Protocols for collecting mouse PDL cells and bone marrow cells, differentiation, and data analysis.

Periodontal ligament cells (PDLCs) and macrophages in bone marrow cells have been widely used to investigate novel therapeutic agents to treat periodontitis. Here, we present a protocol for collecting primary mouse PDLCs and bone marrow cells. We detail steps for culturing and differentiation for both cell types and review data analysis for in vitro experiments using primary PDLCs and bone marrow cells. This protocol can be used to explore the impact of novel therapeutic agents using in vitro experiments. For complete details on the use and execution of this protocol, please refer to Sirisereephap et al.1.

Soybean peptide inhibits the biofilm of periodontopathic bacteria via bactericidal activity.

OBJECTIVE: This study aimed to clarify the antibacterial mechanism and antibiofilm effect of soybean-derived peptide BCBS-11 against periodontopathic bacteria. DESIGN: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of BCBS-11 against Porphyromonas gingivalis (P. gingivalis), Fusobacterium nucleatum (F. nucleatum), and Streptococcus mitis (S. mitis) were determined for the antibacterial mechanism. The effect of BCBS-11 on membrane permeability and depolarization activity were investigated using propidium iodide (PI) staining and 3, 3'-dipropylthiadicarbocyanine iodide (DiSC3-(5)) analysis. Monospecies and multispecies biofilms were cultured on 96-well plates. The amount of biofilm was determined using crystal violet staining to determine the inhibition of biofilm formation and the eradication of established biofilm using BCBS-11. The cytotoxicity of BCBS-11 was evaluated using 3-(4, 5-Dimethylthiazol-2-yl)- 2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The MIC and MBC indicated the bactericidal activity of BCBS-11 against P. gingivalis and F. nucleatum. The PI staining revealed that BCBS-11 disrupted the bacterial membrane integrity. The DiSC3-(5) analysis indicated that BCBS-11 depolarized the bacterial cytoplasmic membrane. These results indicate the antimicrobial action of BCBS-11 through membrane disruption and the collapse of membrane electrochemical gradient. BCBS-11 significantly inhibited the monospecies biofilm formation of P. gingivalis and F. nucleatum and also inhibited dual-species biofilm. BCBS-11 was not cytotoxic toward human oral epithelial cells. CONCLUSIONS: BCBS-11 inhibits the monospecies and multispecies biofilm formation of P. gingivalis and F. nucleatum, and their bactericidal activity results from membrane disruption.

Laminin Isoforms in Human Dental Pulp: Lymphatic Vessels Express Laminin-332, and Schwann Cell-Associated Laminin-211 Modulates CD163 Expression of M2-like Macrophages.

Laminin, a basement membrane heterotrimeric glycoprotein composed of α/ß/γ subunits, has important tissue-specific functions in the control of cellular behavior. Our recent study showed the colocalization of CD163+ M2-like macrophages with Schwann cells in human dental pulp, leading us to hypothesize that the laminin isoform of Schwann cells is associated with CD163 expression. The present study investigated the distribution of laminin isoforms in human dental pulp and the underlying mechanisms that affect macrophage phenotypes. Immunofluorescence analysis indicated that blood vessels were exclusively positive for laminin α4 and α5, whereas laminin α2 was associated with Schwann cells. Unexpectedly, laminin α3/laminin-332 (α3ß3γ2) was detected on lymphatic vessels. In intact and carious teeth, CD163+ cells were associated with laminin α2, whereas CD206 single-positive cells were present inside, outside, and along blood vessels. In vitro incubation of THP-1 macrophages in plates coated with laminin-211/511 or its functionally analogous E8 fragments of α-chain (E8-α) indicated that cell shapes differed between macrophages grown on laminin-211/E8-α2 and macrophages grown on laminin-511/E8-α5. Laminin-211/E8-α2-coated plates upregulated CD163 expression, compared with laminin-511/E8-α5-coated plates. Integrin α3- and integrin α6-neutralizing Abs altered the shape of THP-1 macrophages and upregulated mRNA levels of CD206 and CD163 in macrophages grown on laminin-511; the neutralizing Abs did not affect macrophages grown on laminin-211. These findings suggest that laminin isoforms differentially regulate macrophage behavior via distinct integrin-laminin affinities. Of note, laminin-332 is expressed by pulpal lymphatic vessels, the existence of which has been debated; laminin-211 might have a role in maintaining CD163 expression on macrophages.

