Early Caries in an In Vivo Model: Structural and Nanomechanical Characterization.

The utilization of rat models in cariology research has made substantial contributions to decipher mechanisms of caries formation and to develop preventive treatments. The existing rat models still have potential for improvement toward establishing a more accurate standard caries protocol to utilize in testing and/or developing new dental technologies. The current caries-scoring methods rely on optical microscopy-based techniques, which necessitates formation of highly advanced lesions. Moreover, models that facilitate the implementation of cariogenic bacteria by shifting the balance of oral flora through desalivation and/or antibiotic treatment create a nonnatural environment. Furthermore, there is a paucity of detailed structural and mechanical characterization on the resulting carious lesions. The purpose of this study was to develop a rat model that induces formation of mild carious lesions and to provide comprehensive structural and mechanical characterization. With this aim in mind, an in vivo model promoting progression of mild lesions was established with specific pathogen-free Sprague-Dawley rats. Cariogenic bacteria, Streptococcus mutans, was implemented into the oral flora without the use of antibiotics or desalivation surgery. During caries formation, progression of the infection was monitored by quantifying the relative abundance of S. mutans in oral flora with quantitative real-time polymerase chain reaction. A significant increase in colonization efficacy of S. mutans was detected during cariogenic challenge ( P < 0.01). The resulting carious lesions were analyzed by conventional light optical and scanning electron microscopy. A detailed structural and morphological characterization on fissure caries with different degrees of severity was provided. The changes in the morphology and demineralization state of the sound and carious tissues were quantified by energy-dispersive X-ray spectroscopy, and local mechanical properties were acquired with nanoindentation. The principles laid out in this work can be utilized in cariology research and developed into a standard protocol for future studies.

Influence of histamine on the expression of CCL20 in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Histamine plays an important role during allergic and inflammatory reactions, and it has been suggested to influence periodontal inflammation. The aim of this study was to investigate the effects of histamine on the expression of the antimicrobial peptide C-C chemokine ligand 20 (CCL20) in human gingival fibroblasts (HGFs) when exposed to toll-like receptor (TLR) agonists. MATERIAL AND METHODS: Monolayers of HGFs from three different donors were exposed to histamine, alone, and in combination with Pam3CSK4 (a TLR2 agonist) or lipopolysaccharide (LPS) from Escherichia coli (a TLR4 agonist), for 2, 4, 6 or 12 h. In another experimental group, cells were pretreated with a specific histamine-1 receptor antagonist (H1R) antagonist, cetirizine. Real-time PCR analysis was performed to detect expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), CCL20 and interleukin-8 (IL8) genes. The levels of CCL20 and IL-8 protein were determined by ELISA. RESULTS: In HGFs, histamine induced expression of CCL20 and IL8 genes in a time-dependent manner (p < 0.05). Combined stimulation with histamine and Pam3CSK4 or LPS led to a significant amplification in expression of CCL20 and IL-8 when compared with treatment with each stimulant alone (p < 0.05), and this effect was mediated via pathways involving the H1R (p < 0.05). CONCLUSION: The results of this study suggest a sensitizing effect of histamine on early innate immune responses of HGFs when simultaneously exposed to bacterial virulence factors.

Houttuynia cordata modulates oral innate immune mediators: potential role of herbal plant on oral health.

OBJECTIVE: Epithelial cells play an active role in oral innate immunity by producing various immune mediators. Houttuynia cordata Thunb (H. cordata), a herbal plant found in Asia, possesses many activities. However, its impacts on oral innate immunity have never been reported. The aim of this study was to determine the effects of H. cordata extract on the expression of innate immune mediators produced by oral epithelial cells. MATERIALS AND METHODS: Primary gingival epithelial cells (GECs) were treated with various concentrations of the extract for 18 h. The gene expression of hBD2, SLPI, cytokines, and chemokines was measured using quantitative real-time RT-PCR. The secreted proteins in the culture supernatants were detected by ELISA or Luminex assay. Cytotoxicity of the extract was assessed using CellTiter-Blue Assay. RESULTS: H. cordata significantly induced the expression of hBD2, SLPI, IL-8, and CCL20 in a dose-dependent manner without cytotoxicity. The secreted hBD2 and SLPI proteins were modulated, and the levels of IL-2, IL-6, IL-8, and IFN-γ were significantly induced by the extract. CONCLUSIONS: Our data indicated that H. cordata can modulate oral innate immune mediators. These findings may lead to the development of new topical agents from H. cordata for the prevention and treatment of immune-mediated oral diseases.

Protease inhibitor levels in periodontal health and disease.

