Gingival tissue human beta-defensin levels in relation to infection and inflammation.

AIM: To profile gingival tissue levels of human beta-defensin (hBD)-2 and hBD-3 in relation to gingival inflammation, Th17-related cytokine concentrations, Porphyromonas gingivalis counts, and gingipain and total protease activities. MATERIALS AND METHODS: Gingival tissue and subgingival plaque samples were collected from 21 periodontitis patients including 48 periodontal pocket sites with marginal, mild, or moderate to severe inflammation. hBD levels were determined by immunodetection, P. gingivalis counts with real-time polymerase chain reaction, protease activities with fluorogenic substrates, and cytokine concentrations with Luminex technique. Data were statistically analysed using Kruskal-Wallis and Mann-Whitney U tests and Spearman correlation coefficients. RESULTS: Subgingival plaque counts of P. gingivalis (p = .001) and gingipain activity (p < .001), as well as interleukin (IL)-1ß (p = .012), IL-10 (p = .024), IL-17A (p = .002), IL-17F (p = .006), and IL-23 (p = .036) concentrations were elevated in severely inflamed sites, whereas no change was observed in hBD-2 and hBD-3 levels. Negative correlations were found between protease activity and hBD-2 (p = .033) and hBD-3(p = .003) levels. CONCLUSIONS: Shift in gingival inflammation from marginal to mild stage is related to elevations in subgingival plaque P. gingivalis counts and gingipain activity, but not to tissue hBD levels. Negative correlations between hBDs and total protease activity suggest the degradation of these antimicrobial peptides in progressed inflammation.

Regulatory effects of PRF and titanium surfaces on cellular adhesion, spread, and cytokine expressions of gingival keratinocytes.

Dental implant material has an impact on adhesion and spreading of oral mucosal cells on its surface. Platelet-rich fibrin (PRF), a second-generation platelet concentrate, can enhance cell proliferation and adhesion. The aim was to examine the regulatory effects of PRF and titanium surfaces on cellular adhesion, spread, and cytokine expressions of gingival keratinocytes. Human gingival keratinocytes were cultured on titanium grade 4, titanium grade 5 (Ti5), and HA discs at 37 °C in a CO2 incubator for 6 h and 24 h, using either elutes of titanium-PRF (T-PRF) or leukocyte and platelet-rich fibrin (L-PRF), or mammalian cell culture medium as growth media. Cell numbers were determined using a Cell Titer 96 assay. Interleukin (IL)-1ß, IL-1Ra, IL-8, monocyte chemoattractant protein (MCP)-1, and vascular endothelial growth factor (VEGF) expression levels were measured using the Luminex® xMAP™ technique, and cell adhesion and spread by scanning electron microscopy. Epithelial cell adhesion and spread was most prominent to Ti5 surfaces. L-PRF stimulated cell adhesion to HA surface. Both T-PRF and L-PRF activated the expressions of IL-1 ß, IL-8, IL-1Ra, MCP-1, and VEGF, T-PRF being the strongest activator. Titanium surface type has a regulatory role in epithelial cell adhesion and spread, while PRF type determines the cytokine response.

Quorum sensing molecules regulate epithelial cytokine response and biofilm-related virulence of three Prevotella species.

Quorum sensing (QS) signaling regulates the motility, adhesion, and biofilm formation of bacteria, and at the same time activates immune response in eukaryotic organisms. We recently demonstrated that the QS molecule, dihydroxy-2, 3-pentanedione (DPD), and its analogs significantly inhibit estradiol-regulated virulence of Prevotella aurantiaca, one of the four species in the Prevotella intermedia group. Here, we examined the combined effects of estradiol and QS signaling on 1) cytokine response of human gingival keratinocytes (HMK) against whole cell extract (WCE) of P. intermedia, Prevotella nigrescens, and Prevotella pallens, and 2) biofilm formation of these three Prevotella species. All experiments were performed in the presence or absence of estradiol, and with different QS molecules: DPD and its analogs (ethyl-DPD, butyl-DPD, and isobutyl-DPD). Concentrations of interleukin (IL)-1ß, -6, and -8 were determined by the Luminex multiplex immunoassay, biofilm mass was quantitatively evaluated by measuring protein concentration via the Bradford method, and the microtopography of biofilms was assessed by scanning electron microscopy (SEM) imaging. Concentrations of IL-6 and IL-8 were elevated when HMK cells were incubated with estradiol and WCE of P. intermedia and P. nigrescens, but decreased when incubated with estradiol and WCE of P. pallens. Butyl-DPD neutralized the estradiol- and WCE-induced regulation of HMK interleukin expression and, at the same time, inhibited the biofilm formation of P. intermedia and P. nigrescens. SEM micrographs revealed a decrease in biofilm mass after application of butyl-DPD, which was most detectable among the P. intermedia ATCC 25611 and P. nigrescens ATCC 33563 and AHN 8293 strains. In conclusion, butyl-DPD analog is able to neutralize the WCE-induced epithelial cytokine response and, at the same time, to inhibit the biofilm formation of P. intermedia and P. nigrescens.