Matcha Green Tea Exhibits Bactericidal Activity against Streptococcus pneumoniae and Inhibits Functional Pneumolysin.

Streptococcus pneumoniae is a causative pathogen of several human infectious diseases including community-acquired pneumonia. Pneumolysin (PLY), a pore-forming toxin, plays an important role in the pathogenesis of pneumococcal pneumonia. In recent years, the use of traditional natural substances for prevention has drawn attention because of the increasing antibacterial drug resistance of S. pneumoniae. According to some studies, green tea exhibits antibacterial and antitoxin activities. The polyphenols, namely the catechins epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC) are largely responsible for these activities. Although matcha green tea provides more polyphenols than green tea infusions, its relationship with pneumococcal pneumonia remains unclear. In this study, we found that treatment with 20 mg/mL matcha supernatant exhibited significant antibacterial activity against S. pneumoniae regardless of antimicrobial resistance. In addition, the matcha supernatant suppressed PLY-mediated hemolysis and cytolysis by inhibiting PLY oligomerization. Moreover, the matcha supernatant and catechins inhibited PLY-mediated neutrophil death and the release of neutrophil elastase. These findings suggest that matcha green tea reduces the virulence of S. pneumoniae in vitro and may be a promising agent for the treatment of pneumococcal infections.

Oral Pathobiont-Induced Changes in Gut Microbiota Aggravate the Pathology of Nonalcoholic Fatty Liver Disease in Mice.

Background & Aims: Periodontitis increases the risk of nonalcoholic fatty liver disease (NAFLD); however, the underlying mechanisms are unclear. Here, we show that gut dysbiosis induced by oral administration of Porphyromonas gingivalis, a representative periodontopathic bacterium, is involved in the aggravation of NAFLD pathology. Methods: C57BL/6N mice were administered either vehicle, P. gingivalis, or Prevotella intermedia, another periodontopathic bacterium with weaker periodontal pathogenicity, followed by feeding on a choline-deficient, l-amino acid-defined, high-fat diet with 60 kcal% fat and 0.1% methionine (CDAHFD60). The gut microbial communities were analyzed by pyrosequencing the 16S ribosomal RNA genes. Metagenomic analysis was used to determine the relative abundance of the Kyoto Encyclopedia of Genes and Genomes pathways encoded in the gut microbiota. Serum metabolites were analyzed using nuclear magnetic resonance-based metabolomics coupled with multivariate statistical analyses. Hepatic gene expression profiles were analyzed via DNA microarray and quantitative polymerase chain reaction. Results: CDAHFD60 feeding induced hepatic steatosis, and in combination with bacterial administration, it further aggravated NAFLD pathology, thereby increasing fibrosis. Gene expression analysis of liver samples revealed that genes involved in NAFLD pathology were perturbed, and the two bacteria induced distinct expression profiles. This might be due to quantitative and qualitative differences in the influx of bacterial products in the gut because the serum endotoxin levels, compositions of the gut microbiota, and serum metabolite profiles induced by the ingested P. intermedia and P. gingivalis were different. Conclusions: Swallowed periodontopathic bacteria aggravate NAFLD pathology, likely due to dysregulation of gene expression by inducing gut dysbiosis and subsequent influx of gut bacteria and/or bacterial products.

Rice bran-derived protein fractions enhance sulforaphane-induced anti-oxidative activity in gingival epithelial cells.

