Differential and coordinated expression of defensins and cytokines by gingival epithelial cells and dendritic cells in response to oral bacteria.

BACKGROUND: Epithelial cells and dendritic cells (DCs) both initiate and contribute to innate immune responses to bacteria. However, much less is known about the coordinated regulation of innate immune responses between GECs and immune cells, particularly DCs in the oral cavity. The present study was conducted to investigate whether their responses are coordinated and are bacteria-specific in the oral cavity. RESULTS: The beta-defensin antimicrobial peptides hBD1, hBD2 and hBD3 were expressed by immature DCs as well as gingival epithelial cells (GECs). HBD1, hBD2 and hBD3 are upregulated in DCs while hBD2 and hBD3 are upregulated in GECs in response to bacterial stimulation. Responses of both cell types were bacteria-specific, as demonstrated by distinctive profiles of hBDs mRNA expression and secreted cytokines and chemokines in response to cell wall preparations of various bacteria of different pathogenicity: Fusobacterium nucleatum, Actinomyces naeslundii and Porphyromonas gingivalis. The regulation of expression of hBD2, IL-8, CXCL2/GRObeta and CCL-20/MIP3alpha by GECs was greatly enhanced by conditioned medium from bacterially activated DCs. This enhancement was primarily mediated via IL-1beta, since induction was largely attenuated by IL-1 receptor antagonist. In addition, the defensins influence DCs by eliciting differential cytokine and chemokine secretion. HBD2 significantly induced IL-6, while hBD3 induced MCP-1 to approximately the same extent as LPS, suggesting a unique role in immune responses. CONCLUSIONS: The results suggest that cytokines, chemokines and beta-defensins are involved in interaction of these two cell types, and the responses are bacteria-specific. Differential and coordinated regulation between GECs and DCs may be important in regulation of innate immune homeostasis and response to pathogens in the oral cavity.

PAR1- and PAR2-induced innate immune markers are negatively regulated by PI3K/Akt signaling pathway in oral keratinocytes.

BACKGROUND: Protease-Activated Receptors (PARs), members of G-protein-coupled receptors, are activated by proteolytic activity of various proteases. Activation of PAR1 and PAR2 triggers innate immune responses in human oral keratinocytes (HOKs), but the signaling pathways downstream of PAR activation in HOKs have not been clearly defined. In this study, we aimed to determine if PAR1- and PAR2-mediated signaling differs in the induction of innate immune markers CXCL3, CXCL5 and CCL20 via ERK, p38 and PI3K/Akt. RESULTS: Our data show the induction of innate immunity by PAR1 requires both p38 and ERK MAP kinases, while PAR2 prominently signals via p38. However, inhibition of PI3K enhances expression of innate immune markers predominantly via suppressing p38 phosphorylation signaled by PAR activation. CONCLUSION: Our data indicate that proteases mediating PAR1 and PAR2 activation differentially signal via MAP kinase cascades. In addition, the production of chemokines induced by PAR1 and PAR2 is suppressed by PI3K/Akt, thus keeping the innate immune responses of HOK in balance. The results of our study provide a novel insight into signaling pathways involved in PAR activation.

Age-related heterogeneity in dental caries and associated risk factors in individuals with cystic fibrosis ages 6-20 years: A pilot study.

BACKGROUND: The literature conflicts regarding dental caries risk in cystic fibrosis (CF) relative to controls. METHODS: Prospective, observational study of age-related heterogeneity in caries rates and potential risk factors in individuals with CF ages 6-20 at a single clinic in Washington state (N=85). Caries rates for enrolled CF participants and historical controls from NHANES were compared using cubic spline regression models. Generalized linear regression models identified correlates of age and caries in CF. RESULTS: Children ages 6-9 with CF had significantly lower caries than controls (Holm's P<0.05). There was no difference for ages 10-20 by CF status (Holm's P>0.05). Various biological/intraoral, medical, and behavioral factors were associated with caries and age in CF. CONCLUSIONS: Younger children with CF may be protected from caries, but there is apparent loss of protection in early adolescence associated with multiple risk factors. Additional studies are needed to confirm these findings.

