Interferon activated gene 204 protects against bone loss in experimental periodontitis.

BACKGROUND: Periodontal destruction can be the result of different known and yet-to-be-discovered biological pathways. Recent human genetic association studies have implicated interferon-gamma inducible protein 16 (IFI16) and absent in melanoma 2 (AIM2) with high periodontal interleukin (IL)-1ß levels and more destructive disease, but mechanistic evidence is lacking. Here, we sought to experimentally validate these observational associations and better understand IFI16 and AIM2's roles in periodontitis. METHODS: Periodontitis was induced in Ifi204-/- (IFI16 murine homolog) and Aim2-/- mice using the ligature model. Chimeric mice were created to identify the main source cells of Ifi204 in the periodontium. IFI16-silenced human endothelial cells were treated with periodontal pathogens in vitro. Periodontal tissues from Ifi204-/- mice were evaluated for alveolar bone (micro-CT), cell inflammatory infiltration (MPO+ staining), Il1b (qRT-PCR), and osteoclast numbers (cathepsin K+ staining). RESULTS: Ifi204-deficient mice> exhibited >20% higher alveolar bone loss than wild-type (WT) (P < 0.05), while no significant difference was found in Aim2-/- mice. Ifi204's effect on bone loss was primarily mediated by a nonbone marrow source and was independent of Aim2. Ifi204-deficient mice had greater neutrophil/macrophage trafficking into gingival tissues regardless of periodontitis development compared to WT. In human endothelial cells, IFI16 decreased the chemokine response to periodontal pathogens. In murine periodontitis, Ifi204 depletion elevated gingival Il1b and increased osteoclast numbers at diseased sites (P < 0.05). CONCLUSIONS: These findings support IFI16's role as a novel regulator of inflammatory cell trafficking to the periodontium that protects against bone loss and offers potential targets for the development of new periodontal disease biomarkers and therapeutics.

The oral bacterial microbiome of occlusal surfaces in children and its association with diet and caries.

Dental caries is the most prevalent disease in humans globally. Efforts to control it have been invigorated by an increasing knowledge of the oral microbiome composition. This study aimed to evaluate the bacterial diversity in occlusal biofilms and its relationship with clinical surface diagnosis and dietary habits. Anamneses were recorded from thirteen 12-year-old children. Biofilm samples collected from occlusal surfaces of 46 permanent second molars were analyzed by 16S rRNA amplicon sequencing combined with the BLASTN-based search algorithm for species identification. The overall mean decayed, missing and filled surfaces modified index [DMFSm Index, including active white spot lesions (AWSL)] value was 8.77±7.47. Biofilm communities were highly polymicrobial collectively, representing 10 bacterial phyla, 25 classes, 29 orders, 58 families, 107 genera, 723 species. Streptococcus sp_Oral_Taxon_065, Corynebacterium matruchotii, Actinomyces viscosus, Actinomyces sp_Oral_Taxon_175, Actinomyces sp_Oral_Taxon_178, Actinomyces sp_Oral_Taxon_877, Prevotella nigrescens, Dialister micraerophilus, Eubacterium_XI G 1 infirmum were more abundant among surfaces with AWSL, and Streptococcus gordonii, Streptococcus sp._Oral_Taxon_058, Enterobacter sp._str._638 Streptococcus australis, Yersinia mollaretii, Enterobacter cloacae, Streptococcus sp._Oral_Taxon_71, Streptococcus sp._Oral_Taxon_F11, Centipeda sp._Oral_Taxon_D18 were more abundant among sound surfaces. Streptococcus mutans was detected on all surfaces in all patients, while Streptococcus sobrinus was detected only in three patients (mean relative abundances 7.1% and 0.6%, respectively). Neither species differentiated healthy from diseased sites. Diets of nine of the subjects were scored as high in fermentable carbohydrates (≧2X/day between meals). A direct association between relative abundances of bacteria and carbohydrate consumption was observed among 18 species. High consumption of fermentable carbohydrates and sound surfaces were associated with a reduction in bacterial diversity. PCoA plots displayed differences in bacterial community profiles between sound and diseased surfaces. Our study showed that, in addition to mutans streptococci, other species may be associated with the initiation of dental caries on occlusal surfaces, and that biofilm diversity of tooth surfaces is influenced by carbohydrate consumption and a surface's health status.

Comparison of suction device with saliva ejector for aerosol and spatter reduction during ultrasonic scaling.