OBJECTIVE: Food-derived bioactive peptides have been reported to exhibit various beneficial effects, including anti-microbial, anti-inflammatory, and anti-oxidant properties. Oxidative stress has been implicated in the development of several inflammatory diseases such as periodontal disease. However, the anti-oxidative effect of food-derived bioactive peptides in gingival epithelial cells (GECs) is unknown. Therefore, we examined the bioactivity of the peptides in GECs. DESIGN: Food-derived peptide fractionations derived from rice bran, rice endosperm, corn, and soy were screened for anti-oxidative effects using anti-oxidant response element (ARE)-luciferase-transfected HEK 293 cells. The induction of anti-oxidation-related genes and proteins in GECs by the fractions were examined by quantitative PCR and Western blotting, respectively. Then, the fraction-mediated anti-oxidative effects were examined by measuring intracellular reactive oxygen species (ROS) levels using flow cytometry. Furthermore, the anti-oxidative response-related cellular signaling pathways were analyzed via Western blotting. RESULTS: Although treatment with the food-derived peptides alone did not activate anti-oxidative responses, co-treatment with sulforaphane (SFN; a potent anti-oxidant) and certain food-derived peptides enhanced anti-oxidative responses in ARE-luciferase-transfected HEK 293 cells. The fractions augmented heme oxygenase-1 mRNA and protein expression in GECs. The percentage of ROS-positive cells was significantly decreased by co-treatment with SFN and peptide fractions derived from rice bran. Furthermore, the involvement of both nuclear factor erythroid 2-related factor 2 (Nrf2) and extracellular signal-regulated kinase (ERK) in the enhancement of anti-oxidative responses was demonstrated by Western blotting. CONCLUSIONS: Peptides derived from rice bran enhances SFN-induced anti-oxidative responses in GECs through ERK-Nrf2-ARE signaling.

Rice peptide with amino acid substitution inhibits biofilm formation by Porphyromonas gingivalis and Fusobacterium nucleatum.

OBJECTIVE: Rice peptide has antibacterial properties that have been tested in planktonic bacterial culture. However, bacteria form biofilm at disease sites and are resistant to antibacterial agents. The aim of this study was to clarify the mechanisms of action of rice peptide and its amino acid substitution against periodontopathic bacteria and their antibiofilm effects. DESIGN: Porphyromonas gingivalis and Fusobacterium nucleatum were treated with AmyI-1-18 rice peptide or its arginine-substituted analog, G12R, under anaerobic conditions. The amount of biofilm was evaluated by crystal violet staining. The integrity of the bacteria cytoplasmic membrane was studied in a propidium iodide (PI) stain assay and transmission electron microscopy (TEM). RESULTS: Both AmyI-1-18 and G12R inhibited biofilm formation of P. gingivalis and F. nucleatum; in particular, G12R inhibited F. nucleatum at lower concentrations. However, neither peptide eradicated established biofilms significantly. According to the minimum inhibitory concentration and minimum bactericidal concentration against P. gingivalis, AmyI-1-18 has bacteriostatic properties and G12R has bactericidal activity, and both peptides showed bactericidal activity against F. nucleatum. PI staining and TEM analysis indicated that membrane disruption by G12R was enhanced, which suggests that the replacement amino acid reinforced the electostatic interaction between the peptide and bacteria by increase of cationic charge and α-helix content. CONCLUSIONS: Rice peptide inhibited biofilm formation of P. gingivalis and F. nucleatum, and bactericidal activity via membrane destruction was enhanced by amino acid substitution.

Erythromycin inhibits neutrophilic inflammation and mucosal disease by upregulating DEL-1.

