Microbial biomarkers as a predictor of periodontal treatment response: A systematic review.

To evaluate the prognostic accuracy of microbial biomarkers and their associations with the response to active periodontal treatment (APT) and supportive periodontal therapy (SPT). Microbial dysbiosis plays a crucial role in the disease processes of periodontitis. Biomarkers based on microbial composition may offer additional prognostic value, supplementing the limitations of current clinical parameters. While these microbial biomarkers have been clinically evaluated, there is a lack of consensus regarding their prognostic accuracy. A structured search strategy was applied to MEDLINE (PubMed), Cochrane Library, and Embase on 1/11/2022 to identify relevant publications. Prospective clinical studies involving either APT or SPT, with at least 3-month follow-up were included. There were no restrictions on the type of microbial compositional analysis. 1918 unique records were retrieved, and 13 studies (comprising 943 adult patients) were included. Heterogeneity of the studies precluded a meta-analysis, and none of the included studies had performed the sequence analysis of the periodontal microbiome. Seven and six studies reported on response to APT and SPT, respectively. The prognostic accuracy of the microbial biomarkers for APT and SPT was examined in only two and four studies, respectively. Microbial biomarkers had limited predictive accuracy for APT and inconsistent associations for different species across studies. For SPT, elevated abundance of periodontal pathogens at the start of SPT was predictive of subsequent periodontal progression. Similarly, persistent high pathogen loads were consistently associated with progressive periodontitis, defined as an increased pocket probing depth or clinical attachment loss. While there was insufficient evidence to support the clinical use of microbial biomarkers as prognostic tools for active periodontal treatment outcomes, biomarkers that quantify periodontal pathogen loads may offer prognostic value for predicting progressive periodontitis in the subsequent supportive periodontal therapy phase. Additional research will be required to translate information regarding subgingival biofilm composition and phenotype into clinically relevant prognostic tools.

Toll-like receptor-4 activation by subgingival biofilm and periodontal treatment response.

OBJECTIVES: This study aims to investigate longitudinally the activation of Toll-like receptor-4 (TLR-4) by subgingival biofilm samples before and after nonsurgical periodontal therapy (NSPT). MATERIALS AND METHODS: Forty periodontitis patients received NSPT and were reviewed 3 and 6 months post-treatment. Subgingival biofilm was sampled from 4 teeth per patient, at baseline and each follow-up time point. TLR-4 activation was determined using the HEK-BLUE™/hTLR4 system. Changes in TLR-4 activation and probing pocket depths (PPDs) were evaluated using generalised linear models, and the association between TLR-4 activation and pocket reduction (defined as 6-month PPDs ≤ 3mm) was determined using generalised estimating equations. RESULTS: At 6 months, the mean TLR-4 activation by subgingival biofilm samples was significantly reduced from 11.2AU (95%CI 7.1AU, 15.4AU) to 3.6AU (95%CI 2.3AU, 4.8AU, p < 0.001), paralleling significant reductions in mean PPDs at sampled sites. The response to NSPT was associated with longitudinal TLR-4 activation profiles, with significantly higher TLR-4 activation by subgingival biofilm obtained from sites that did not achieve pocket reduction, compared to sites at which pocket reduction was achieved. CONCLUSIONS: The activation of TLR-4 by subgingival biofilm samples was reduced after NSPT, and this reduction was significantly associated with the clinical improvements (PPD reductions) at sampled sites. CLINICAL RELEVANCE: This study demonstrated an association between the longitudinal profile of TLR-4 activation by subgingival biofilm and periodontal treatment response. Longitudinal monitoring of TLR-4 activation by subgingival biofilm may potentially identify non-responsive sites, enabling targeted additional treatment.

Correlation between microbial host factors and caries among older adults.

