred in 35 patients (incidence rate = 2.77/1,000 persons/year). In the pravastatin continued group, 9 causes of fatal and nonfatal myocardial infarction occurred (0.84/1,000 persons/year), whereas in the discontinued group, 4 cases occurred (2.06/1,000 persons/ year). The risk ratio for cardiac events was correlated with the number of risks. In the low-risk group (< or = 1 risk), decreased rates of LDL-cholesterol were less in the cardiac event group than the non-cardiac event group (LDL-cholesterol; 16% vs 25%, p = 0.04). These results suggested the following; 1) Pravastatin maintained a cholesterol lowering effect long-term without serious complications. 2) Pravastatin administration might reduce the mortality rate and myocardial infarction. 3) The combination of multiple risks is a strong factor for a cardiac event in addition to hypercholesterolemia.

Surface-initiated atom transfer radical polymerization from chitin nanofiber macroinitiator film.

This paper reports the preparation of chitin nanofiber-graft-poly(2-hydroxyethyl acrylate) (CNF-g-polyHEA) films by surface-initiated atom transfer radical polymerization (ATRP) of HEA monomer from a CNF macroinitiator film. First, a CNF film was prepared by regeneration from a chitin ion gel with an ionic liquid. Then, acylation of the CNF surface with α-bromoisobutyryl bromide was carried out to obtain the CNF macroinitiator film having the initiating moieties (α-bromoisobutyrate group). The surface-initiated graft polymerization of HEA from the CNF macroinitiator film by ATRP was performed to produce the CNF-g-polyHEA film. The IR, XRD, and SEM measurements of the resulting film indicated the progress of the graft polymerization of HEA on surface of CNFs. The molecular weights of the grafted polyHEAs increased with prolonged polymerization times, which affected the mechanical properties of the films under tensile mode.