Macrolide antibiotics exert antiinflammatory effects; however, little is known regarding their immunomodulatory mechanisms. In this study, using 2 distinct mouse models of mucosal inflammatory disease (LPS-induced acute lung injury and ligature-induced periodontitis), we demonstrated that the antiinflammatory action of erythromycin (ERM) is mediated through upregulation of the secreted homeostatic protein developmental endothelial locus-1 (DEL-1). Consistent with the anti-neutrophil recruitment action of endothelial cell-derived DEL-1, ERM inhibited neutrophil infiltration in the lungs and the periodontium in a DEL-1-dependent manner. Whereas ERM (but not other antibiotics, such as josamycin and penicillin) protected against lethal pulmonary inflammation and inflammatory periodontal bone loss, these protective effects of ERM were abolished in Del1-deficient mice. By interacting with the growth hormone secretagogue receptor and activating JAK2 in human lung microvascular endothelial cells, ERM induced DEL-1 transcription that was mediated by MAPK p38 and was CCAAT/enhancer binding protein-ß dependent. Moreover, ERM reversed IL-17-induced inhibition of DEL-1 transcription, in a manner that was dependent not only on JAK2 but also on PI3K/AKT signaling. Because DEL-1 levels are severely reduced in inflammatory conditions and with aging, the ability of ERM to upregulate DEL-1 may lead to a novel approach for the treatment of inflammatory and aging-related diseases.

Epithelial TRPV1 channels: Expression, function, and pathogenicity in the oral cavity.

BACKGROUND: The oral cavity serves as an entrance to the body and is therefore exposed to various exogenous stimuli, including mechanical forces, chemical agents, and bacterial components. The oral mucosa responds to these stimuli to maintain homeostasis and good oral health. The transient receptor potential vanilloid 1 (TRPV1) ion channel functions as an environment-sensing protein and is involved in a wide variety of cellular responses. Recent studies have revealed that epithelial TRPV1 ion channels in the oral cavity play pivotal roles in several pathophysiological conditions. In this review, we summarize the features of epithelial TRPV1 channels in the oral cavity and focus on their cellular function and pathogenicity with reference to related findings in other organs and tissues. HIGHLIGHT: TRPV1 channels are widely expressed in epithelial cells in the oral cavity and play pivotal roles in fundamental cellular processes and disease progression. CONCLUSION: This review suggests that oral epithelial TRPV1 contributes to several cellular functions such as cell proliferation, barrier function, and inflammation. Further understanding of the characteristics of epithelial TRPV1 in the oral cavity may provide new insights into the prevention or treatment of diseases.

Mutual inhibition between Prkd2 and Bcl6 controls T follicular helper cell differentiation.

T follicular helper cells (TFH) participate in germinal center (GC) development and are necessary for B cell production of high-affinity, isotype-switched antibodies. In a forward genetic screen, we identified a missense mutation in Prkd2, encoding the serine/threonine kinase protein kinase D2, which caused elevated titers of immunoglobulin E (IgE) in the serum. Subsequent analysis of serum antibodies in mice with a targeted null mutation of Prkd2 demonstrated polyclonal hypergammaglobulinemia of IgE, IgG1, and IgA isotypes, which was exacerbated by the T cell-dependent humoral response to immunization. GC formation and GC B cells were increased in Prkd2-/- spleens. These effects were the result of excessive cell-autonomous TFH development caused by unrestricted Bcl6 nuclear translocation in Prkd2-/- CD4+ T cells. Prkd2 directly binds to Bcl6, and Prkd2-dependent phosphorylation of Bcl6 is necessary to constrain Bcl6 to the cytoplasm, thereby limiting TFH development. In response to immunization, Bcl6 repressed Prkd2 expression in CD4+ T cells, thereby committing them to TFH development. Thus, Prkd2 and Bcl6 form a mutually inhibitory positive feedback loop that controls the stable transition from naïve CD4+ T cells to TFH during the adaptive immune response.

A bacterial metabolite induces Nrf2-mediated anti-oxidative responses in gingival epithelial cells by activating the MAPK signaling pathway.