Increased Levels of Dissolved Titanium Are Associated With Peri-Implantitis - A Cross-Sectional Study.

BACKGROUND: Peri-implantitis represents a disruption of the biocompatible interface between the titanium dioxide layer of the implant surface and the peri-implant tissues. Increasing preclinical data suggest that peri-implantitis microbiota not only triggers an inflammatory immune response but also causes electrochemical alterations of the titanium surfaces, i.e., corrosion, that aggravate this inflammatory response. Thus, it was hypothesized that there is an association between dissolution of titanium from dental implants, which suggests corrosion, and peri-implantitis in humans. The objective of this study is to compare levels of dissolved titanium in submucosal plaque collected from healthy implants and implants with peri-implantitis. METHODS: Submucosal plaque from 20 implants with peri-implantitis and 20 healthy implants was collected with sterile curets from 30 participants. Levels of titanium were quantified using inductively coupled plasma mass spectrometry and normalized for mass of bacterial DNA per sample to exclude confounding by varying amounts of plaque per site. Statistical analysis was performed using generalized estimated equations to adjust for clustering of implants per participant. RESULTS: Implants with peri-implantitis harbored significantly higher mean levels of titanium (0.85 ± 2.47) versus healthy implants (0.07 ± 0.19) after adjusting for amount of plaque collected per site (P = 0.033). CONCLUSIONS: Greater levels of dissolved titanium were detected in submucosal plaque around implants with peri-implantitis compared with healthy implants, indicating an association between titanium dissolution and peri-implantitis. Factors triggering titanium dissolution, as well as the role of titanium corrosion in the peri-implant inflammatory process, warrant further investigation.

Antimicrobial barrier of an in vitro oral epithelial model.

OBJECTIVE: Oral epithelia function as a microbial barrier and are actively involved in recognizing and responding to bacteria. Our goal was to examine a tissue engineered model of buccal epithelium for its response to oral bacteria and proinflammatory cytokines and compare the tissue responses with those of a submerged monolayer cell culture. DESIGN: The tissue model was characterized for keratin and beta-defensin expression. Altered expression of beta-defensins was evaluated by RT-PCR after exposure of the apical surface to oral bacteria and after exposure to TNF-alpha in the medium. These were compared to the response in traditional submerged oral epithelial cell culture. RESULTS: The buccal model showed expression of differentiation specific keratin 13, hBD1 and hBD3 in the upper half of the tissue; hBD2 was not detected. hBD1 mRNA was constitutively expressed, while hBD2 mRNA increased 2-fold after exposure of the apical surface to three oral bacteria tested and hBD3 mRNA increased in response to the non-pathogenic bacteria tested. In contrast, hBD2 mRNA increased 3-600-fold in response to bacteria in submerged cell culture. HBD2 mRNA increased over 100-fold in response to TNF-alpha in the tissue model and 50-fold in submerged cell culture. Thus, the tissue model is capable of upregulating hBD2, however, the minimal response to bacteria suggests that the tissue has an effective antimicrobial barrier due to its morphology, differentiation, and defensin expression. CONCLUSIONS: The oral mucosal model is differentiated, expresses hBD1 and hBD3, and has an intact surface with a functional antimicrobial barrier.

Antibacterial activity of gold-titanates on Gram-positive cariogenic bacteria.