Dipeptidyl peptidase IV and quorum sensing signaling in biofilm-related virulence of Prevotella aurantiaca.

Biofilm formation and dipeptidyl peptidase IV (DPPIV) enzyme activity contribute to the virulence of oral bacteria, and these virulence factors are partly regulated by quorum sensing signaling system. We recently demonstrated that estradiol regulates growth properties and DPPIV activity of Prevotella intermedia, Prevotella nigrescens, and Prevotella pallens. Here, we examined the DPPIV dependency of biofilm formation of Prevotella aurantiaca. Three strains (two clinical strains AHN 37505 and 37552 and the type strain CCUG 57723) were incubated in three estradiol concentrations (30, 90, and 120 nmol/L). Regulation of DPPIV activity, biofilm and fimbria formation, and coaggregation of bacterial strains were analyzed after incubation with four concentrations (10 nM, 100 nM, 1 µM, 10 µM) of dihydroxy-2,3-pentaedione (DPD), the universal precursor of autoinducer -2 (AI-2), and analogs (ethyl-DPD, butyl-DPD, and isobutyl-DPD) for 24 h. Estradiol enhanced the planktonic growth, coaggregation, and biofilm formation of P. aurantiaca strains. The whole cell extract of AHN 37505 had the highest DPPIV activity, followed by CCUG 57723 and AHN 37552. Inhibition of DPPIV activity with di-isopropylfluorophosphate suppressed the effect of estradiol on biofilm formation. At 100 nM and 10 µM concentrations of DPD, butyl DPD, and isobutyl DPD, biofilm formation of P. aurantiaca was significantly inhibited. Fimbriae formation was enhanced up to concentrations of 100 nM and 1 µM followed by a significant inhibition at higher concentrations of DPD and all analogs. A slight but significant inhibitory effect of DPD and analogs on DPPIV activity was observed. Our results indicate that DPPIV plays a key role in the estradiol-regulated biofilm formation of P. aurantiaca. Quorum sensing autoinducer DPD and C1-alkyl analogs could inhibit biofilm-related virulence of P. aurantiaca.

Morphological and functional adaptations of Fusobacterium nucleatum exposed to human neutrophil Peptide-1.

BACKGROUND AND OBJECTIVE: We recently demonstrated that Fusobacterium nucleatum can resist to human neutrophil peptide (HNP)-1 by decreasing its membrane permeability and increasing its proliferation and biofilm formation. In this continuation study, we aimed to further evaluate and explain these resistance properties by determining the morphological and functional adaptations of F. nucleatum, using transmission electron microscopy (TEM). MATERIALS AND METHODS: Cultures of the type strain of F. nucleatum (ssp. nucleatum ATCC 25586) and two clinical strains (ssp. polymorphum AHN 9910 and ssp. nucleatum AHN 9508) were incubated without (0 µg/ml) or with four different test concentrations of recombinant HNP-1 (1, 5, 10 and 20 µg/ml). Membrane morphology and thickness, and cell (visualized by TEM), planktonic growth (measured in colony forming units), and biofilm formation (measured as total mass) were analyzed. Scrambled HNP-1 was used in planktonic growth and biofilm formation studies as a negative control. RESULTS: TEM analyses revealed a decrease in the outer membrane surface corrugations and roughness of the strain AHN 9508 with increasing HNP-1 concentrations. In higher concentrations of HNP-1, the strain AHN 9910 showed thicker outer membranes with a number of associated rough vesicles attached to the outer surface. Intracellular granules became increasingly visible in the strain ATCC 25586 with increasing peptide concentrations. With increased concentrations of HNP-1, planktonic growth of the two clinical strains was significantly enhanced (P < 0.001) and of the type strain significantly suppressed (P < 0.01). HNP-1 decreased the biofilm formation of the two clinical strains, AHN 9910 (P < 0.01) and 9508 (P < 0.001) significantly. Scrambled HNP-1 showed no effect on planktonic growth or biofilm formation of the tested strains. DISCUSSION: F. nucleatum has the ability to withstand the lethal effects of HNP-1, and the ultrastructural changes on bacterial membrane and cytoplasm may play role in this adaptive process.