OBJECTIVE: Oxidative stress, which is defined as an imbalance between pro-oxidant and antioxidant systems, has been implicated in the development and/or progression of several inflammatory diseases, including periodontal disease. The reactive oxygen species (ROS) are the primary inducers of oxidative stress. In the induction of cytoprotective enzymes, the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling in antioxidant systems takes a main role. Notably, 10-oxo-trans-11-octadecenoic acid (KetoC), known as a bioactive metabolite generated by intestinal microorganisms, has been reported to have beneficial effects on several biological responses. Therefore, we investigated the antioxidant effect of KetoC on gingival epithelial cells (GECs) in this present study. METHODS: An SV40-T antigen-transformed human gingival epithelial cell line (Epi4) was used for experiments. The alteration of anti-oxidative stress related genes was analyzed by qPCR. The cellular ROS levels were evaluated by flow cytometry. To explore its molecular mechanisms, ARE promotor activity was analyzed by luciferase assay; the involvement of mitogen-activated protein kinase (MAPK) and G protein-coupled receptor 120 (GPR120) were evaluated by Western blotting and luciferase assay, respectively. RESULTS: KetoC significantly increased the expression of antioxidant-related genes in GECs. The level of ROS was significantly inhibited by the pretreatment of KetoC. Extracellular signal-regulated kinase (ERK) phosphorylation by KetoC promoted both the nuclear translocation of Nrf2 and its binding to the ARE in GECs. Further, GPR120 regulated the activation of KetoC induced-Nrf2-ARE signaling. CONCLUSION: KetoC exerts a protective function against the oxidative stress in GECs through GPR120-dependent ERK-Nrf2-ARE signaling.

Gingival epithelial barrier: regulation by beneficial and harmful microbes.

The gingival epithelium acts as a physical barrier to separate the biofilm from the gingival tissue, providing the first line of defense against bacterial invasion in periodontal disease. Disruption of the gingival epithelial barrier, and the subsequent penetration of exogenous pathogens into the host tissues, triggers an inflammatory response, establishing chronic infection. Currently, more than 700 different bacterial species have been identified in the oral cavity, some of which are known to be periodontopathic. These bacteria contribute to epithelial barrier dysfunction in the gingiva by producing several virulence factors. However, some bacteria in the oral cavity appear to be beneficial, helping gingival epithelial cells maintain their integrity and barrier function. This review aims to discuss current findings regarding microorganism interactions and epithelial barrier function in the oral cavity, with reference to investigations in the gut, where this interaction has been extensively studied.

Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin-dependent neutrophil lysis.

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA-treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA-treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA-induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.

Indirect regulation of PCSK9 gene in inflammatory response by Porphyromonas gingivalis infection.

Pro-protein convertase subtilisin/kexin type 9 (PCSK9), a secreted serine protease, regulates serum low-density lipoprotein (LDL) cholesterol levels by targeting the degradation of LDL receptor (LDLR) in the liver. Although previous reports describe elevated levels of PCSK9 in patients with periodontitis, the mechanisms that trigger this increase in serum PCSK9 levels and induce the related inflammatory response remain unclear. In an unc93b1-deficient mouse of Porphyromonas gingivalis infection, nucleic acid antigen recognition via Toll-like receptors was found to promote PCSK9 production, suggesting an indirect role for tumor necrosis factor-α as an inducer of PCSK9 in contrast to that reported in previous studies. Furthermore, PCSK9 production was independent of the TIR domain-containing adapter-inducing interferon-ß-dependent signaling pathway. These results indicate that changes in LDLR expression precede an increase in the serum PCSK9 level in the context of an infectious disease such as periodontitis.

Peptides from rice endosperm protein restrain periodontal bone loss in mouse model of periodontitis.

