Effects of a Berry Polyphenolic Fraction on the Pathogenic Properties of Porphyromonas gingivalis.

Porphyromonas gingivalis expresses a broad array of virulence factors that enable it to play a central role in the etiopathogenesis of periodontitis. The objective of the present study was to assess the effects of a berry polyphenolic fraction (Orophenol®) composed of extracts from cranberry, wild blueberry, and strawberry on the main pathogenic determinants of P. gingivalis. Orophenol® attenuated the growth of P. gingivalis and decreased its hemolytic activity, its adherence to a basement membrane matrix model, and its proteinase activities. The berry polyphenolic fraction also impaired the production of reactive oxygen species (ROS) by oral keratinocytes stimulated with P. gingivalis. Lastly, using an in vitro model of oral keratinocyte barrier, the fraction exerted a protective effect against the damages mediated by P. gingivalis. In conclusion, the berry polyphenolic fraction investigated in the present study attenuated several pathogenic properties of P. gingivalis. Although future clinical investigations are required, our study provided evidence that the polyphenols contained in this fraction may represent bioactive molecules of high interest for the prevention and/or treatment of periodontal disease.

A Phenolic-rich Extract of Cocoa (Theobroma cacao L.) Beans Impairs the Pathogenic Properties of Porphyromonas gingivalis and Attenuates the Activation of Nuclear Factor Kappa B in a Monocyte Model.

Periodontitis, an inflammatory disease that affects tooth-supporting tissues, is the result of a polymicrobial infection involving mainly Gram negative anaerobic bacteria. The aim of the present study was to investigate the effects of a phenolic-rich extract of cocoa (Theobroma cacao L.) beans on the pathogenic properties of Porphyromonas gingivalis, which is well-known as a keystone pathogen in the development of periodontitis. The effect of the cocoa extract on P. gingivalis-induced activation of the nuclear factor kappa B (NF-κB) transcription factor in a monocyte model was also assessed. The cocoa extract, whose major phenolic compound was epicatechin, inhibited the growth, hemolytic activity, proteolytic activities, and adherence properties (basement membrane matrix, erythrocytes) of P. gingivalis in a dose-dependent manner. It also protected the barrier function of a keratinocyte model against the deleterious effects mediated by P. gingivalis, and attenuated reactive oxygen species (ROS) production by oral keratinocytes treated with P. gingivalis. Lastly, the cocoa extract showed an anti-inflammatory property by preventing P. gingivalis-induced NF-κB activation in monocytes. In conclusion, this in vitro study highlighted the potential value of an epicatechin-rich extract of cocoa beans for preventing and/or treating periodontal diseases.

Quebecol Shows Potential to Alleviate Periodontal Tissue Damage and Promote Bone Formation in In Vitro Models.

Quebecol is a polyphenolic compound initially isolated from Canadian maple syrup in 2011. Recently, our group demonstrated in a macrophage model that quebecol inhibits the secretion of pro-inflammatory cytokines and reduces the activation of the NF-κB transcription factor. In this study, we further explored the therapeutic potential of quebecol against periodontal disease, an inflammatory disorder of bacterial origin affecting tooth-supporting tissues. More specifically, the effects of this natural compound on matrix metalloproteinase (MMP) activity and macrophage secretion, as well as on the mineralization activity of osteoblasts (bone-forming cells), were investigated. Results showed that exposing lipopolysaccharide (LPS)-treated macrophages to quebecol led to a significant decrease in the secretion of MMP-8 and MMP-9. In addition, quebecol dose dependently inhibited the catalytic activity of MMP-9. Quebecol also enhanced the mineralization activity of osteoblasts. This study brought forward additional evidence to support the potential of quebecol as a nutraceutical agent against periodontitis.

A cocoa (Theobroma cacao L.) extract impairs the growth, virulence properties, and inflammatory potential of Fusobacterium nucleatum and improves oral epithelial barrier function.