BACKGROUND AND OBJECTIVE: Our previous study showed that protease inhibitors were attenuated by the periodontal pathogen Porphyromonas gingivalis in cultured gingival epithelial cells. We hypothesize that fewer protease inhibitors would be present in more advanced periodontal disease sites, where the level of P. gingivalis may be high. The goal of this study was to investigate the relationship between the protease inhibitor [secretory leukocyte protease inhibitor (SLPI), elastase-specific inhibitor (ELAFIN) and squamous cell carcinoma antigen (SCCA)] levels in gingival crevicular fluid and the number of P. gingivalis micro-organisms in subgingival plaque. MATERIAL AND METHODS: Plaque samples from subjects without (n = 18) and with moderate to advanced periodontitis (n = 41) were used to quantify P. gingivalis using real-time PCR. Protease inhibitor levels in the gingival crevicular fluid of all the subjects were determined by ELISA. RESULTS: P. gingivalis was detected in 68.3% of patients with periodontitis, while 16.7% of subjects without periodontitis had a detectable level of P. gingivalis. Patients with periodontitis and P. gingivalis in their plaque exhibited lower SLPI and ELAFIN levels (p < 0.001) compared with control subjects without periodontitis. Secretory leukocyte protease inhibitor was also reduced (p < 0.05) in gingival crevicular fluid of periodontitis patients without a detectable level of P. gingivalis. Periodontitis patients with high vs. low levels of P. gingivalis exhibited reciprocal mean levels of SLPI and ELAFIN concentrations. CONCLUSION: The reduced concentrations of SLPI and ELAFIN may contribute to the loss of host protective capacity and increase susceptibility to breakdown from chronic infection. The work of this investigation may aid in finding diagnostic and prognostic markers in periodontal health and disease and may also help in finding pharmacological targets directed against periodontal inflammation.

Epigenetic regulation of human ß-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria.

Gingival epithelia utilize multiple signaling pathways to regulate innate immune responses to various oral bacteria, but little is understood about how these bacteria alter epithelial epigenetic status. In this study we report that DNA methyltransferase (DNMT1) and histone deacetylase expression were decreased in gingival epithelial cells treated with oral pathogen Porphyromonas gingivalis and nonpathogen Fusobacterium nucleatum. Pretreatment with trichostatin A and sodium butyrate, which increase acetylation of chromatin histones, significantly enhanced the gene expression of antimicrobial proteins human ß-defensin 2 (hBD2) and CC chemokine ligand 20 (CCL20) in response to both bacterial challenges. Pretreatment with DNMT inhibitor 5'-azacytidine increased hBD2 and CCL20 expression in response to F. nucleatum, but not to P. gingivalis. Furthermore, we observed a differential pattern of protein levels of H3K4me3, which has been associated with chromatin remodeling and activation of gene transcription, in response to P. gingivalis vs. F. nucleatum. This study provides a new insight into the bacteria-specific innate immune responses via epigenetic regulation.

Differential utilization of nuclear factor-kappaB signaling pathways for gingival epithelial cell responses to oral commensal and pathogenic bacteria.

INTRODUCTION: Human beta-defensin-2 (hBD-2) is an antimicrobial peptide, induced by bacterial stimuli and inflammation, that plays a role in mucosal and skin innate immune defense. The nuclear factor-kappaB (NF-kappaB) transcription factor family is important in innate and adaptive immune responses to bacteria and proinflammatory cytokines. NF-kappaB operates via the traditional IKKbeta signaling, as well as an alternative pathway utilizing IKKalpha signaling, which is important in keratinocyte differentiation. Our previous studies showed that pathogenic, but not commensal, bacteria used NF-kappaB signaling in hBD-2 induction. The objective of this study was to understand which arm of the NF-kappaB pathway is involved in gingival epithelial cell responses to pathogenic bacteria, including hBD-2 induction. METHODS: Cultured oral epithelial cells were transfected with synthetic small interfering RNAs (siRNAs) specific for various steps in each pathway, namely IKKbeta, TRAF6 and MyD88 in the canonical, and IKKalpha and TRAF3 in the alternative pathway, and subsequently stimulated with various oral bacteria. RESULTS: The hBD-2 induction level was reduced to 21-61% in cells in which the alternative NF-kappaB pathway was blocked and subsequently stimulated with pathogenic bacteria, while cells in which the canonical pathway was blocked showed reduction to 78-99%. Cells stimulated with commensals showed little change in hBD-2 induction level regardless of the siRNA used. Microarray analysis showed that oral epithelia differentially regulated numerous innate immune markers in response to pathogens and commensals. CONCLUSION: Our data suggest a role for the IKKalpha/TRAF3 pathway in NF-kappaB activation by pathogenic bacteria, while commensal bacteria do not utilize either NF-kappaB pathway, for hBD-2 induction.