OBJECTIVE: Food-derived peptides have been reported to exhibit antibacterial activity against periodontal pathogenic bacteria. However, no effect has been shown on inflammation and bone resorption in periodontal pathology. The overall objective of the current study was to investigate how rice peptides influence biological defense mechanisms against periodontitis-induced inflammatory bone loss, and identify their novel functions as a potential anti-inflammatory drug. DESIGN: The expression of inflammatory and osteoclast-related molecules was examined in mouse macrophage-derived RAW 264.7 cell cultures using qPCR. Subsequently, the effect of these peptides on inflammatory bone loss in mouse periodontitis was examined using a mouse model of tooth ligation. Briefly, periodontal bone loss was induced for 7 days in mice by ligating the maxillary second molar and leaving the contralateral tooth un-ligated (baseline control). The mice were microinjected daily with the peptide in the gingiva until the day before euthanization. One week after the ligation, TRAP-positive multinucleated cells (MNCs) were enumerated from five random coronal sections of the ligated sites in each mouse. RESULTS: Rice peptides REP9 and REP11 significantly inhibited transcription activity of inflammatory and osteoclast-related molecules. Local treatment with the rice peptides, in mice subjected to ligature-induced periodontitis, inhibited inflammatory bone loss, explaining the decreased numbers of osteoclasts in bone tissue sections. CONCLUSION: Therefore, these data suggested that the rice peptides possess a protective effect against periodontitis.

A bacterial metabolite ameliorates periodontal pathogen-induced gingival epithelial barrier disruption via GPR40 signaling.

Several studies have demonstrated the remarkable properties of microbiota and their metabolites in the pathogenesis of several inflammatory diseases. 10-Hydroxy-cis-12-octadecenoic acid (HYA), a bioactive metabolite generated by probiotic microorganisms during the process of fatty acid metabolism, has been studied for its protective effects against epithelial barrier impairment in the intestines. Herein, we examined the effect of HYA on gingival epithelial barrier function and its possible application for the prevention and treatment of periodontal disease. We found that GPR40, a fatty acid receptor, was expressed on gingival epithelial cells; activation of GPR40 by HYA significantly inhibited barrier impairment induced by Porphyromonas gingivalis, a representative periodontopathic bacterium. The degradation of E-cadherin and beta-catenin, basic components of the epithelial barrier, was prevented in a GPR40-dependent manner in vitro. Oral inoculation of HYA in a mouse experimental periodontitis model suppressed the bacteria-induced degradation of E-cadherin and subsequent inflammatory cytokine production in the gingival tissue. Collectively, these results suggest that HYA exerts a protective function, through GPR40 signaling, against periodontopathic bacteria-induced gingival epithelial barrier impairment and contributes to the suppression of inflammatory responses in periodontal diseases.

Pneumococcal DNA-binding proteins released through autolysis induce the production of proinflammatory cytokines via toll-like receptor 4.

Streptococcus pneumoniae is a leading cause of bacterial pneumonia. Our previous study suggested that S. pneumoniae autolysis-dependently releases intracellular pneumolysin, which subsequently leads to lung injury. In this study, we hypothesized that pneumococcal autolysis induces the leakage of additional intracellular molecules that could increase the pathogenicity of S. pneumoniae. Liquid chromatography tandem-mass spectrometry analysis identified that chaperone protein DnaK, elongation factor Tu (EF-Tu), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were released with pneumococcal DNA by autolysis. We demonstrated that recombinant (r) DnaK, rEF-Tu, and rGAPDH induced significantly higher levels of interleukin-6 and tumor necrosis factor production in peritoneal macrophages and THP-1-derived macrophage-like cells via toll-like receptor 4. Furthermore, the DNA-binding activity of these proteins was confirmed by surface plasmon resonance assay. We demonstrated that pneumococcal DnaK, EF-Tu, and GAPDH induced the production of proinflammatory cytokines in macrophages, and might cause host tissue damage and affect the development of pneumococcal diseases.

Profiling biomarkers in gingival crevicular fluid using multiplex bead immunoassay.