Objective: Gram-positive cariogenic bacteria are etiological agents in dental caries; therefore, strategies to inhibit these bacteria to reduce the incident of this disease have intensified. In this study, we investigated antibacterial activities of titanates and gold-titanates against Lactobacillus casei (Lc) and Streptococcus mutans (Sm). Materials and methods: Monosodium titanate (MST), nanomonosodium titanate (nMST) and amorphous peroxo-titanate (APT), which are inorganic compounds with high-binding affinity for specific metal ions, were used. Total bacterial proteins were measured to represent bacterial cell mass after 24 h incubation with gold-titanates. We further examined the effect of nMST-Au(III) concentrations (10,200,400 mg/L) on Lc and Sm cell viability over time via Live/Dead fluorescent staining and colony forming units (CFUs). Transmission electron microscopy (TEM) was used to determine specific locations on the bacterial cells affected by the nMST-Au(III). Results: We found all gold-titanates and APT alone reduced bacterial protein for Lc (p value <0.001) while only MST-Au(III) and nMST-Au(III) affected Sm growth (p value <0.001). Overall, nMST-Au(III) showed the most effectiveness against both Lc and Sm at 400 mg/L. The Live/Dead staining showed all concentrations of nMST-Au(III) affected Lc growth but only 200 and 400 mg/L nMST-Au(III) interrupted Sm growth. The growth curves based on CFUs/mL showed all nMST-Au(III) concentrations affected growth of both Lc and Sm. TEM images showed nMST-Au(III) attached to Lc and Sm cell wall and were internalized into both cells.Conclusions: nMST-Au(III) demonstrated potential antimicrobial activity against Gram-positive cariogenic bacteria. These results support further development of nMST-Au(III) as a potential novel material to prevent dental caries.

Nickel alloys in the oral environment.

The use of nickel casting alloys for long-term restorations in dentistry has long been controversial. A 'tug-of-war' between economic, engineering and biological considerations is central to this controversy; nickel-casting alloys have low costs and favorable physical properties, but are corrosion-prone in the oral environment. Clinicians and researchers have questioned the safety of nickel-containing dental alloys because several nickel compounds are known to cause adverse biological effects in vivo and in vitro in contexts outside of dentistry. The debate revolves around the extent to which corrosion products from oral restorations cause intraoral or systemic biological problems. Current evidence suggests that nickel alloys may be used successfully and safely in dentistry if clinical risks are taken into account. However, these alloys may cause significant clinical problems, primarily allergenic and inflammatory, if the risks are ignored.

Effect of Ni(II) on inflammatory gene expression in THP1 monocytic cells.

Nickel-containing alloys are in common use for dental restorations, but tend to corrode and release Ni(II) in service. Ni(II) increases secretion of several inflammatory cytokines from activated monocytic cells, suggesting that nickel alloys may exaggerate inflammatory responses in adjacent periodontal tissues. In this work, the effects of Ni(II) on expression of inflammatory cytokine and receptor genes as well as nuclear factor-kappa B (NFκB)-related genes were assessed using quantitative real-time polymerase chain reaction (PCR) and PCR-based arrays in the human THP1 monocytic cell line pre-exposed to Ni(II) for 72 h, then activated by lipopolysaccharide. The expression of 10 inflammatory genes was down-regulated ≥50% by Ni(II) versus non-Ni(II) controls, whereas some genes like IL8 were up-regulated significantly by Ni(II). Expression of seven NFκB-related genes was up-regulated by Ni(II) by ≥50%, and HMOX1 expression, a redox protein regulated by NRF2, was increased by >500%. The current results suggest that Ni(II) has diverse effects on inflammatory gene expression, which may partly account for previous reports of Ni(II)-induced changes in inflammatory cytokine secretion from monocytes and alterations in NFκB regulation. Further work is needed to verify these effects in primary cells and to ascertain how Ni(II) alters gene expression.

Ni(II) alters the NFκB signaling pathway in monocytic cells.