Fusobacterium nucleatum is associated with many conditions and diseases, including periodontal diseases that affect tooth-supporting tissues. The aim of the present study was to investigate the effects of a cocoa extract (Theobroma cacao L.) on F. nucleatum with respect to growth, biofilm formation, adherence, and hydrogen sulfide (H2S) production. The anti-inflammatory properties and the effect on epithelial barrier function of the cocoa extract were also assessed. The cocoa extract, whose major phenolic compound is epicatechin, dose-dependently inhibited the growth, biofilm formation, adherence properties (basement membrane matrix, oral epithelial cells), and H2S production of F. nucleatum. It also decreased IL-6 and IL-8 production by F. nucleatum-stimulated oral epithelial cells and inhibited F. nucleatum-induced NF-κB activation in monocytes. Lastly, the cocoa extract enhanced the barrier function of an oral epithelial model by increasing the transepithelial electrical resistance. We provide evidence that the beneficial properties of an epicatechin-rich cocoa extract may be useful for preventing and/or treating periodontal diseases.

A green tea extract and epigallocatechin-3-gallate attenuate the deleterious effects of irinotecan in an oral epithelial cell model.

OBJECTIVE: To investigate the ability of a green tea extract and epigallocatechin-3-gallate (EGCG) to protect oral epithelial cells against the deleterious effects of the chemotherapeutic agent irinotecan, with respect to cytotoxicity; reactive oxygen species (ROS) generation; cytokine and matrix metalloproteinase (MMP) production; and cell proliferation and migration. METHODS: The B11 oral keratinocyte and GMSM-K oral epithelial cell lines were used in this study. Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. A fluorometric assay was used to quantify ROS production. Cell proliferation was assessed using a fluorescent cell tracker dye, while a migration assay kit was used to monitor cell migration. Cytokine and MMP secretion was quantified by an enzyme-linked immunosorbent assay. RESULTS: The green tea extract and EGCG reduced the cytotoxicity of irinotecan toward oral keratinocyte and epithelial cell lines. Irinotecan-induced intracellular ROS generation by oral keratinocytes was reduced by the green tea extract and EGCG. Irinotecan negatively affected the proliferation and migration of oral keratinocytes in a dose-dependent manner. However, these effects were not neutralized by the green tea extract, while EGCG showed a trend to attenuate the irinotecan-induced decrease in cell migration. The green tea extract and EGCG also had a dose-dependent inhibitory effect on irinotecan-induced secretion of interleukin-6 and interleukin-8 by oral epithelial cells. Lastly, the irinotecan-induced decrease in the secretion of MMP-2 and MMP-9 by oral epithelial cells was partially restored by the green tea extract and EGCG. CONCLUSIONS: The green tea extract and EGCG, through anti-cytotoxic, anti-oxidative, and anti-inflammatory properties, may protect the oral mucosa against the deleterious effects of the chemotherapeutic agent irinotecan and may be of interest for treating oral mucositis.

A Dual Zinc plus Arginine formulation protects against tumor necrosis factor-alpha-induced barrier dysfunction and enhances cell proliferation and migration in an in vitro gingival keratinocyte model.