Does estradiol have an impact on the dipeptidyl peptidase IV enzyme activity of the Prevotella intermedia group bacteria?

Initiation and development of pregnancy-associated gingivitis is seemingly related to the microbial shift towards specific gram-negative anaerobes in subgingival biofilms. It is known that Prevotella intermedia sensu lato is able to use estradiol as an alternative source of growth instead of vitamin K. The aim of the present study was to investigate the impact of estradiol on the bacterial dipeptidyl peptidase IV (DPPIV) enzyme activity in vitro as a virulent factor of the Prevotella intermedia group bacteria, namely P. intermedia, Prevotella nigrescens, Prevotella pallens, and Prevotella aurantiaca. In all experiments, 2 strains of each Prevotella species were used. Bacteria were incubated with the concentrations of 0, 30, 90, and 120 nmol/L of estradiol and were allowed to build biofilms at an air-solid interface. DPPIV activities of biofilms were measured kinetically during 20 min using a fluorometric assay. The enzyme activity was later related to the amount of protein produced by the same biofilm, reflecting the biofilm mass. Estradiol significantly increased DPPIV activities of the 8 Prevotella strains in a strain- and dose-dependent manner. In conclusion, our in vitro experiments indicate that estradiol regulates the DPPIV enzyme activity of P. intermedia, P. nigrescens, P. pallens, and P. aurantiaca strains differently. Our results may, at least partly, explain the role of estradiol to elicit a virulent state which contributes to the pathogenesis of pregnancy-related gingivitis.

Effect of estradiol on planktonic growth, coaggregation, and biofilm formation of the Prevotella intermedia group bacteria.

Alterations in the quantity and quality of biofilms at gingival margin are considered to play a role in the initiation and development of pregnancy-related gingivitis. Prevotella intermedia sensu lato is able to consume estradiol, the major sex hormone secreted during pregnancy, in the absence of vitamin K. The aim of the study was to examine the effect of estradiol on the planktonic growth, coaggregation, polysaccharide production, and biofilm formation of the P. intermedia group bacteria, namely P. intermedia, Prevotella nigrescens, and Prevotella pallens. In all experiments, the type strain (ATCC) and a clinical strain (AHN) of P. intermedia, P. nigrescens, and P. pallens were incubated with the concentrations of 0, 30, 90, and 120 nmol/L of estradiol. Planktonic growth was assessed by means of the colony forming unit method, while coaggregation and biofilm formation were assessed by spectrophotometric methods. In the determination of protein and polysaccharide levels, the Bradford and phenol-sulfuric acid methods were used, respectively. P. pallens AHN 9283 and P. nigrescens ATCC 33563 increased their numbers at planktonic stage with increasing estradiol concentrations. In 48-h biofilm tests, elevated protein levels were found for both strains of P. intermedia, and the strains P. nigrescens ATCC 33563 and P. pallens AHN 9283 in the presence of estradiol. The P. intermedia strains also increased the levels of polysaccharide formation in the biofilm. Coaggregation of the P. intermedia group organisms with Fusobacterium nucleatum was enhanced only in P. intermedia AHN 8290. In conclusion, our in vitro experiments indicate that estradiol regulates planktonic growth, coaggregation, polysaccharide production, and biofilm formation characteristics of P. intermedia, P. nigrescens, and P. pallens differently. These results may, at least partly, explain the differences seen in their contribution to the pathogenesis of pregnancy-related gingivitis.