Nickel-based alloys are used for dental restorations because of their strength, high moduli, and relatively low cost. However, these alloys corrode significantly in use, particularly in lower pH environments that are common under oral biofilms. Ni(II) corrosion products increase inflammatory cytokine secretion from activated monocytes, suggesting that nickel alloys may exacerbate inflammatory responses in adjacent periodontal tissues caused by dental plaque. Because inflammatory cytokine secretion is regulated in part by the NFκB signaling pathway, our goal in the current work was to determine whether Ni(II) altered cellular levels or nuclear localization of NFκB-family subtypes. THP1 monocytes were exposed to Ni(II) for 72 h, and activated with lipopolysaccharide for the last 30 min to 6 h. Secretion of IL6 and TNFα were measured using ELISA, and NFκB levels and localization was measured using SDS-PAGE with immunoblots and digital analysis. We observed that Ni(II) did not alter the levels of secreted TNFα from activated monocytes, but increased secreted IL6 levels about 30% over controls. Ni(II) did not alter whole-cell levels of any NFκB subtype, but increased nuclear persistance of p65 and c-Rel. Our results suggest that Ni(II) may increase inflammatory cytokine secretion by increasing nuclear localization of some NFκB subtypes. Further studies should be done to determine the prominence of this mechanism in clinical environments.

Peroxotitanate- and monosodium metal-titanate compounds as inhibitors of bacterial growth.

Sodium titanates are ion-exchange materials that effectively bind a variety of metal ions over a wide pH range. Sodium titanates alone have no known adverse biological effects but metal-exchanged titanates (or metal titanates) can deliver metal ions to mammalian cells to alter cell processes in vitro. In this work, we test a hypothesis that metal-titanate compounds inhibit bacterial growth; demonstration of this principle is one prerequisite to developing metal-based, titanate-delivered antibacterial agents. Focusing initially on oral diseases, we exposed five species of oral bacteria to titanates for 24 h, with or without loading of Au(III), Pd(II), Pt(II), and Pt(IV), and measuring bacterial growth in planktonic assays through increases in optical density. In each experiment, bacterial growth was compared with control cultures of titanates or bacteria alone. We observed no suppression of bacterial growth by the sodium titanates alone, but significant (p < 0.05, two-sided t-tests) suppression was observed with metal-titanate compounds, particularly Au(III)-titanates, but with other metal titanates as well. Growth inhibition ranged from 15 to 100% depending on the metal ion and bacterial species involved. Furthermore, in specific cases, the titanates inhibited bacterial growth 5- to 375-fold versus metal ions alone, suggesting that titanates enhanced metal-bacteria interactions. This work supports further development of metal titanates as a novel class of antibacterials.

Differential effects of periopathogens on host protease inhibitors SLPI, elafin, SCCA1, and SCCA2.

OBJECTIVE: Secretory leukocyte peptidase inhibitors (SLPI), elafin, squamous cell carcinoma antigen 1 and 2 (SCCA1 and SCCA2) are specific endogenous serine protease inhibitors expressed by epithelial cells that prevent tissue damage from excessive proteolytic enzyme activity due to inflammation. To determine the effects of various periopathogens on these protease inhibitors, we utilized human gingival epithelial cells (GECs) challenged with cell-free bacteria supernatants of various periopathogens Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. DESIGN: The gene expression and secretion of SLPI, elafin, SCCA1, and SCCA2 were determined using real-time PCR and ELISA, respectively. The direct effects of periopathogens and P. gingivalis gingipain mutants on these inhibitors were examined in vitro by Western Blot. The effect on the innate immune response of GECs was measured by expression of antimicrobial peptides: human beta-defenisin-2 (hBD2) and chemokine (C-C motif) ligand 20 (CCL20). RESULTS: We found that SLPI, SCCA2, elafin, hBD2, and CCL20 gene expression levels were significantly induced (p<0.001) in response to P. gingivalis, whose virulence factors include cysteine proteases, but not in response to stimulation by other bacteria. P. gingivalis reduced the secretion of SLPI and elafin significantly in GECs, and degraded recombinant SLPI, elafin, SCCA1, and SCCA2. Differential degradation patterns of SLPI, elafin, SCCA1, and SCCA2 were observed with different bacteria as well as P. gingivalis mutants associated with the loss of specific gingipains secreted by P. gingivalis. In addition, pretreatment of GECs with SLPI, SCCA1, or SCCA2 partially blocked hBD2 and CCL20 mRNA expression in response to P. gingivalis, suggesting a protective effect. CONCLUSION: Our results suggest that different periopathogens affect the host protease inhibitors in a different manner, suggesting host susceptibility may differ in the presence of these pathogens. The balance between cellular protease inhibitors and their degradation may be an important factor in susceptibility to periodontal infection.