OBJECTIVE: To investigate the effects of Dual Zinc plus Arginine formulations (aqueous solution and dentifrice) on tumor necrosis factor-alpha (TNF-α)-induced barrier dysfunction as well as on cell proliferation and migration in an in vitro gingival keratinocyte model. DESIGN: Gingival keratinocytes were seeded onto the membrane of a double-chamber system in the absence and presence of recombinant TNF-α and the formulations under investigation. The barrier function was assessed by determination of transepithelial electrical resistance (TER) and paracellular transport of fluorescein isothiocyanate (FITC)-dextran. The distribution of zonula occludens-1 (ZO-1) and occludin was visualized by immunofluorescence microscopy. The effects of the formulations on keratinocyte cell proliferation were determined using a fluorescent cell tracker dye, while a migration assay kit was used to investigate their effects on cell migration. RESULTS: Under conditions where TNF-α induces loss of keratinocyte barrier integrity, the Dual Zinc plus Arginine formulations (aqueous solution and dentifrice) protected the keratinocyte tight junction against the damages since they prevented the TNF-α-induced drop in TER and increase in FITC-dextran paracellular flux in the in vitro model. The treatment of keratinocytes with the formulations markedly mitigated the altered distribution of ZO-1 and occludin. Both formulations increased the proliferation of keratinocytes and alleviated the negative impact caused by TNF-α. Lastly, the formulations increased the migration capacity of keratinocytes. CONCLUSIONS: The ability of the Dual Zinc plus Arginine formulations to protect the barrier integrity of gingival keratinocytes from TNF-α-induced damage and to promote their proliferation and migration suggests that they may offer benefits for oral health.

Production of TNF-𝛼 by macrophages stimulated with endodontic pathogens and its effect on the biological properties of stem cells of the apical papilla.

OBJECTIVES: The first objective of the present study was to investigate TNF-𝛼 secretion by macrophages stimulated with endodontic pathogens and bacterial cell surface components. The second objective was to assess the in vitro effects of TNF-𝛼 on periostin, cytokine, and matrix metalloproteinase (MMP) secretion by and the viability, proliferation rate, and mineralization potential of stem cells of the apical papilla (SCAP). METHODS: TNF-𝛼 secretion by macrophages stimulated with either endodontic pathogens or bacterial surface components was assessed using an enzyme-linked immunosorbent assay (ELISA). The viability and proliferation rate of SCAP treated with TNF-𝛼 were assessed using a colorimetric MTT assay. The mineralization potential of TNF-𝛼-treated SCAP was determined by Alizarin Red staining. Periostin secretion by SCAP was determined by ELISA while cytokine and MMP secretion were assessed using a multiplexing laser bead assay. RESULTS: TNF-𝛼 secretion by macrophages increased following a stimulation with Gram-negative and Gram-positive endodontic pathogens. Lipopolysaccharide and lipoteichoic acid also dose-dependently increased the secretion of TNF-𝛼 by macrophages. The viability, proliferation rate, and mineralization activity of SCAP were negatively affected by a TNF-𝛼 treatment. Treating SCAP with TNF-𝛼 attenuated the secretion of periostin and upregulated the secretion of several cytokines and MMPs. CONCLUSIONS: TNF-𝛼 exerts deleterious effects on SCAP by affecting their viability, proliferation rate, and mineralization potential. By its ability to induce the secretion of pro-inflammatory cytokines and MMPs by SCAP, TNF-𝛼 can contribute to creating an inflammatory environment, promoting tissue destruction, and consequently interfering with the success of regenerative endodontic therapy. CLINICAL RELEVANCE: TNF-𝛼 has deleterious impacts on stem cells of the apical papilla and may compromise the outcome of regenerative endodontic therapy.

Effects of the Licorice Isoflavans Licoricidin and Glabridin on the Growth, Adherence Properties, and Acid Production of Streptococcus mutans, and Assessment of Their Biocompatibility.

Pharmacological studies have linked a number of human health benefits with licorice due to its anticancer, anti-inflammatory, anti-oxidant, and antimicrobial properties. The aim of this study was to investigate the effects of licoricidin and glabridin, two major licorice isoflavans, on growth and virulence properties (biofilm formation, acid production, dextran production, adherence) of the cariogenic bacterium Streptococcus mutans. Moreover, the biocompatibility of these licorice compounds was assessed in an in vitro model of oral keratinocytes. We used a broth microdilution assay to show that licoricidin and glabridin exhibit a marked antibacterial activity against S. mutans. Glabridin and, to a lesser extent, licoricidin reduced the biofilm viability of S. mutans. In addition, glabridin decreased the production of dextran by S. mutans. The two licorice isoflavans attenuated the adherence of S. mutans to a saliva-coated hydroxylapatite surface, and reduced acid production from glucose. Lastly, depending on the concentrations tested, the two licorice isoflavans showed no or low toxicity toward oral keratinocytes. Within the limitations of this study, our data suggest that licoricidin and glabridin may be promising agents for controlling dental caries.