Salivary macrophage activation-related chemokines and mitogen-activated kinase kinase-degrading proteolytic activity in type 1 diabetes mellitus.

BACKGROUND: This cross-sectional study aimed to evaluate salivary concentrations of macrophage activation-related chemokines and mitogen-activated kinase kinase (MAPKK)-degrading proteolytic activity in children and adolescents with and without type 1 diabetes mellitus (T1DM). METHODS: A total of 122 children and adolescents (65 T1DM patients, 50.8% female, mean age:10.9 years; 57 systemically healthy controls, 36.8% female, mean age: 9.5 years) were included in the study. Salivary concentrations of interferon gamma inducible protein-10 (IP-10), monocyte chemoattractant protein (MCP)-1, MCP-2, MCP-3, MCP-4, macrophage-derived chemokine (MDC), macrophage migration inhibitory factor (MIF), monokine induced by interferon gamma (MIG), and macrophage inflammatory protein-1 alpha (MIP-1α) were quantified using a bead-based technique. MAPKK-degrading proteolytic activity was detected using fluorescent peptide substrates. RESULTS: The T1DM group had higher plaque index (PI%, p = 0.032) and bleeding on probing (BOP%, p = 0.045) scores, and lower decayed, missing, filled teeth (dmft/DMFT, p = 0.002) index scores compared to the healthy controls. Compared to the controls, salivary MCP-1 (p = 0.007), MCP-3 (p < 0.001), MIG (p = 0.007), and MIP-1α (p = 0.033) concentrations were elevated whereas MCP-4 concentrations decreased (p < 0.001) in the T1DM group. After adjusting for age, PI%, BOP%, and dmft/DMFT scores, significant differences in salivary concentrations of MIG (p = 0.033) and MIP-1α (p = 0.017) were observed between the groups. Moreover, protease activities directed to the cleavage sites of MEK23-18 (p = 0.001), MKK6b7-22 (p = 0.007), MKK451-66 (p = 0.005), MKK7b37-52 (p = 0.034), and MKK7b69-84 (p = 0.009) were elevated in the T1DM group. CONCLUSION: T1DM disrupts the salivary macrophage activation-related chemokine profile and dysregulates proteolytic MAPKK cleavage. These findings can be an outcome of the impaired systemic immune response in T1DM.

Prevotella species as oral residents and infectious agents with potential impact on systemic conditions.

Oral Prevotella are known as anaerobic commensals on oral mucosae and in dental plaques from early life onwards, including pigmented P. melaninogenica, P. nigrescens, and P. pallens and non-pigmented Prevotella species. Many Prevotella species contribute to oral inflammatory processes, being frequent findings in dysbiotic biofilms of periodontal diseases (P. intermedia, P. nigrescens), cariotic lesions (P. denticola, Alloprevotella (formerly Prevotella) tannerae), endodontic infections (P. baroniae, P. oris, P. multisaccharivorax), and other clinically relevant oral conditions. Over the years, several novel species have been recovered from the oral cavity without knowledge of their clinical relevance. Within this wide genus, virulence properties and other characteristics like biofilm formation seemingly vary in a species- and strain-dependent manner, as shown for the P. intermedia group organisms (P. aurantiaca, P. intermedia, P. nigrescens, and P. pallens). Oral Prevotella species are identified in various non-oral infections and chronic pathological conditions. Here, we have updated the knowledge of the genus Prevotella and the role of Prevotella species as residents and infectious agents of the oral cavity, as well as their detection in non-oral infections, but also gathered information on their potential link to cancers of the head and neck, and other systemic disorders.

Elevated Baseline Salivary Protease Activity May Predict the Steadiness of Gingival Inflammation During Periodontal Healing: A 12-Week Follow-Up Study on Adults.