Phospholipase C, p38/MAPK, and NF-kappaB-mediated induction of MIP-3alpha/CCL20 by Porphyromonas gingivalis.

Macrophage inflammatory protein-3alpha/C-C chemokine ligand 20 (MIP-3alpha/CCL20) is an antimicrobial peptide that plays an important role in innate immunity. In addition to direct microbicidal effects, MIP-3alpha/CCL20 also exhibits cytokine-like functions that are critical during dendritic cell activation. The aim of the present study was to investigate further which signaling pathways are involved in the MIP-3alpha/CCL20 mRNA expression in response to whole-cell Porphyromonas gingivalis. Primary gingival epithelial cells (GECs) and the immortalized oral keratinocyte cell-line OKF6/TERT-2 were stimulated with whole-cell P. gingivalis. Prior to stimulation, GECs and OKF6/TERT-2 cells were pretreated with specific inhibitors for nuclear-factor-kappaB (NF-kappaB), mitogen-activated protein kinase (MAPK), phospholipase C (PLC), and phosphatidylinositol-3-kinase (PI3K). In GECs and OKF6/TERT-2 cells, activation of NF-kappaB was examined after exposure to P. gingivalis. The gene expression of MIP-3alpha/CCL20 was significantly induced in response to P. gingivalis (P

Modulation of expression of innate immunity markers CXCL5/ENA-78 and CCL20/MIP3alpha by protease-activated receptors (PARs) in human gingival epithelial cells.

Protease-activated receptors (PARs) are G-protein-coupled receptors with an active role in host defense. The two most highly expressed members of the PAR family in gingival epithelial cells (GECs) are PAR1 and PAR2. The major virulence factors of periodontal pathogen Porphyromonas gingivalis are its proteases which can activate PAR2. However, little is known about the function of PARs in GECs when they are activated by their endogenous agonist enzymes. The purpose of this study was to characterize how the expression of innate immune markers is modulated when PAR1 and PAR2 are activated by their agonist enzymes, thrombin and trypsin, respectively. Here, we report that activation of PAR1 and PAR2 induces cell proliferation at low concentration. Activation of PAR via proteolytic activity of thrombin and trypsin induces expression of CXCL5/ENA-78 and CCL20/MIP3alpha in a concentration-dependent manner. Induction of CXCL5 via PAR1 was inhibited in the presence of PAR1 cleavage blocking antibodies and by PAR1 siRNA. The induction of CXCL5 and CCL20 via PAR2 was inhibited by PAR2 siRNA. These findings indicate an active role in innate immune responses by PAR1 and PAR2 in GECs. Modulation of innate immunity by PARs may contribute to co-ordinated and balanced immunosurveillance in GECs.

Interplay of protease-activated receptors and NOD pattern recognition receptors in epithelial innate immune responses to bacteria.

Protease-activated receptors (PARs), nucleotide-binding oligomerization domain (NOD) receptors and Toll-like receptors (TLRs) play a role in innate immunity, but little is known about interaction between these receptors. The goal of this study was to investigate how silencing one receptor affects the expression of other receptors and downstream innate immune markers in response to bacteria. Human gingival epithelial cells (GECs) were transfected with siRNA specific for PAR1 or PAR2, then stimulated with periopathogen Porphyromonas gingivalis, bridging organism between pathogens and non-pathogens Fusobacterium nucleatum, or non-pathogen Streptococcus gordonii. PAR1 or PAR2 knock-down resulted in up-regulated NOD1 and NOD2 expression with P. gingivalis or F. nucleatum stimulation (p<0.01), as well as enhanced TLR2 and TLR4 expression when cells were stimulated by bacteria that utilize TLR2 or TLR4, respectively. Involvement of PARs for induction of CC chemokine ligand 20 (CCL20), a cytokine with antimicrobial properties, was observed following stimulation of the three bacterial species. Furthermore, results from multiple cytokine ELISA array showed receptors utilized in the induction of various innate immune markers are tailored to individual bacterium tested. Our data suggest complex interplay of several receptors is required for appropriate innate immune responses to the different types of bacteria present within the oral cavity and that receptor expression itself is altered depending on which organism the cell encounters.