A polyphenolic cinnamon fraction exhibits anti-inflammatory properties in a monocyte/macrophage model.

Periodontal diseases are bacteria-induced inflammatory disorders that lead to the destruction of the tooth-supporting tissues. Active compounds endowed with a capacity to regulate the inflammatory response are regarded as potential therapeutic agents for the treatment of periodontal diseases. The aim of this study was to characterize the anti-inflammatory properties of a polyphenolic cinnamon fraction. Chromatographic and mass spectrometry analyses of the polyphenolic composition of the cinnamon fraction revealed that phenolic acids, flavonoids (flavonols, anthocyanins, flavan-3-ols), and procyanidins make up 9.22%, 0.72%, and 10.63% of the cinnamon fraction, respectively. We used a macrophage model stimulated with lipopolysaccharides (LPS) from either Aggregatibacter actinomycetemcomitans or Escherichia coli to show that the cinnamon fraction dose-dependently reduced IL-6, IL-8, and TNF-α secretion. Evidence was brought that this inhibition of cytokine secretion may result from the ability of the fraction to prevent LPS-induced NF-κB activation. We also showed that the cinnamon fraction reduces LPS binding to monocytes, which may contribute to its anti-inflammatory properties. Lastly, using a competitor assay, it was found that the cinnamon fraction may represent a natural PPAR-γ ligand. Within the limitations of this in vitro study, the cinnamon fraction was shown to exhibit a therapeutic potential for the treatment of periodontal diseases due to its anti-inflammatory properties.

Effect of a Berry Polyphenolic Fraction on Biofilm Formation, Adherence Properties and Gene Expression of Streptococcus mutans and Its Biocompatibility with Oral Epithelial Cells.

The ability of Streptococcus mutans to adhere to oral surfaces and form biofilm is a key step in the tooth decay process. The aim of this study was to investigate a berry (wild blueberry, cranberry, and strawberry) polyphenolic fraction, commercialized as Orophenol®, for its antibacterial, anti-biofilm, and anti-adhesion properties on S. mutans. Moreover, the biocompatibility of the fraction with human oral epithelial cells was assessed. Phenolic acids, flavonoids (flavonols, anthocyanins, flavan-3-ols), and procyanidins made up 10.71%, 19.76%, and 5.29% of the berry polyphenolic fraction, respectively, as determined by chromatography and mass spectrometry. The berry polyphenolic preparation dose-dependently inhibited S. mutans biofilm formation while not reducing bacterial growth. At concentrations ranging from 250 to 1000 µg/mL, the fraction inhibited the adhesion of S. mutans to both saliva-coated hydroxyapatite and saliva-coated nickel-chrome alloy. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that incubating S. mutans with the berry polyphenolic fraction was associated with a reduced expression of luxS gene, which regulates quorum sensing in S. mutans. The berry fraction did not show any significant cytotoxicity in an oral epithelial cell model. In conclusion, Orophenol®, which is a mixture of polyphenols from wild blueberry, cranberry and strawberry, possesses interesting anti-caries properties while being compatible with oral epithelial cells.

Effects of a tart cherry (Prunus cerasus L.) phenolic extract on Porphyromonas gingivalis and its ability to impair the oral epithelial barrier.