Aim was to profile salivary total protease, Porphyromonas gingivalis gingipain, and neutrophil elastase activities in relation to the resolution of periodontal inflammation, salivary macrophage-derived chemokine (MDC), and macrophage inflammatory protein-1α concentrations. Nonsurgical periodontal treatment was performed in 24 periodontitis patients in a prospective interventional study design. Periodontal clinical parameters were recorded, and stimulated saliva samples were collected at baseline and 2, 6, and 12 weeks after treatment. Salivary total protease and gingipain activities were determined using fluorogenic substrates, elastase activity by chromogenic substrates, and cytokine concentrations by Luminex immunoassay. For statistical analyses, generalized linear mixed models for repeated measures were used. Salivary total protease activity elevated, while gingival inflammation and plaque accumulation decreased 2 and 6 weeks after periodontal therapy. Salivary MDC concentration was elevated 12 weeks after periodontal treatment. Patients with elevated protease activities at baseline in comparison to patients with low baseline total protease activities, had higher levels of gingival inflammation before and after periodontal treatment. In conclusion, elevations in salivary total protease activity seem to be part of periodontal healing at its early phases. Higher levels of salivary total protease activities before periodontal treatment may predict the severity and steadiness of unresolved gingival inflammation.

Human neutrophil peptide-1 affects matrix metalloproteinase-2, -8 and -9 secretions of oral squamous cell carcinoma cell lines in vitro.

OBJECTIVES: The present study aimed to investigate the effect of HNP-1 on the matrix metalloproteinase (MMP)-2, -8 and -9 secretions of two oral squamous cell carcinoma (OSCC) cell lines (UT-SCC-43A and UT-SCC-43B). DESIGN: In all experiments, the two OSCC cell lines were incubated with graded concentrations (0, 1, 5, and 10 µg/ml) of HNP-1 for 24 and 48 h. Cell viability was measured using a colorimetric proliferation test and cell death was analyzed with a colorimetric cytotoxicity detection kit. Enzyme activity of MMP-2 and MMP-9 was detected by using gelatin zymography, and molecular weight forms of MMP-8 were determined by Western-blot and a densitometric quantitation method. RESULTS: Both cell lines showed a significant increase in LDH toxicity at 24h (UT-SCC-43A: p=0.005 & UT-SCC-43B: p=0.014). Reduced gelatinolytic activities of proMMP-2 were detected in UT-SCC-43B cell line after 24 and 48 h of incubation with HNP-1 (1 µg/ml: p<0.001, 5 µg/ml: p<0.001, and 10 µg/ml: p=0.0225). MMP-8 levels of both cell lines decreased at 200-250 kDa after 24h of incubation, while after 48 h only UT-SCC-43B decreased at 45-50 kDa. CONCLUSIONS: Our results indicate that HNP-1 suppresses the secretion of MMP-2, -8, and -9 in OSCC cell lines.

Comparison of physical quality assurance between Scanora 3D and 3D Accuitomo 80 dental CT scanners.

BACKGROUND: The use of cone beam computed tomography (CBCT) in dentistry has proven to be useful in the diagnosis and treatment planning of several oral and maxillofacial diseases. The quality of the resulting image is dictated by many factors related to the patient, unit, and operator. MATERIALS AND METHODS: In this work, two dental CBCT units, namely Scanora 3D and 3D Accuitomo 80, were assessed and compared in terms of quantitative effective dose delivered to specific locations in a dosimetry phantom. Resolution and contrast were evaluated in only 3D Accuitomo 80 using special quality assurance phantoms. RESULTS: Scanora 3D, with less radiation time, showed less dosing values compared to 3D Accuitomo 80 (mean 0.33 mSv, SD±0.16 vs. 0.18 mSv, SD±0.1). Using paired t-test, no significant difference was found in Accuitomo two scan sessions (p>0.05), while it was highly significant in Scanora (p>0.05). The modulation transfer function value (at 2 lp/mm), in both measurements, was found to be 4.4%. The contrast assessment of 3D Accuitomo 80 in the two measurements showed few differences, for example, the grayscale values were the same (SD=0) while the noise level was slightly different (SD=0 and 0.67, respectively). CONCLUSIONS: The radiation dose values in these two CBCT units are significantly less than those encountered in systemic CT scans. However, the dose seems to be affected more by changing the field of view rather than the voltage or amperage. The low doses were at the expense of the image quality produced, which was still acceptable. Although the spatial resolution and contrast were inferior to the medical images produced in systemic CT units, the present results recommend adopting CBCTs in maxillofacial imaging because of low radiation dose and adequate image quality.