BACKGROUND: There is little knowledge about factors which may affect oral health among older adults. The objective of this study was to determine the relationship between Streptococcus mutans (MS) and Lactobacilli (LB) counts and caries among older adults. METHODS: In this community-based observation study, 141 participants aged 60 years and above were recruited from the west district of Singapore. Alongside the clinical examination, saliva samples were collected to determine Streptococcus mutans (MS) and Lactobacilli (LB) counts, as well as to record salivary flow rate and buffering capacity of saliva. RESULTS: Of the 141 participants, 63.8% were female and 94.3% were of Chinese ethnicity. The mean DMFT was 11.08 (s.d. 8.27). 9.9% of participants had at least one decayed tooth, 52.5% had minimum one missing tooth and 86.5% had at least one filled tooth. 67.4% had MS counts of ≥ 105 while LB counts were ≥ 105 for 48.2%. 83.7% had normal salivary flow or hypersalivation (> = 1 mL/min), the buffering capacity of the saliva was alkaline in 61% of the participants. Multivariate analysis showed that participants who had high MS counts were less likely to have a DMFT < 12 [OR (95% CI), 0.29 (0.11-0.77)] whereas participants who had high LB counts were less likely to have a DMFT ≤ 14 [OR (95% CI), 0.45 (0.20-1.002)]. CONCLUSION: Our study showed a positive correlation between MS and LB counts and caries experience in older adults. The mean DMFT was on the low side in our sample despite having a relatively high MS count. This suggests that there are many other factors which vary according to host environment, physiological and biological conditions that may affect MS and LB counts in the oral cavity. CLINICAL RELEVANCE: Our study supports the knowledge that the aetiology of dental caries among older adults is a complex process and it would be wrong to consider caries as a same problem with the same solution for all age groups.

Telomere length is regulated by FGF-2 in human embryonic stem cells and affects the life span of its differentiated progenies.

The ability of human embryonic stem cells (hESCs) to proliferate indefinitely is attributed to its high telomerase activity and associated long telomere. However, factors regulating telomere length in hESCs remain largely uncharacterized. The aims of this study were, to identify factors which modulate telomere length of hESCs, and to determine if the telomere length of hESCs influences cellular senescence of its differentiated progeny cells. Telomerase reverse transcriptase (TERT) gene expression, telomerase activity and telomere length of hESCs cultured in different culture systems were compared. Genetically identical hESCs of different telomere lengths were differentiated into fibroblasts simultaneously, and the population doubling and cellular senescence levels were determined. We found that telomere lengths were significantly different in different culture systems and Fibroblast growth factor-2 (FGF-2) upregulated TERT expression, telomerase activity and telomere length via Wnt/ß-catenin signaling pathway in hESCs in a significant manner. We also provide evidence that fibroblast differentiated from hESCs with longer telomere exhibited significant more population doublings and longer life span than those derived from hESCs with shorter telomeres. Thus, FGF-2 levels in hESCs culture systems can be manipulated to generate cells with longer telomere which would be advantageous in the applications of hESCs in regenerative medicine.

Reduced Glutathione Mediates Resistance to H2S Toxicity in Oral Streptococci.

Periodontal disease is associated with changes in the composition of the oral microflora, where health-associated oral streptococci decrease while Gram-negative anaerobes predominate in disease. A key feature of periodontal disease-associated anaerobes is their ability to produce hydrogen sulfide (H2S) abundantly as a by-product of anaerobic metabolism. So far, H2S has been reported to be either cytoprotective or cytotoxic by modulating bacterial antioxidant defense systems. Although oral anaerobes produce large amounts of H2S, the potential effects of H2S on oral streptococci are currently unknown. The aim of this study was to determine the effects of H2S on the survival and biofilm formation of oral streptococci. The growth and biofilm formation of Streptococcus mitis and Streptococcus oralis were inhibited by H2S. However, H2S did not significantly affect the growth of Streptococcus gordonii or Streptococcus sanguinis. The differential susceptibility of oral streptococci to H2S was attributed to differences in the intracellular concentrations of reduced glutathione (GSH). In the absence of GSH, H2S elicited its toxicity through an iron-dependent mechanism. Collectively, our results showed that H2S exerts antimicrobial effects on certain oral streptococci, potentially contributing to the decrease in health-associated plaque microflora.

Quantifying periodontitis-associated oral dysbiosis in tongue and saliva microbiomes-An integrated data analysis.