Expression of defensins in gingiva and their role in periodontal health and disease.

Oral epithelium is a stratified squamous epithelium that functions as the barrier between the outside environment and the host. In the oral cavity, epithelial tissues are constantly exposed to a variety of bacteria, but most individuals maintain healthy homeostasis. Epithelial cells contribute to the innate host response, and antimicrobial peptide expression in all human epithelia, including oral epithelia, is an important part of this epithelial function. These antimicrobial peptides have a broad spectrum of activity against both Gram-negative and Gram-positive bacteria as well as against yeast and viruses. In humans these antimicrobial peptides include defensins and a cathelicidin family member LL-37 in skin and oral mucosa and other epithelia. The human defensins include the alpha-defensins of intestinal and neutrophil origin, and the beta-defensins of skin and oral mucosa and other epithelia. Present studies have identified specific signaling routes that pathogens and commensals take in stimulating these innate immune responses, and this may open the way for development of new therapeutic agents for periodontal diseases.

Intra- and interspecies regulation of gene expression by Actinobacillus actinomycetemcomitans LuxS.

The cell density-dependent control of gene expression is employed by many bacteria for regulating a variety of physiological functions, including the generation of bioluminescence, sporulation, formation of biofilms, and the expression of virulence factors. Although periodontal organisms do not appear to secrete acyl-homoserine lactone signals, several species, e.g., Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum, have recently been shown to secrete a signal related to the autoinducer II (AI-2) of the signal system 2 pathway in Vibrio harveyi. Here, we report that the periodontal pathogen Actinobacillus actinomycetemcomitans expresses a homolog of V. harveyi luxS and secretes an AI-2-like signal. Cell-free conditioned medium from A. actinomycetemcomitans or from a recombinant Escherichia coli strain (E. coli AIS) expressing A. actinomycetemcomitans luxS induced luminescence in V. harveyi BB170 >200-fold over controls. AI-2 levels peaked in mid-exponential-phase cultures of A. actinomycetemcomitans and were significantly reduced in late-log- and stationary-phase cultures. Incubation of early-log-phase A. actinomycetemcomitans cells with conditioned medium from A. actinomycetemcomitans or from E. coli AIS resulted in a threefold induction of leukotoxic activity and a concomitant increase in leukotoxin polypeptide. In contrast, no increase in leukotoxin expression occurred when cells were exposed to sterile medium or to conditioned broth from E. coli AIS(-), a recombinant strain in which luxS was insertionally inactivated. A. actinomycetemcomitans AI-2 also induced expression of afuA, encoding a periplasmic iron transport protein, approximately eightfold, suggesting that LuxS-dependent signaling may play a role in the regulation of iron acquisition by A. actinomycetemcomitans. Finally, A. actinomycetemcomitans AI-2 added in trans complemented a luxS knockout mutation in P. gingivalis by modulating the expression of the luxS-regulated genes uvrB and hasF in this organism. Together, these results suggest that LuxS-dependent signaling may modulate aspects of virulence and the uptake of iron by A. actinomycetemcomitans and induce responses in other periodontal organisms in mixed-species oral biofilm.

Signaling system in Porphyromonas gingivalis based on a LuxS protein.

The luxS gene of quorum-sensing Vibrio harveyi is required for type 2 autoinducer production. We identified a Porphyromonas gingivalis open reading frame encoding a predicted peptide of 161 aa that shares 29% identity with the amino acid sequence of the LuxS protein of V. harveyi. Conditioned medium from a late-log-phase P. gingivalis culture induced the luciferase operon of V. harveyi, but that from a luxS insertional mutant did not. In P. gingivalis, the expression of luxS mRNA was environmentally controlled and varied according to the cell density and the osmolarity of the culture medium. In addition, differential display PCR showed that the inactivation of P. gingivalis luxS resulted in up-regulation of a hemin acquisition protein and an arginine-specific protease and reduced expression of a hemin-regulated protein, a TonB homologue, and an excinuclease. The data suggest that the luxS gene in P. gingivalis may function to control the expression of genes involved in the acquisition of hemin.

Inhibition of epithelial cell apoptosis by Porphyromonas gingivalis.