Periodontal diseases, including gingivitis and periodontitis, are a global oral health problem. Porphyromonas gingivalis, a key pathogen involved in the onset of periodontitis, is able to colonize the subgingival epithelium and invade the underlying connective tissue due to the contribution of cysteine proteases known as gingipains. In this study, we investigated the effects of a phenolic extract prepared from tart cherry (Prunus cerasus L.) juice on the growth, adherence, and protease activity of P. gingivalis. We also assessed the protective effect of the tart cherry extract on the disruption of the oral epithelial barrier induced by P. gingivalis. The tart cherry extract that contains procyanidins and quercetin and its derivatives (rutinoside, glucoside) as the most important phenolic compounds attenuated P. gingivalis growth, reduced adherence to an experimental basement membrane matrix model, and decreased the protease activities of P. gingivalis. The tart cherry extract also exerted a protective effect on the integrity of the oral epithelial barrier in an in vitro model infected with P. gingivalis. More specifically, the extract prevented a decrease in transepithelial electrical resistance as well as the destruction of tight junction proteins (zonula occludens-1 and occludin). These results suggest that the tart cherry phenolic extract may be a promising natural product for the treatment of periodontitis through its ability to attenuate the virulence properties of P. gingivalis and curtail the ability of this pathogen to impair the oral epithelial barrier.

Effects of Saliva From Periodontally Healthy and Diseased Subjects on Barrier Function and the Inflammatory Response in in vitro Models of the Oral Epithelium.

Background: Periodontitis is a multifactorial, bacteria-mediated chronic inflammatory disease that results in the progressive destruction of the tooth-supporting tissues. It is well-known that saliva from subjects suffering from this disease generally contains higher levels of pro-inflammatory mediators, matrix metalloproteinases (MMP), and bacteria-derived toxic products. The aim of this study was to investigate and compare the effects of saliva from periodontally healthy and diseased subjects on the barrier function and inflammatory response in in vitro models of the oral epithelium. Methods: Unstimulated saliva samples from two groups of subjects, one with a healthy periodontium (n = 12) and one with severe generalized periodontitis (n = 11), were filter-sterilized. All the saliva samples were analyzed using an immunological multiplex assay to determine the levels of various cytokines and MMPs relevant to periodontitis. The impact of saliva on epithelial barrier integrity was assessed by monitoring transepithelial electrical resistance (TER) in an oral epithelium model using the B11 keratinocyte cell line. GMSM-K oral epithelial cells were treated with saliva from both groups to determine their ability to induce the secretion of interleukin-6 (IL-6) and interleukin-8 (IL-8), as determined by an enzyme-linked immunosorbent assay (ELISA). Results: Saliva from the periodontitis subjects contained significantly higher concentrations of matrix metalloproteinase-8 (MMP-8), matrix metalloproteinase-9 (MMP-9), IL-8, and C-X-C motif chemokine ligand 1 (CXCL1) compared to saliva from the healthy subjects. Saliva from the healthy and periodontitis subjects affected cytokine secretion and TER in a similar manner. More specifically, saliva from both groups increased TER and induced IL-6 and IL-8 secretion in the in vitro oral epithelium models used. Conclusion: Independently of the presence or absence of periodontitis, saliva can increase the relative TER and the secretion of IL-6 and IL-8 in in vitro models of the oral epithelium.

Effects of Labrador Tea, Peppermint, and Winter Savory Essential Oils on Fusobacterium nucleatum.

Bad breath or halitosis is an oral condition caused by volatile sulfur compounds (VSC) produced by bacteria found in the dental and tongue biofilms. Fusobacterium nucleatum is a Gram-negative anaerobic bacterium that has been strongly associated with halitosis. In this study, essential oils (EO) from three plants, Labrador tea (Rhododendron groenlandicum [Oeder] Kron & Judd), peppermint (Mentha x piperita L.), and winter savory (Satureja montana L.), were investigated for their effects on growth, biofilm formation and killing, and VSC production by F. nucleatum. Moreover, their biocompatibility with oral keratinocytes was investigated. Using a broth microdilution assay, winter savory EO and to a lesser extent Labrador tea and peppermint EO showed antibacterial activity against F. nucleatum. A treatment of pre-formed biofilms of F. nucleatum with EO also significantly decreased bacterial viability as determined by a luminescence assay monitoring adenosine triphosphate production. The EO were found to permeabilize the bacterial cell membrane, suggesting that it represents the target of the tested EO. The three EO under investigation were able to dose-dependently reduce VSC production by F. nucleatum. Lastly, no significant loss of cell viability was observed when oral keratinocytes were treated with the EO at concentrations effective against F. nucleatum. This study supports the potential of Labrador tea, peppermint, and winter savory EO as promising agents to control halitosis and promote oral health.