BACKGROUND: Periodontitis is primarily driven by subgingival biofilm dysbiosis. However, the quantification and impact of this periodontal dysbiosis on other oral microbial niches remain unclear. This study seeks to quantify the dysbiotic changes in tongue and salivary microbiomes resulting from periodontitis by applying a clinically relevant dysbiosis index to an integrated data analysis. METHODS: The National Center for Biotechnology Information (NCBI) database was searched to identify BioProjects with published studies on salivary and tongue microbiomes of healthy and periodontitis subjects. Raw sequence datasets were processed using a standardized bioinformatic pipeline and categorized by their ecological niche and periodontal status. The subgingival microbial dysbiosis index (SMDI), a dysbiosis index originally developed using the subgingival microbiome, was computed at species and genus levels and customized for each niche. Its diagnostic accuracy for periodontitis was evaluated using receiver operating characteristic curves. RESULTS: Four studies, contributing 328 microbiome samples, were included. At both species and genus levels, periodontitis samples had a higher SMDI, but the differences were only significant for subgingival biofilm and saliva (p < 0.001). However, SMDI showed good diagnostic accuracy for periodontitis status for all three niches (area under curve ranging from 0.76 to 0.90, p < 0.05). The dysbiosis index of subgingival biofilm was positively correlated with saliva consistently (p < 0.001) and with the tongue at the genus level (p = 0.036). CONCLUSIONS: While the impact on the tongue microbiome requires further investigation, periodontitis-associated dysbiosis affects the salivary microbiome and is quantifiable using the dysbiosis index. The diagnostic potential of salivary microbial dysbiosis as a convenient periodontal biomarker for assessing periodontal status has potential public health and clinical applications. PLAIN LANGUAGE SUMMARY: Periodontitis, a severe inflammation of the gums which causes bone loss, is a disease caused by an imbalance of good and bad bacteria under the gums. However, it is unclear how this bacterial imbalance in the gums affects the bacterial balance of other distinct parts of the mouth, such as the saliva and tongue. This study uses bacteria datasets of four previously published studies, contributing a total of 328 bacterial samples. The data were processed using a uniform data analysis workflow, and a bacterial score, the subgingival microbial dysbiosis index (SMDI), previously shown to capture periodontitis-associated bacteria imbalance, was calculated separately for samples from under the gums, the saliva, and the tongue. The SMDI was able to distinguish between health and periodontitis within each oral location, and in general, the scores were higher for periodontitis samples, though this difference was significant only for bacteria under the gums and in saliva. Saliva scores were also consistently correlated with bacteria under the gums. This study shows that periodontitis-associated bacterial imbalances are observed in oral locations beyond just under the gums, particularly the saliva. Thus, saliva bacteria may be used as a convenient biomarker for assessing gum disease, allowing for potential public health and clinical applications.

Boron-containing coating yields enhanced antimicrobial and mechanical effects on translucent zirconia.

OBJECTIVES: To evaluate the mechanical and antimicrobial properties of boron-containing coating on translucent zirconia (5Y-PSZ). METHODS: 5Y-PSZ discs (Control) were coated with a glaze (Glaze), silver- (AgCoat), or boron-containing (BCoat) glasses. The coatings' antimicrobial potential was characterized using S. mutans biofilms after 48 h via viable colony-forming units (CFU), metabolic activity (CV) assays, and quantification of extracellular polysaccharide matrix (EPS). Biofilm architectures were imaged under scanning electron and confocal laser scanning microscopies (SEM and CLSM). The cytocompatibility was determined at 24 h via WST-1 and LIVE&DEAD assays using periodontal ligament stem cells (PDLSCs). The coatings' effects on properties were characterized by Vickers hardness, biaxial bending tests, and fractography analysis. Statistical analyses were performed via one-way ANOVA, Tukey's tests, Weibull analysis, and Pearson's correlation analysis. RESULTS: BCoat significantly decreased biofilm formation, having the lowest CFU and metabolic activity compared with the other groups. BCoat and AgCoat presented the lowest EPS, followed by Glaze and Control. SEM and CLSM images revealed that the biofilms on BCoat were thin and sparse, with lower biovolume. In contrast, the other groups yielded robust biofilms with higher biovolume. The cytocompatibility was similar in all groups. BCoat, AgCoat, and Glaze also presented similar hardness and were significantly lower than Control. BCoat had the highest flexural strength, characteristic strength and Weibull parameters (σF: 625 MPa; σ0: 620 MPa; m = 11.5), followed by AgCoat (σF: 464 MPa; σ0: 478 MPa; m = 5.3). SIGNIFICANCE: BCoat is a cytocompatible coating with promising antimicrobial properties that can improve the mechanical properties and reliability of 5Y-PSZ.

Human embryonic stem cells derived keratinocyte as an in vitro research model for the study of immune response.