OBJECTIVE: Biomarkers in gingival crevicular fluid (GCF) have been investigated; however, measurements were limited by the small sample volume available. The aim of this study was to determine the levels of 40 different cytokines and chemokines in GCF samples. DESIGN: Eleven patients with generalised chronic periodontitis participating in a supportive periodontal therapy programme with remaining probing pocket depths (PDs) of >5mm were enrolled. One healthy and two diseased sites were sampled in each subject. Forty biomarkers in GCF were examined using a multiplex bead immunoassay. Porphyromonas gingivalis from the diseased sites was quantified by real-time polymerase chain reaction. RESULTS: Twenty-six biomarkers were detected in the GCF samples using the multiplex bead immunoassay. The levels of nine biomarkers were significantly different between the diseased and healthy sites after adjustment with Bonferroni's correction. The level of 26 biomarkers in diseased sites was compared between bleeding on probing (BOP)-positive and BOP-negative sites. Interleukin (IL)-1ß and interferon-inducible protein (IP)-10 levels were significantly higher in BOP-positive diseased sites than BOP-negative diseased sites after adjustment for multiple comparisons (IL-1ß, p=0.0007, IP-10; p=0.0009). In addition, the levels of IL-1ß in GCF were found to be strongly correlated with the P. gingivalis ratio (r=0.646, p=0.0012). CONCLUSION: IL-1ß levels in GCF correlate with the PDs, BOP and the presence of P. gingivalis in subgingival plaque. Multiplex bead assays can be useful in GCF studies. These findings can help in identifying new diagnostic methods in the diagnosis of periodontal disease.

Increased serum PCSK9 concentrations are associated with periodontal infection but do not correlate with LDL cholesterol concentration.

BACKGROUND: Periodontal disease increases the risk of atherothrombotic disease, and high concentrations of low density lipoprotein (LDL) cholesterol are considered to be involved; however, the underlying mechanisms are largely unknown. Recent studies demonstrated that proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role in circulating LDL cholesterol concentrations. The aim of the present study is to analyze serum PCSK9 concentrations and their relation to lipoprotein concentrations in periodontitis patients. METHODS: Sera were obtained from 40 periodontitis patients and 30 control subjects. PCSK9 concentrations, high-sensitivity C-reactive protein (hs-CRP), IL-6, TNF-α and Porphyromonas gingivalis antibodies were measured by ELISA, and lipid profiles were determined by a commercial laboratory. RESULTS: Periodontitis patients demonstrated significantly higher serum antibody titer to P. gingivalis and hs-CRP concentrations than control subjects, suggesting infection with P. gingivalis and a systemic inflammatory response. PCSK9 concentrations in periodontitis patients were significantly higher than those in control subjects. However, the concentrations of total and LDL cholesterols were not significantly different between periodontitis patients and control subjects. Moreover, no correlations were observed between PCSK9 concentrations and lipid profiles. CONCLUSION: Periodontal infection upregulates PCSK9 production. However, further studies are required to elucidate how periodontal infection affects PCSK9 concentrations and subsequent lipid metabolism.

Effect of interleukin-17 on the expression of chemokines in gingival epithelial cells.

The role of interleukin (IL)-17 in cellular communication in inflammation has been well described, and a positive correlation between the severity of periodontitis and the level of IL-17 was reported. Although epithelial cells are a major target of IL-17, little is known about the effect of IL-17 on the production of chemokines by human gingival epithelial cells (HGECs). We evaluated the effects of IL-17 on the expression of CXCL8 and CCL2 by HGECs using quantitative real-time PCR and ELISA. In addition, the role of the nuclear factor (NF)-κB signalling pathway in the IL-17-mediated expression of chemokines was assessed using a specific inhibitor. Stimulation with IL-17 up-regulated the expression of CXCL8 mRNA but not of CCL2 mRNA in HGECs, whereas tumour necrosis factor-α (TNF-α) elevated the expression of mRNA for both chemokines. Stimulation with IL-17 up-regulated the secretion of CXCL8 protein, but not the secretion of CCL2 protein. The effect of IL-17 on CXCL8 production was suppressed using an anti-IL-17R Ig, suggesting a role for a specific receptor-ligand interaction. Inhibition of the NF-κB signalling pathway demonstrated that NF-κB activation is required for the CXCL8 expression in HGECs. In conclusion, IL-17 is involved in the regulation of the innate immune response in HGECs by inducing CXCL8 production.