Porphyromonas gingivalis is periodontal pathogen that is capable of invading gingival epithelial cells (GECs). Apoptotic responses of primary cultures of GECs to P. gingivalis were investigated with a DNA fragmentation ELISA assay. P. gingivalis induced a transient increase in GEC DNA fragmentation; however, after prolonged incubation GECs did not undergo apoptosis. Furthermore, P. gingivalis blocked apoptosis in GECs following stimulation with camptothecin. Immunoblotting of GECs with Bcl-2 or Bax antibodies showed that P. gingivalis up-regulated Bcl-2 levels in GECs, whereas Bax levels were transiently elevated and declined after 24 h stimulation. Streptococcus gordonii did not affect levels of either molecule. RT-PCR demonstrated that induction of Bcl-2 occurs at the transcriptional level. The results suggest that P. gingivalis can inhibit apoptosis in GECs by up-regulation of the anti-apoptotic molecule Bcl-2. The prevention of host cell apoptosis may represent a strategy for P. gingivalis survival within invaded GECs.

Serum antibody titers to Bacteroides forsythus in elderly subjects with gingivitis or periodontitis.

BACKGROUND: The risks for periodontal disease appear to increase with age. STUDY PURPOSE: To determine associations between clinical findings, the presence of specific bacteria in periodontal pockets, and serum antibody titers. 10 older subjects (mean age=73.0 years SD+/-4.9) with confirmed gingivitis only (gingivitis group) and 10 subjects with periodontitis (mean age: 76.1 years, SD+/-10.4) (periodontitis group) were studied. RESULTS: The mean group differences for probing depth and clinical attachment levels were 4.1 mm and 5.6 mm, respectively, and were significantly different (p<0.001). Both groups had high plaque scores (>60% surfaces with plaque). DNA probes demonstrated that B. forsythus was present in 8/10 samples from the periodontitis group and in 7/10 samples from the gingivitis group. The B. forsythus isolates studied were found in four of the subjects with periodontitis and from 2 of the subjects with gingivitis. Serum antibody titers to 6 ribotypes of B. forsythus were studied. Western blots, gradient gels, and pulsed field gel electrophoresis concurrently demonstrated that the B. forsythus isolates were genotypically, and phenotypically unique for each subject. Antibody titers to two selected B. forsythus isolates were significantly higher in the periodontitis group (p<0.01, Mann-Whitney test). The study confirmed that antibody serum titers to the six different ribotypes of B. forsythus varied greatly between older individuals with gingivitis or periodontitis. Not all strains of B. forsythus elicited higher titers in periodontitis affected subjects. CONCLUSIONS: The results of the present study suggest genotype variation of B. forsythus that is unique to the individual and that serotype variation can be expected. It is possible that B. forsythus under specific host conditions can modulate surface antigen factors to evade the host immune response.

Regulation of the Porphyromonas gingivalis fimA (Fimbrillin) gene.

In common with many bacterial virulence genes, the fimbrillin (fimA) gene of Porphyromonas gingivalis is modulated in response to environmental fluctuation. The trans-acting components that comprise the regulatory system for transcriptional activity of the fimA gene in P. gingivalis were investigated. Three major proteins were found to bind to the upstream region of the fimA promoter. One of these proteins was fimbrillin itself, and the other two were a major arginine protease (Rgp) and lysine protease (Kgp). Production of these proteins was necessary for maximal fimA transcription. An exogenous fimA promoter-lacZ reporter was inactive when introduced into a strain of P. gingivalis carrying a mutation in the indigenous fimA gene. Furthermore, fimA mRNA levels were significantly decreased in rgp and kgp mutant strains. These data indicate that P. gingivalis has evolved multiple levels of control of fimbrial gene expression to enhance its survival in hostile environments.

Identification of a Porphyromonas gingivalis receptor for the Streptococcus gordonii SspB protein.

Colonization of the plaque biofilm by the oral pathogen Porphyromonas gingivalis is favored by the presence of antecedent organisms such as Streptococcus gordonii. Coadhesion between P. gingivalis and S. gordonii can be mediated by the SspB protein of S. gordonii; however, the P. gingivalis cognate receptor for this protein has not been identified. In this study, we identified a surface protein of P. gingivalis that interacts with the SspB protein. Coprecipitation between P. gingivalis outer membrane proteins and purified SspB protein demonstrated that a 100-kDa P. gingivalis protein bound to SspB. The 100-kDa protein also bound to an engineered strain of Enterococcus faecalis that expresses the SspB protein on the cell surface. Monospecific polyclonal antibodies to the 100-kDa protein inhibited the binding between P. gingivalis and S. gordonii in a dose-dependent manner up to 86%. Amino acid sequencing of the 100-kDa protein showed homology to a protein previously identified as the P. gingivalis minor fimbria. The minor fimbrial protein may exist as a complex with a hemagglutinin-like protein since the genes encoding these proteins are adjacent on the chromosome and are cotranscribed. Thus, the P. gingivalis receptor for S. gordonii SspB is a 100-kDa protein that structurally may be a minor fimbria-protein complex and functionally effectuates coadhesion.