BACKGROUND: Aerosols and spatter are concerns in health care owing to their potential adverse health effects. The Isolite illuminated isolation system (Isolite Systems) and a saliva ejector were compared for aerosol and spatter reduction during and after ultrasonic scaling. METHODS: Fifty participants were randomized to control (n = 25, saliva ejector) or test (n = 25, Isolite) groups and received a prophylaxis with an ultrasonic scaler. Aerosols were collected in a petri dish containing transport media, dispersed, and plated to anaerobic blood agar to determine colony-forming units (CFUs). The authors analyzed the data using a t test. RESULTS: No significant difference occurred between groups in aerosol and spatter reduction (P = .25). Mean (standard deviation) of log10 CFUs per milliliter collected during ultrasonic scaling in the control and test groups were 3.61 (0.95) and 3.30 (0.88), respectively. All samples contained α-hemolytic streptococci, and many samples contained strictly oral anaerobes. CONCLUSIONS: A significant amount of contamination occurred during ultrasonic scaling in both groups, as indicated by high numbers of CFUs and the identification of strictly oral anaerobes in all plates. PRACTICAL IMPLICATIONS: Neither device reduced aerosols and spatter effectively, and there was no significant difference in reduction between the 2 devices. Additional measures should be taken with these devices to reduce the likelihood of disease transmission.

Localization of Burkholderia cepacia complex bacteria in cystic fibrosis lungs and interactions with Pseudomonas aeruginosa in hypoxic mucus.

The localization of Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) lungs, alone or during coinfection with Pseudomonas aeruginosa, is poorly understood. We performed immunohistochemistry for Bcc and P. aeruginosa bacteria on 21 coinfected or singly infected CF lungs obtained at transplantation or autopsy. Parallel in vitro experiments examined the growth of two Bcc species, Burkholderia cenocepacia and Burkholderia multivorans, in environments similar to those occupied by P. aeruginosa in the CF lung. Bcc bacteria were predominantly identified in the CF lung as single cells or small clusters within phagocytes and mucus but not as "biofilm-like structures." In contrast, P. aeruginosa was identified in biofilm-like masses, but densities appeared to be reduced during coinfection with Bcc bacteria. Based on chemical analyses of CF and non-CF respiratory secretions, a test medium was defined to study Bcc growth and interactions with P. aeruginosa in an environment mimicking the CF lung. When test medium was supplemented with alternative electron acceptors under anaerobic conditions, B. cenocepacia and B. multivorans used fermentation rather than anaerobic respiration to gain energy, consistent with the identification of fermentation products by high-performance liquid chromatography (HPLC). Both Bcc species also expressed mucinases that produced carbon sources from mucins for growth. In the presence of P. aeruginosa in vitro, both Bcc species grew anaerobically but not aerobically. We propose that Bcc bacteria (i) invade a P. aeruginosa-infected CF lung when the airway lumen is anaerobic, (ii) inhibit P. aeruginosa biofilm-like growth, and (iii) expand the host bacterial niche from mucus to also include macrophages.

Antibacterial surface properties of fluoride-containing resin-based sealants.

OBJECTIVES: The aim of the present study was to determine the antibacterial properties of three resin-based pit and fissure sealant products: Clinpro (3M ESPE), Embrace (Pulpdent), and UltraSeal XT plus (Ultradent). METHODS: The antibacterial effects of the sealants were tested in both an agar diffusion assay and a planktonic growth inhibition assay using Streptococcus mutans and Lactobacillus acidophilus. The materials were applied to paper and enamel disks in the former and on the side walls of 96-well microtiter plates on the latter. RESULTS: All materials showed either diffusible or contact antibacterial effects in the agar diffusion assays. The effect was diminished when enamel disks were used as substrate. In the planktonic growth inhibition assay, Clinpro had its effect reduced, but retained activity against both bacteria over time. L. acidophilus was more sensitive than S. mutans to UltraSeal. Embrace retained antibacterial activity against both bacteria over time. CONCLUSIONS: Within the limitations of this in vitro study it can be concluded that all materials are capable of contact inhibition of L. acidophilus and S. mutans growth. Embrace has the longer lasting antibacterial activity when in solution, especially against S. mutans.

Periapical inflammation and bacterial penetration after coronal inoculation of dog roots filled with RealSeal 1 or Thermafil.