Protease-activated receptor 2 mediates human beta-defensin 2 and CC chemokine ligand 20 mRNA expression in response to proteases secreted by Porphyromonas gingivalis.

The oral pathogen Porphyromonas gingivalis secretes proteases such as Arg-gingipain B (RgpB) that activate protease-activated receptors (PARs). Human beta-defensins (hBDs) and the macrophage inflammatory protein 3alpha/CC chemokine ligand 20 (CCL20) produced by epithelial cells are antimicrobial peptides that provide cytokine function and play an important role in innate immunity. The aim of the present study was to determine whether specific members of the PAR family mediate the expression of these innate immunity markers in gingival epithelial cells (GECs) when exposed to P. gingivalis cell-free culture supernatant or purified RgpB. hBD-2 mRNA in GECs was induced in response to supernatant and purified RgpB from P. gingivalis (P = 0.02 and P = 0.016, respectively). This effect was abrogated by the protease inhibitor tosyl-l-lysine chloromethyl ketone (TLCK) (P < 0.05). In response to P. gingivalis supernatant and to purified RgpB, the hBD-2 mRNA expression was significantly decreased in PAR-2 gene knockdown cells, whereas no change was detected in PAR-1 gene knockdown cells. CCL20 mRNA expression also increased in response to the supernatant of P. gingivalis, and this effect was blocked by the protease inhibitor, TLCK (P = 0.05 and P = 0.024, respectively), and was blocked in PAR-2 gene knockdown cells. Our data indicate that hBD-2 and CCL20 mRNA up-regulation by P. gingivalis supernatant and purified RgpB was mediated via PAR-2, but not via PAR-1, and that proteases play a role in the regulation of innate immune responses in GECs. GECs use PARs to recognize P. gingivalis and mediate cell responses involved in innate immunity.

Innate immune response of oral and foreskin keratinocytes: utilization of different signaling pathways by various bacterial species.

The innate immune response is critical for the epithelial antimicrobial barrier. The human beta-defensins are small, cationic antimicrobial peptides that are made by epithelial cells and that play a role in mucosal and skin defenses. Human beta-defensin 1 (hBD-1) is expressed constitutively in epithelial tissues, whereas hBD-2 and hBD-3 are expressed in response to bacterial stimuli or inflammation. Previous studies showed that hBD-2 was induced by Fusobacterium nucleatum cell wall extract without the involvement of the NF-kappaB transcription factors, which typically are associated with innate immunity and inflammation. The goal of this study was to characterize signaling pathways involved in hBD-2 induction in response to commensal and pathogenic bacteria. Cultured human oral and foreskin keratinocytes were treated separately with inhibitors of NF-kappaB, c-Jun N-terminal kinase (JNK), and p38 and then stimulated with oral commensal Streptococcus gordonii, oral pathogens Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, skin commensal Staphylococcus epidermidis, or skin pathogen Streptococcus pyogenes. Different bacteria induced different levels of hBD-2 and in response to the various inhibitors tested, although certain common patterns were observed for commensal- and pathogen-stimulated cells. hBD-2 induction by all bacteria tested was partially or completely blocked by inhibitors of the JNK and p38 pathways. However, in addition, hBD-2 induction by pathogenic bacteria in both oral and foreskin keratinocytes was blocked by inhibitors of NF-kappaB. The results indicate that commensal and pathogenic bacteria utilize different pathways in hBD-2 induction and suggest that epithelial cells from different body sites have common signaling mechanisms to distinguish between commensal and pathogenic bacteria.