Highbush blueberry proanthocyanidins alleviate Porphyromonas gingivalis-induced deleterious effects on oral mucosal cells.

Strong evidence points to Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, as a keystone species in the development of the chronic form of periodontitis. The aim of the present study was to investigate the ability of highbush blueberry proanthocyanidins (PACs) to alleviate the P. gingivalis-induced deleterious effects on oral mucosal cells. We first showed that highbush blueberry PACs protect the integrity of the gingival keratinocyte barrier against P. gingivalis-mediated damage, as determined by measuring the transepithelial electrical resistance and paracellular flux of FITC-conjugated dextran. Moreover, the PACs prevented the translocation of P. gingivalis across the gingival keratinocyte barrier model. The proteinase activity of P. gingivalis was inhibited by the PACs suggesting that they may exert beneficial effects by reducing proteolytic degradation of the epithelial tight junctions. Regulation of gingival fibroblast inflammatory reactions may be one of the ways to prevent and control periodontal disease progression and severity. We showed that PACs significantly reduce IL-6 and IL-8 secretion by P. gingivalis-stimulated gingival fibroblasts. The present study showed the capacity of highbush blueberry PACs to protect the integrity of an in vitro model of gingival keratinocyte barrier against P. gingivalis, and to attenuate the secretion of pro-inflammatory cytokines by gingival fibroblasts infected with P. gingivalis. These results suggest beneficial effects of blueberry PACs thus supporting the need for future clinical trials on the potential of these bioactive molecules for periodontal disease prevention and/or treatment.

A Dual Zinc plus Arginine formulation attenuates the pathogenic properties of Porphyromonas gingivalis and protects gingival keratinocyte barrier function in an in vitro model.

BACKGROUND AND OBJECTIVES: Porphyromonas gingivalis, a late colonizer of the periodontal biofilm, has been strongly associated with the chronic form of periodontitis. The aim of this study was to investigate the effects of a Dual Zinc plus Arginine formulation (aqueous solution and dentifrice) on the pathogenic properties of P. gingivalis and the barrier function of an in vitro gingival epithelium model. RESULTS: The Dual Zinc plus Arginine aqueous solution and dentifrice inhibited the hemolytic and proteolytic activities of P. gingivalis. The Dual Zinc plus Arginine aqueous solution and dentifrice enhanced the barrier function of an in vitro gingival epithelium model as determined by a time-dependent increase in transepithelial electrical resistance and decrease in paracellular permeability. This was associated with an increased immunolabeling of two important tight junction proteins: zonula occludens-1 and occludin. The deleterious effects of P. gingivalis on keratinocyte barrier function as well as the ability of the bacterium to translocate through a gingival epithelium model were attenuated in the presence of either Dual Zinc plus Arginine aqueous solution or dentifrice. CONCLUSIONS: The Dual Zinc plus Arginine formulation may offer benefits for patients affected by periodontal disease through its ability to attenuate the pathogenic properties of P. gingivalis and promote keratinocyte barrier function.

Biocompatible combinations of nisin and licorice polyphenols exert synergistic bactericidal effects against Enterococcus faecalis and inhibit NF-κB activation in monocytes.