INTRODUCTION: The purpose of this study was to subject 2 carrier-based root filling products to a 4-month microbial challenge in a dog model with histologic markers to assess periapical inflammation and bacterial penetration of the 2 filling materials. Histologic evidence of bacterial penetration and periapical inflammation were the outcome parameters used to compare the products. METHODS: Teeth were aseptically prepared and then filled with carrier-based Resilon (RealSeal 1 [RS-1], n = 25) or with carrier-based gutta-percha (Thermafil, n = 25) and were left exposed for 4 months. The first control group received a coronal seal over either RS-1 or Thermafil root fillings (n = 8). A second control group was instrumented and left completely empty (n = 8). RESULTS: Histologic evidence of periapical inflammation was observed in 29% of the Thermafil group and in 9% of the RS-1 group. This difference was only significant when controlling for a possible tooth position effect on inflammation presence (P < .05). Histologic evidence of bacterial penetration was present in 9% of the RS-1 group and in 70% of the Thermafil group. The difference in penetration rates between RS-1 and Thermafil was statistically significant when controlling for any dog or tooth position effects on bacterial penetration (P < .001). Furthermore, there was a statistically significant correlation between histologic evidence of inflammation and histologic evidence of infection (P = .002). CONCLUSIONS: RS-1 appeared to resist bacterial penetration more effectively than Thermafil under the conditions of this study.

Critical role of apoptotic speck protein containing a caspase recruitment domain (ASC) and NLRP3 in causing necrosis and ASC speck formation induced by Porphyromonas gingivalis in human cells.

Periodontal disease is a chronic inflammatory disorder that leads to the destruction of tooth-supporting tissue and affects 10-20 million people in the U.S. alone. The oral pathogen Porphyromonas gingivalis causes inflammatory host response leading to periodontal and other secondary inflammatory diseases. To identify molecular components that control host response to P. gingivalis in humans, roles for the NLR (NBD-LRR) protein, NLRP3 (cryopyrin, NALP3), and its adaptor apoptotic speck protein containing a C-terminal caspase recruitment domain (ASC) were studied. P. gingivalis strain A7436 induces cell death in THP1 monocytic cells and in human primary peripheral blood macrophages. This process is ASC and NLRP3 dependent and can be replicated by P. gingivalis LPS and Escherichia coli. P. gingivalis-induced cell death is caspase and IL-1 independent and exhibits morphological features consistent with necrosis including loss of membrane integrity and release of cellular content. Intriguingly, P. gingivalis-induced cell death is accompanied by the formation of ASC aggregation specks, a process not previously described during microbial infection. ASC specks are observed in P. gingivalis-infected primary human mononuclear cells and are dependent on NLRP3. This work shows that P. gingivalis causes ASC- and NLRP3-dependent necrosis, accompanied by ASC speck formation.

Synergistic pathogenicity of Porphyromonas gingivalis and Fusobacterium nucleatum in the mouse subcutaneous chamber model.

Porphyromonas gingivalis and Fusobacterium nucleatum are often coisolated from sites of infection, such as suppurative apical periodontitis. The synergistic pathogenicity of mixed infection of P. gingivalis HG 405 with F. nucleatum PK 1594 was studied in the mouse subcutaneous chamber model in groups of seven animals. The minimal dose for P. gingivalis HG 405 that was required to infect 100% of the chambers was reduced by 1,000-fold when animals were inoculated in the same chamber with 1 x 10(9)F. nucleatum PK 1594 (p < 0.001). To benefit from the presence of the fusobacteria, P. gingivalis HG 405 had to be coinoculated; inoculation in separate chambers for the same animal had no such effect (p < 0.001). Subinfective F. nucleatum inocula also benefited from the association with P. gingivalis HG 405 and uniformly established an infection when this partner was present (p < 0.001). These results suggest that the frequent and natural coexistence of P. gingivalis and F. nucleatum in diseased sites may express such a synergism in successful establishment and survival of small inocula.

Effect of resin adhesive systems on root caries formation in vitro.

OBJECTIVE: To evaluate the capability of different adhesive systems to inhibit in vitro caries formation. METHOD AND MATERIALS: Four materials were tested: Gluma Comfort Bond (GL; Heraeus Kulzer), Gluma Comfort Bond + Desensitizer (GL+; Heraeus Kulzer), iBond (iB; Heraeus Kulzer), and One-up Bond F (OUB; Tokuyama). Bovine roots were ground to obtain flat mesial and distal dentin surfaces. Nail varnish was applied to the entire root surface except for two 10 mm X 2 mm windows on the flattened surfaces. The adhesives were applied to the exposed areas according to the manufacturers' instructions. The roots were incubated at 37 degrees C for 1 week in a suspension of StreptococcuS mutans and Lactobacillus acidophilus. Control groups either received no root surface treatment or were etched with 37% phosphoric acid (without immersion in the bacterial suspension). Specimens were sectioned perpendicular to the long axis of the roots, stained for 24 hours with Rhodamine B, and analyzed with confocal laser microscopy. The data were analyzed by 1-way ANOVA and Fisher's PLSD test at 95% confidence level. RESULTS: Lesions in the OUB, iB, and GL+ groups were significantly shallower than those in the no-treatment group. Acid etching did not produce any measurable lesions. Mean lesion depth in the GL group was similar to that in the no-treatment group. There was no significant difference between mean lesion depths in the GL+, GL, and iB groups. CONCLUSION: Fluoride- and glutaraldehyde-containing adhesive systems might be an aid in root caries prevention.

