BACKGROUND AND OBJECTIVE:Plaque-induced gingival inflammation (gingivitis) is ubiquitous in humans. The epithelial barrier reacts to the presence of oral bacteria and induces inflammatory cascades. The objective of this study was to investigate the mechanism by which the small molecule micronutrient curcumin could decrease inflammatory response in vitro to oral bacterium heat-killed Fusobacterium nucleatum as curcumin could be a useful compound for combatting gingivitis already consumed by humans.METHODS:H400 oral epithelial cell line was pre-conditioned with curcumin and the production of cytokines was measured by enzyme-linked immunosorbent assay (ELISA) and translocation of transcription factors was used to monitor inflammatory responses. Haem oxygenase (HO-1) expression and molecules that HO-1 releases were evaluated for their potential to reduce the quantity of cytokine production. Immunofluorescence microscopy and Western blotting were used to evaluate changes in transcription factor and enzyme location.RESULTS:Pre-conditioning of H400 cells with a sub-apoptotic concentration of curcumin (20 μM) attenuated secretion of Granulocyte-Macrophage - Colony-Stimulating Factor (GM-CSF) and reduced NFkB nuclear translocation. This pre-conditioning caused an increase in nuclear Nrf2; an initial drop (at 8 h) followed by an adaptive increase (at 24 h) in glutathione; and an increase in haem oxygenase (HO-1) expression. Inhibition of HO-1 by SnPPIX prevented the curcumin-induced attenuation of GM-CSF production. HO-1 catalyses the breakdown of haem to carbon monoxide, free iron and biliverdin: the HO-1/CO anti-inflammatory pathway. Elevations in carbon monoxide, achieved using carbon monoxide releasing molecule-2 (CORM2) treatment alone abrogated F. nucleatum-induced cytokine production. Biliverdin is converted to bilirubin by biliverdin reductase (BVR). This pleiotropic protein was found to increase in cell membrane expression upon curcumin treatment.CONCLUSION:Curcumin decreased inflammatory cytokine production induced by Fusobacterium nucleatum in H400 oral epithelial cells. The mechanism of action appears to be driven by the increase of haem oxygenase and the production of carbon monoxide.

AimThe aim of this study was to investigate the association of serum vitamin B12 with the progression of periodontitis and risk of tooth loss in a prospective cohort study.Materials and MethodsIn the Study of Health in Pomerania, 1648 participants were followed from 2002-2006 to 2008-2012 (mean duration 5.9years). Serum vitamin B12 was measured by chemiluminescent enzyme immunoassay. Probing pocket depth (PD) and clinical attachment loss (CAL) were measured to reflect periodontal status on a half-mouth basis at each survey cycle. Tooth numbers are based upon a full-mouth tooth count.Results and ConclusionsIn multivariate regression models, baseline vitamin B12 was inversely associated with changes in mean PD (P-trend=0.06) and mean CAL (P-trend=0.01), and risk ratios of tooth loss (TL; P-trend=0.006) over time. Compared to participants in the highest vitamin B12 quartile, those in the lowest quartile had 0.10mm (95%CI: 0.03, 0.17; P-difference=0.007) greater increase in mean PD, 0.23mm (95%CI: 0.09, 0.36; P-difference=0.001) greater increase in mean CAL and a relative risk of 1.57 (95%CI: 1.22, 2.03; P-difference<0.001) for TL. Stratified analyses showed stronger associations between vitamin B12 and changes in mean CAL among never smokers (P-interaction=0.058). Further studies are needed to understand the potential mechanisms of these findings.

BACKGROUND Previous experimental models suggest that vitamin E may ameliorate periodontitis. However, epidemiologic studies show inconsistent evidence in supporting this plausible association. OBJECTIVE We investigated the association between serum α-tocopherol (αT) and γ-tocopherol (γT) and periodontitis in a large cross-sectional US population. METHODS This study included 4708 participants in the 1999-2001 NHANES. Serum tocopherols were measured by HPLC and values were adjusted by total cholesterol (TC). Periodontal status was assessed by mean clinical attachment loss (CAL) and probing pocket depth (PPD). Total periodontitis (TPD) was defined as the sum of mild, moderate, and severe periodontitis. All measurements were performed by NHANES. RESULTS Means ± SDs of serum αT:TC ratio from low to high quartiles were 4.0 ± 0.4, 4.8 ± 0.2, 5.7 ± 0.4, and 9.1 ± 2.7 μmol/mmol. In multivariate regression models, αT:TC quartiles were inversely associated with mean CAL (P-trend = 0.06), mean PPD (P-trend < 0.001), and TPD (P-trend < 0.001) overall. Adjusted mean differences (95% CIs) between the first and fourth quartile of αT:TC were 0.12 mm (0.03, 0.20; P-difference = 0.005) for mean CAL and 0.12 mm (0.06, 0.17; P-difference < 0.001) for mean PPD, whereas the corresponding OR for TPD was 1.65 (95% CI: 1.26, 2.16; P-difference = 0.001). In a dose-response analysis, a clear inverse association between αT:TC and mean CAL, mean PPD, and TPD was observed among participants with relatively low αT:TC. No differences were seen in participants with higher αT:TC ratios. Participants with γT:TC ratio in the interquartile range showed a significantly lower mean PPD than those in the highest quartile. CONCLUSIONS A nonlinear inverse association was observed between serum αT and severity of periodontitis, which was restricted to adults with normal but relatively low αT status. These findings warrant further confirmation in longitudinal or intervention studies.