Distribution of erm(F) and tet(Q) genes in 4 oral bacterial species and genotypic variation between resistant and susceptible isolates.

BACKGROUND: Bacteroides forsythus, Porphyromonas gingivalis and Prevotella intermedia are Gram-negative anaerobic bacteria that are currently considered potential periopathogens. Prevotella nigrescens has recently been separated from P. intermedia and its rôle in periodontitis is unknown. The erm(F) gene codes for an rRNA methylase, conferring resistance to macrolides, lincosamides and streptogramin B (MLSB), and the tet(Q) gene for a ribosomal protection protein, conferring resistance to tetracycline. The presence of these resistance genes could impair the use of antibiotics for therapy. PURPOSE: The aim of this study was to determine the carriage of erm(F) and tet(Q), and genetic variability of 12 Porphyromonas gingivalis, 10 Prevotella intermedia, 25 Prevotella nigrescens and 17 Bacteroides forsythus isolates from 9 different patient samples. METHODS: We used polymerase chain reaction (PCR) for detecting antibiotic resistance genes, and pulsed-field gel electrophoresis (PFGE) for detecting genetic variability among the isolates. RESULTS: Thirty-one (48%) isolates were resistant to both erythromycin and tetracycline and carried the erm(F) and tet(Q) genes, eight (13%) were tetracycline resistant and carried the tet(Q) gene, 9 (14%) were erythromycin resistant and carried the erm(F) gene, and 12 (19%) isolates did not carry antibiotic resistance genes. PFGE was used to compare isolates from the same patient and isolates from different patient samples digested with XbaI. No association was found between antibiotic resistance gene carriage and PFGE patterns in any species examined. All isolates of the same species from the same patient had highly related or identical PFGE patterns. Isolates of same species from different patients had unique PFGE pattern for each species tested. CONCLUSION: All isolates of the same species from any one patient were genetically related to each other but distinct from isolates from other patients, and 66% of the patients carried antibiotic resistant isolates, which could impair antibiotic therapy.

Protease-activated receptor signaling increases epithelial antimicrobial peptide expression.

Epithelial tissues provide both a physical barrier and an antimicrobial barrier. Antimicrobial peptides of the human beta-defensin (hBD) family are part of the innate immune responses that play a role in mucosal defense. hBDs are made in epithelia including oral epithelium where the bacterial load is particularly great. hBD-2 and hBD-3 are up-regulated in response to bacterial stimuli. Previous studies show that hBD-2 expression in human gingival epithelial cells (GEC) is stimulated by both nonpathogenic and pathogenic bacteria, including Porphyromonas gingivalis, a Gram-negative pathogen associated with periodontitis. Present evidence suggests that hBD-2 expression in GEC uses several signaling pathways, including an NF-kappaB-mediated pathway but without apparent LPS-TLR4 signaling. Protease-activated receptors (PAR) are G-protein-coupled receptors that mediate cellular responses to extracellular proteinases. P. gingivalis secretes multiple proteases that contribute to its virulence mechanisms. To determine whether PAR signaling is used in hBD-2 induction, GEC were stimulated with wild-type P. gingivalis or mutants lacking one or more proteases. hBD-2 mRNA expression was reduced in GEC stimulated with single protease mutants (11-67% compared with wild type), strongly reduced in double mutants (0.1-16%), and restored to wild-type levels (93%) in mutant with restored protease activity. Stimulation by wild type was partially blocked by inhibitors of phospholipase C, a main signaling pathway for PARs. Expression of hBD-3 was unaffected. Peptide agonist of PAR-2, but not PAR-1 activator, also induced hBD-2 in GEC. Thus, P. gingivalis proteases are directly involved in regulation of hBD-2 in cultured GEC, and this induction partially uses the PAR-2 receptor and signaling pathway.