Enterococcus faecalis is one of the bacterial species most frequently isolated from persistent endodontic and apical periodontal infections. The aim of the present study was to evaluate the synergistic antibacterial effects of nisin and selected licorice polyphenols (glabridin, licoricidin, licochalcone A) against planktonic and biofilm-embedded E. faecalis cells. The biocompatibility and anti-inflammatory properties of the nisin/licorice polyphenol combinations were also investigated. The lantibiotic bacteriocin (nisin), the two isoflavonoids (glabridin, licoricidin), and the chalcone (licochalcone A) efficiently inhibited the growth of E. faecalis, with MICs ranging from 6.25 to 25 µg/mL. Combining nisin with each licorice polyphenol individually resulted in a significant synergistic antibacterial effect. Following a 30-min contact, nisin in combination with either glabridin, licoricidin, or licochalcone A caused significant biofilm killing. The nisin/licorice polyphenol combinations had no cytotoxic effects (oral epithelial cells, gingival fibroblasts, and stem cells of the apical papilla), with the exception of nisin/glabridin, when used at their MICs. Lastly, we showed that nisin/glabridin, nisin/licoricidin, and nisin/licochalcone A inhibit NF-κB activation induced by E. faecalis in a monocyte model, suggesting that these combinations possess anti-inflammatory properties. The present study provides evidence that combinations of nisin and glabridin, licoricidin, or licochalcone A show promise as root canal disinfection agents.

Tart cherry (Prunus cerasus L.) fractions inhibit biofilm formation and adherence properties of oral pathogens and enhance oral epithelial barrier function.

Dental caries, candidiasis, and periodontal disease are the most common oral infections affecting a wide range of the population worldwide. The present study investigated the effects of two tart cherry (Prunus cerasus L.) fractions on important oral pathogens, including Candida albicans, Streptococcus mutans, and Fusobacterium nucleatum, as well as on the barrier function of oral epithelial cells. Procyanidins and quercetin and its derivatives were the most important constituents found in the tart cherry fractions. Although the fractions showed poor antimicrobial activity, they inhibited biofilm formation by the three oral pathogens in a dose-dependent manner. The tart cherry fractions also attenuated the adherence of C. albicans and S. mutans to a hydroxylapatite surface as well as the adherence of F. nucleatum to oral epithelial cells. Treating oral epithelial cells with the tart cherry fractions significantly enhanced the barrier function as determined by monitoring the transepithelial electrical resistance. In conclusion, this study showed that the tart cherry fractions and their bioactive constituents could be promising antiplaque compounds by targeting biofilm formation and adherence properties of oral pathogens. Furthermore, its property of increasing the epithelial barrier function may protect against microbial invasion of the underlying connective tissue.

Cranberry Proanthocyanidins Neutralize the Effects of Aggregatibacter actinomycetemcomitans Leukotoxin.

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium that has been strongly associated with localized aggressive periodontitis. The capacity of A. actinomycetemcomitans to produce a leukotoxin (LtxA) that activates pyroptosis in macrophages and induces the release of endogenous danger signals is thought to play a key role in the disease process. The aim of the present study was to investigate the effects of cranberry proanthocyanidins (PACs) on gene expression and cytotoxic activities of LtxA. We showed that cranberry PACs dose-dependently attenuate the expression of genes making up the leukotoxin operon, including ltxB and ltxC, in the two strains of A. actinomycetemcomitans tested. Cranberry PACs (≥62.5 µg/mL) protected macrophages against the cytotoxic effect of purified LtxA. Moreover, cranberry PACs reduced caspase-1 activation in LtxA-treated macrophages and consequently decreased the release of both IL-1ß and IL-18, which are known as damage-associated molecular patterns (DAMPs) and contribute to the progression of periodontitis by increasing cell migration and osteoclastogenesis. In addition, cranberry PACs reduced the expression of genes encoding the P2X7 receptor and NALP3 (NACHT, LRR and PYD domains-containing protein 3), which play key roles in pore formation and cell death. Lastly, cranberry PACs blocked the binding of LtxA to macrophages and consequently reduced the LtxA-mediated cytotoxicity. In summary, the present study showed that cranberry PACs reduced LtxA gene expression in A. actinomycetemcomitans and neutralized the cytolytic and pro-inflammatory responses of human macrophages treated with LtxA. Given these properties, cranberry PACs may represent promising molecules for prevention and treatment of the aggressive form of periodontitis caused by A. actinomycetemcomitans.