A. Glasgow1, A. Scott1, M. McCrudden1, D. McLean1, F. Lundy1, C. Irwin2, D. Timson3, M. Tunney4, J.S. Elborn1, S. Weldon1, C. Taggart1. 1Queen’s University Belfast, Centre for Infection and Immunity, Belfast, United Kingdom; 2Queen’s University Belfast, Centre for Dental Education, Belfast, United Kingdom; 3Queen’s University Belfast, School of Biological Sciences, Belfast, United Kingdom; 4Queen’s University Belfast, School of Pharmacy, Belfast, United Kingdom

Periodontitis, a ubiquitous chronic inflammatory disease, is associated with reduced antioxidant defences and neutrophil hyperactivity in terms of reactive oxygen species (ROS) generation. Its phenotype is thus characterized by oxidative stress. We have determined the effect of antioxidant micronutrients ascorbate and α-tocopherol on neutrophil ROS generation. Peripheral neutrophils from periodontally-healthy individuals (n = 20) were challenged with phorbol myristate acetate, IgG-opsonised Staphylococcus aureus, Fusobacterium nucleatum or PBS in the presence and absence of micronutrients (50 µM). Total and extracellular ROS were measured by luminol and isoluminol chemiluminescence respectively. Total and extracellular unstimulated, baseline ROS generation was unaffected by α-tocopherol, but inhibited by ascorbate and a combination of both micronutrients. Fcγ-receptor (Fcγ-R)-stimulated total or extracellular ROS generation was not affected by the presence of individual micronutrients. However, the combination significantly reduced extracellular FcγR-stimulated ROS release. Neither micronutrient inhibited TLR-stimulated total ROS, but the combination caused inhibition. Ascorbate and the micronutrient combination, but not α-tocopherol, inhibited extracellular ROS release by TLR-stimulated cells. Such micronutrient effects in vivo could be beneficial in reducing collateral tissue damage in chronic inflammatory diseases, such as periodontitis, while retaining immune-mediated neutrophil function.

AIM:To characterize and map temporal changes in the biological and clinical phenotype during a 21-day experimental gingivitis study.MATERIALS AND METHODS:Experimental gingivitis was induced over 21 days in healthy human volunteers (n = 56), after which normal brushing was resumed (resolution phase). Gingival and plaque indices were assessed. Gingival crevicular fluid was collected from four paired test and contra-lateral control sites in each volunteer during induction (Days 0, 7, 14 and 21) and resolution (Days 28 and 42) of experimental gingivitis. Fluid volumes were measured and a single analyte was quantified from each site-specific, 30s sample. Data were evaluated by analysis of repeated measurements and paired sample tests.RESULTS:Clinical indices and gingival crevicular fluid volumes at test sites increased from Day 0, peaking at Day 21 (test/control differences all p < 0.0001) and decreased back to control levels by Day 28. Levels of four inflammatory markers showed similar patterns, with significant differences between test and control apparent at Day 7 (substance P, cathepsin G, interleukin-1β, elastase: all p < 0.03) and peaking at Day 21 (all p < 0.002). Levels of α-1-antitrypsin showed no pattern.CONCLUSIONS:Levels of substance P, cathepsin G, interleukin-1β and neutrophil elastase act as objective biomarkers of gingival inflammation induction and resolution that typically precede phenotypical changes.

The 21-day experimental gingivitis model, an established noninvasive model of inflammation in response to increasing bacterial accumulation in humans, is designed to enable the study of both the induction and resolution of inflammation. Here, we have analyzed gingival crevicular fluid, an oral fluid comprising a serum transudate and tissue exudates, by LC-MS/MS using Fourier transform ion cyclotron resonance mass spectrometry and iTRAQ isobaric mass tags, to establish meta-proteomic profiles of inflammation-induced changes in proteins in healthy young volunteers. Across the course of experimentally induced gingivitis, we identified 16 bacterial and 186 human proteins. Although abundances of the bacterial proteins identified did not vary temporally, Fusobacterium outer membrane proteins were detected. Fusobacterium species have previously been associated with periodontal health or disease. The human proteins identified spanned a wide range of compartments (both extracellular and intracellular) and functions, including serum proteins, proteins displaying antibacterial properties, and proteins with functions associated with cellular transcription, DNA binding, the cytoskeleton, cell adhesion, and cilia. PolySNAP3 clustering software was used in a multilayered analytical approach. Clusters of proteins that associated with changes to the clinical parameters included neuronal and synapse associated proteins.

Aim: To demonstrate that linoleic acid, a constituent of sunflower oil, can become incorporated into human gingival tissue after application in vivo and, in so doing, ‘nourish’ human gingiva. Method: 14C-linoleic acid was topically applied to discrete gingival sites of healthy adult male volunteers. Tissue was recovered by biopsy at intervals up to 48 hours after administration and analysed for the presence of radiolabel in the lipid species using high performance thin layer chromatography with selected lipids further analysed using gas chromatography. Results: Incorporation of the 14C-radiolabel into human gingival lipids was demonstrated at 4 hours, 24 hours and 48 hours after topical application of 14C-linoleic acid. Conclusion: Topical application of free-fatty acids to gingival surfaces results in their absorption and localised incorporation into the tissue lipids of healthy volunteers.

Aim: To demonstrate metabolism of vitamin E acetate to vitamin E by human gingival and buccal reconstituted epithelial cell cultures. Method: Human gingival and buccal reconstituted epithelial cell cultures (SkinEthic, France) were exposed to vitamin E acetate and maintained for up to seven days at 37°C (5% CO2) in a humidified incubator. Following organic extraction of the cultures, reverse phase HPLC was used to analyse the quantities of vitamin E and vitamin E acetate in the cultures. Results: The ratio of vitamin E to vitamin E acetate increased up to 30 fold more than the solution control in gingival cultures after 7 days. Conclusion: This paper demonstrates that human gingival and buccal epithelial cells can metabolise vitamin E acetate to vitamin E, thus delivering the more active vitamin E species under the control of the activity of endogenous host enzymes.