Antimicrobial activity and local release characteristics of chlorhexidine diacetate loaded within the dental copolymer matrix, ethylene vinyl acetate.

In vitro results are presented for a novel oral drug-delivery system ultimately intended for treatment of oral infections in immunocompromised patients. Test samples of ethylene vinyl acetate copolymer (EVA) containing chlorhexidine diacetate (CDA) showed desirable antimicrobial properties and steady, slow release into aqueous and other media after an initial burst of drug release in the first day of liquid exposure. By washing away this initial burst, the proposed mouthguard device should be capable of sustained delivery of locally effective CDA concentrations far below systemically toxic levels. A prolonged room temperature shelf-life of at least 1 year, and effectivity against a wide range of oral bacteria and Candida species was demonstrated. Drug loaded films showed a top-to-bottom asymmetry in drug release, but good lateral homogeneity, and a linear relationship between initial CDA loading concentration (from 0.63 to 10 wt %) and days 3-14 release rates in a static aqueous environment. The EVA matrix containing CDA appears to possess many suitable properties for localized oral delivery of sustained antimicrobial activity.

NF-kappaB signaling, elastase localization, and phagocytosis differ in HIV-1 permissive and nonpermissive U937 clones.

To identify positive or negative factors for HIV-1 infectivity, clones from the U937 promonocytic cell line that express similar levels of CD4 and CXCR4, but differ in HIV-1 susceptibility, were compared. In contrast to HIV-1 permissive clone 10 (plus), nonpermissive clone 17 (minus) was adherent to coverslips coated with chemokines, was phagocytic, killed bacteria, and expressed human leukocyte elastase (HLE) in a granule-like compartment (HLEG) that was never detected at the cell surface (HLECS). In contrast to the minus clone, the plus clone expressed HLE on the cell surface and was adherent to coverslips coated with the HLECS ligands alpha1proteinase inhibitor (alpha1PI, alpha1antitrypsin) and the HIV-1 fusion peptide. The phosphorylation status of several important signaling proteins was studied at the single cell level. Tumor suppressor p53, NF-kappaB p65, and Akt were constitutively phosphorylated in the plus clone, but not in the minus clone. Surprisingly, both alpha1PI and LPS induced phosphorylation of NF-kappaB p65 Ser-536 in both clones, but induced dephosphorylation of Ser-529 in the plus clone only. HIV-1 permissivity was conferred to the minus clone in a manner that required stimulation by both alpha1PI and LPS and was coincident to NF-kappaB p65 phosphorylation/dephosphorylation events as well as translocation of HLE to the cell surface. Even when stimulated, the minus clone exhibited greater reverse transcriptase activity, but less p24, than the plus clone. Results presented suggest that HIV-1 uptake and production efficiency are influenced by signaling profiles, receptor distribution, and the phagocytic capacity specific to the stage of differentiation of the CD4+ target cell.

Clinical efficiency of 2% chlorhexidine gel in reducing intracanal bacteria.

This study evaluated the clinical efficacy of 2% chlorhexidine (CHX) gel on intracanal bacteria reduction during root canal instrumentation. The additional antibacterial effect of an intracanal dressing (Ca[OH](2) mixed with 2% CHX gel) was also assessed. Forty-three patients with apical periodontitis were recruited. Four patients with irreversible pulpitis were included as negative controls. Teeth were instrumented using rotary instruments and 2% CHX gel as the disinfectant. Bacterial samples were taken upon access (S1), after instrumentation (S2), and after 2 weeks of intracanal dressing (S3). Anaerobic culture was performed. Four samples showed no bacteria growth at S1, which were excluded from further analysis. Of the samples cultured positively at S1, 10.3% (4/39) and 8.3% (4/36) sampled bacteria at S2 and S3, respectively. A significant difference in the percentage of positive culture between S1 and S2 (p < 0.001) but not between S2 and S3 (p = 0.692) was found. These results suggest that 2% CHX gel is an effective root canal disinfectant and additional intracanal dressing did not significantly improve the bacteria reduction on the sampled root canals.