Resveratrol attenuates the pathogenic and inflammatory properties of Porphyromonas gingivalis.

Porphyromonas gingivalis has been strongly associated with chronic periodontitis, which affects tooth-supporting tissues. This Gram-negative anaerobic bacterium produces a repertoire of virulence factors that modulate tissue destruction directly or indirectly by the induction of inflammatory processes. The aim of this study was to investigate the effects of resveratrol, a major polyphenol found in grapes and wine, on the growth and virulence properties of P. gingivalis as well as on gingival keratinocyte tight junction integrity and the host inflammatory response. Resveratrol exhibited antibacterial activity that may result from damage to the bacterial cell membrane. Resveratrol also killed a pre-formed P. gingivalis biofilm and reduced bacterial adherence to matrix proteins. In addition, resveratrol had a protective effect on the integrity of the keratinocyte tight junctions by inhibiting its breakdown by P. gingivalis. This may be related to the ability of resveratrol to inhibit the protease activities of P. gingivalis. Lastly, resveratrol reduced P. gingivalis-mediated activation of the NF-κB signaling pathway and attenuated TREM-1 gene expression as well as soluble TREM-1 secretion in monocytes. The effect on NF-κB activation likely results from the ability of resveratrol to act as a PPAR-γ agonist. In summary, the antibacterial, anti-adherence, and antiprotease properties of resveratrol, as well as its ability to protect the gingival keratinocyte barrier and attenuate the inflammatory response in monocytes suggest that it may be a promising novel therapeutic agent for treating periodontal disease.

Dual action of highbush blueberry proanthocyanidins on Aggregatibacter actinomycetemcomitans and the host inflammatory response.

BACKGROUND: The highbush blueberry (Vaccinium corymbosum) has a beneficial effect on several aspects of human health. The present study investigated the effects of highbush blueberry proanthocyanidins (PACs) on the virulence properties of Aggregatibacter actinomycetemcomitans and macrophage-associated inflammatory responses. METHODS: PACs were isolated from frozen highbush blueberries using solid-phase chromatography. A microplate dilution assay was performed to determine the effect of highbush blueberry PACs on A. actinomycetemcomitans growth as well as biofilm formation stained with crystal violet. Tight junction integrity of oral keratinocytes was assessed by measuring the transepithelial electrical resistance (TER), while macrophage viability was determined with a colorimetric MTT assay. Pro-inflammatory cytokine and MMP secretion by A. actinomycetemcomitans-stimulated macrophages was quantified by ELISA. The U937-3xκB-LUC monocyte cell line transfected with a luciferase reporter gene was used to monitor NF-κB activation. RESULTS: Highbush blueberry PACs reduced the growth of A. actinomycetemcomitans and prevented biofilm formation at sub-inhibitory concentrations. The treatment of pre-formed biofilms with the PACs resulted in a loss of bacterial viability. The antibacterial activity of the PACs appeared to involve damage to the bacterial cell membrane. The PACs protected the oral keratinocytes barrier integrity from damage caused by A. actinomycetemcomitans. The PACs also protected macrophages from the deleterious effect of leukotoxin Ltx-A and dose-dependently inhibited the secretion of pro-inflammatory cytokines (IL-1ß, IL-6, CXCL8, TNF-α), matrix metalloproteinases (MMP-3, MMP-9), and sTREM-1 by A. actinomycetemcomitans-treated macrophages. The PACs also inhibited the activation of the NF-κB signaling pathway. CONCLUSION: The antibacterial and anti-inflammatory properties of highbush blueberry PACs as well as their ability to protect the oral keratinocyte barrier and neutralize leukotoxin activity suggest that they may be promising candidates as novel therapeutic agents.