Mobile elements carrying ermF and tetQ genes in gram-positive and gram-negative bacteria.

Bacteroides spp. conjugative transposon Tn5030 is 150 kb which includes a 43 kb characterized region containing a number of defined genes and an open reading frame (ORF). The 43 kb region is organized with the ORF1 immediately upstream from the ermF gene, coding for an rRNA methylase, then an unknown 20 kb region downstream followed by the tetQ gene (coding for a ribosomal protection protein) then the rteA and rteB genes. The role of ORF1 is unclear; rteA is a putative sensor and rteB a regulator. Thirty-seven (62%) of 60 isolates, representing one gram-positive anaerobic and 13 gram-negative anaerobic species, co-transferred the ermFand tetQ genes to an unrelated Enterococcus faecalis recipient. We used the polymerase chain reaction to show the linkage between ORF1, ermF, tetQ, rteA and rteB. Our data suggest that the ORF1 gene product may participate in the transfer of the ermF gene with or without the ORF1-rteB region and has homology to bacterial transposases. Isolates that co-transferred the ermF and tetQ genes carried and transferred the rteB gene, suggesting that the rteB gene product may be important in transfer of the 43 kb ORF1-rteB region to E. faecalis. The rteB gene product is not required when ermF is transferred independently of tetQ.

Host range of the ermF rRNA methylase gene in bacteria of human and animal origin.

We screened 183 different clinical anaerobic and aerobic bacteria isolated from humans and other animals for the presence of the ermF gene using a polymerase chain reaction (PCR) assay. The ermF gene was detected in 107 (58%) clinical isolates, including 42 (61%) of 69 gram-positive bacteria and 65 (57%) of 114 gram-negative bacteria. Twenty-five ATCC isolates were also tested; 20 (80%) carried the ermF gene. The gene products from the ermF PCR from four isolates were sequenced and showed 95-99% nucleotide homology with the ermF gene and 98-99% amino acid homology with the gene product. Eleven (58%) of the 19 gram-negative donors tested were able to transfer the ermF gene. All nine (100%) of the gram-positive donors tested transferred the ermF gene, using either Enterococcus faecalis or Haemophilus influenzae as the recipients.

Erythromycin-resistant Neisseria gonorrhoeae and oral commensal Neisseria spp. carry known rRNA methylase genes.

Two Neisseria gonorrhoeae isolates from Seattle and two isolates from Uruguay were resistant to erythromycin (MIC, 4 to 16 microg/ml) and had reduced susceptibility to azithromycin (MIC, 1 to 4 microg/ml) due to the presence of the self-mobile rRNA methylase gene(s) ermF or ermB and ermF. The two Seattle isolates and one isolate from Uruguay were multiresistant, carrying either the 25.2-MDa tetM-containing plasmid (Seattle) or a beta-lactamase plasmid (Uruguay). Sixteen commensal Neisseria isolates (10 Neisseria perflava-N. sicca, 2 N. flava, and 4 N. mucosa) for which erythromycin MICs were 4 to 16 microg/ml were shown to carry one or more known rRNA methylase genes, including ermB, ermC, and/or ermF. Many of these isolates also were multiresistant and carried the tetM gene. This is the first time that a complete transposon or a complete conjugative transposon carrying an antibiotic resistance gene has been described for the genus Neisseria.

Antimicrobial susceptibilities of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens spp. isolated in Spain.

The susceptibilities of 143 Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens isolates to 18 antimicrobial agents were tested. All P. gingivalis isolates were susceptible. In contrast, some Prevotella spp. (17%) were resistant to beta-lactams, erythromycin, clindamycin, or tetracycline and carried resistance genes, ermF or tetQ, or beta-lactamases.

Characterization of tetracycline and erythromycin resistance determinants in Treponema denticola.

Treponema denticola isolates were evaluated for the presence of known tetracycline and erythromycin resistance determinants by Southern blot hybridization of whole-cell DNA and PCR assays. We examined all isolates available, which included 12 clinical and 4 American Type Culture Collection isolates. Two isolates carried the Tet B determinant, five isolates carried both the Tet B and Erm F determinants, seven isolates carried the Erm F determinant, and two did not carry any of the Tet or Erm determinants tested. Both the Tet B and Erm F determinants appeared to be associated with the chromosome. Neither of the two T. denticola donors tested could transfer the Tet B determinant, but three of four T. denticola tested transferred the Erm F determinant to an Enterococcus faecalis recipient. This extends the host range of both the tetB and ermF genes into the genus Treponema.