In vitro inhibition of bacterial growth using different dental adhesive systems.

OBJECTIVES: This study evaluated the antibacterial potential of four different adhesive systems. METHODS & MATERIALS: Gluma Comfort Bond + Desensitizer, Gluma Comfort Bond, iBond and One-Up Bond F were tested against Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus and Actinomyces viscosus. The inhibition of growth by calibrated preparations was quantified by the measurement of zones of inhibition on bacterial lawns. Bactericidal activity was determined as reductions in recoverable colony-forming units in bacterial suspensions exposed to test preparations. RESULTS: All the preparations exhibited detectable zones of inhibition for all target bacteria through six months. When the bactericidal action was evaluated, all the materials were able to kill all the tested bacteria when tested immediately after polymerization. After one week of aging, iBond was the only material that continued to kill all of the test strains.

Neutrophil dysfunction in guanosine 3',5'-cyclic monophosphate-dependent protein kinase I-deficient mice.

The regulation of neutrophil functions by Type I cGMP-dependent protein kinase (cGKI) was investigated in wild-type (WT) and cGKI-deficient (cGKI-/-) mice. We demonstrate that murine neutrophils expressed cGKIalpha. Similar to the regulation of Ca2+ by cGKI in other cells, there was a cGMP-dependent decrease in Ca2+ transients in response to C5a in WT, but not cGKI-/- bone marrow neutrophils. In vitro chemotaxis of bone marrow neutrophils to C5a or IL-8 was significantly greater in cGKI-/- than in WT. Enhanced chemotaxis was also observed with cGKI-/- peritoneal exudate neutrophils (PE-N). In vivo chemotaxis with an arachidonic acid-induced inflammatory ear model revealed an increase in both ear weight and myeloperoxidase (MPO) activity in ear punches of cGKI-/- vs WT mice. These changes were attributable to enhanced vascular permeability and increased neutrophil infiltration. The total extractable content of MPO, but not lysozyme, was significantly greater in cGKI-/- than in WT PE-N. Furthermore, the percentage of MPO released in response to fMLP from cGKI-/- (69%) was greater than that from WT PE-N (36%). PMA failed to induce MPO release from PE-N of either genotype. In contrast, fMLP and PMA released equivalent amounts of lysozyme from PE-N. However, the percentage released was less in cGKI-/- (approximately 60%) than in WT (approximately 90%) PE-N. Superoxide release (maximum velocity) revealed no genotype differences in responses to PMA or fMLP stimulation. In summary, these results show that cGKIalpha down-regulates Ca2+ transients and chemotaxis in murine neutrophils. The regulatory influences of cGKIalpha on the secretagogue responses are complex, depending on the granule subtype.

Periapical inflammation after coronal microbial inoculation of dog roots filled with gutta-percha or resilon.

A dog model was used to assess and compare in vivo the efficacy of gutta-percha and AH26 sealer versus Resilon with Epiphany primer and sealer [Resilon "Monoblock" System (RMS)] filled roots in preventing apical periodontitis subsequent to coronal inoculation with oral microorganisms. There were 56 vital roots in the premolars of seven adult beagle dogs aseptically instrumented, filled, and temporized. The roots were randomly divided into four experimental groups (Coronal Leakage Model) and one negative control group and filled as follows: group 1-lateral condensation of gutta-percha and AH26 sealer (n=12); group 2-vertical condensation of gutta-percha and AH26 sealer (n=12); group 3-lateral condensation of RMS (n=12); group 4-vertical condensation of RMS (n=10); negative control (n=10)-gutta-percha and AH26 sealer or RMS root fillings using lateral or vertical condensation techniques as in groups 1 to 4. Positive control-57 additional premolar roots were instrumented, infected and not filled (beginning of the Entombment Model experiment). The premolars in groups 1 to 4 were accessed again, inoculated with dental plaque scaled from the dog's teeth, and temporized. This fresh innoculum of microorganisms was repeated on two more occasions at monthly intervals. The teeth in the negative control group were not accessed again and remained undisturbed. On the 14-wk postcoronal inoculation, dogs were euthanized, and jaw blocks prepared for histologic evaluation under a light microcope. Mild inflammation was observed in 82% (18 of 22) of roots filled with gutta-percha and AH26 sealer that was stastistically more than roots filled with RMS (19% or 4 of 21) and roots in the negative control (22% or 2 of 9) (McNemar paired analysis, p < 0.05). The Resilon "Monoblock" System was associated with less apical periodontitis, which may be because of its superior resistance to coronal microleakage.