BACKGROUND: The innate immune response (IMR) is critical for the oral mucosa due to their continuous exposure to various oral pathogens. Keratinocytes play important role in IMR. Therefore, to date, keratinocytes from different sources have been used as in vitro research model for the study of IMR. However, current keratinocyte research models are hampered by the limited supply, patients' dependency and batch to batch variation. Therefore, in this study, we demonstrated the use of human embryonic stem cells (hESCs) derived keratinocytes (H9-Kert) as an alternative research model for the study of IMR. METHODS: The expression kinetics of toll-like receptor (TLR) 2, TLR 4, interleukin (IL) -6, IL-8, inducible nitric oxide synthase (iNOS) and tumour necrosis factor-alpha (TNF-α), in H9-Kert and immortalized human keratinocyte cell line (HaCaT) were analysed at mRNA levels by both reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. The activation of the inflammatory transcription factor nuclear factor kappa-b (NFĸB) was assayed in these cells by transiently transfecting the cells with NFĸB reporter plasmid. Activation of NFĸB following treatment with heat-killed Porphyromonas gingivalis (P. gingivalis), an oral pathogen, was determined by assaying for the reporter, secreted alkaline phosphatase activity. RESULTS: The expression of TLRs, cytokines and activation of NFĸB following bacterial stimulation showed in both H9-Kert and the widely used HaCaT keratinocyte cell line was similar. CONCLUSION: Overall, our results support the potential application of hESCs as an alternative limitless cell source for primary keratinocytes which can be used as consistent and dependable research tool with minimum variations and no donor's dependency.

Modeling periodontal host-microbe interactions using vascularized gingival connective tissue equivalents.

Gingival connective tissue and its vasculature play a crucial role in the host's immune response against the periodontal microbiome and serve as a bridge between the oral and systemic environments. However, there is a lack of representative models that mimic the complex features of vascularized gingival connective tissue and its interaction with the periodontal microbiome, hindering our understanding of periodontal health and disease. Towards this pursuit, we present the characterization of vascularized gingival connective tissue equivalents (CTEs) as a model to study the interactions between oral biofilm colonizers and gingival tissues in healthy and diseased states. Whole-mount immunolabeling and label-free confocal reflectance microscopy of human fibrin-based matrix embedded with gingival fibroblasts and microvascular endothelial cells demonstrated the generation of bi-cellular vascularized gingival CTEs. Next, we investigated the response of the vascularized gingival CTEs to early, intermediate, and late oral biofilm colonizers. Despite colonization, the early colonizers did not elicit any significant change in the production of the cytokines and chemokines by the CTEs representative of the commensal and homeostatic state. In contrast, intermediate and late colonizers representing a transition to a diseased state exhibited connective tissue and vascular invasion, and elicited a differential immune response accompanied by increased monocyte migration. The culture supernatants produced by the vascularized gingival CTEs in response to early and intermediate colonizers polarized macrophages towards an immunomodulatory M2-like phenotype which activates and protects the host, while the late colonizers polarized towards a pro-inflammatory M1-like phenotype. Lastly,in silicoanalysis showed a high strength of associations between the proteins and transcripts investigated with periodontitis and vascular diseases. In conclusion, the vascularized gingival CTEs provide a biomimeticin vitroplatform to study host-microbiome interactions and innate immune response in periodontal health and diseased states, which potentially paves the way toward the development and assessment of novel periodontal therapeutics.

Modeling Crevicular Fluid Flow and Host-Oral Microbiome Interactions in a Gingival Crevice-on-Chip.

Gingival crevice and gingival crevicular fluid (GCF) flow play a crucial role at the gingiva-oral microbiome interface which contributes toward maintaining the balance between gingival health and periodontal disease. Interstitial flow of GCF strongly impacts the host-microbiome interactions and tissue responses. However, currently available in vitro preclinical models largely disregard the dynamic nature of gingival crevicular microenvironment, thus limiting the progress in the development of periodontal therapeutics. Here, a proof-of-principle "gingival crevice-on-chip" microfluidic platform to culture gingival connective tissue equivalent (CTE) under dynamic interstitial fluid flow mimicking the GCF is described. On-chip co-culture using oral symbiont (Streptococcus oralis) shows the potential to recapitulate microbial colonization, formation of biofilm-like structures at the tissue-microbiome interface, long-term co-culture, and bacterial clearance secondary to simulated GCF (s-GCF) flow. Further, on-chip exposure of the gingival CTEs to the toll-like receptor-2 (TLR-2) agonist or periodontal pathogen Fusobacterium nucleatum demonstrates the potential to mimic early gingival inflammation. In contrast to direct exposure, the induction of s-GCF flow toward the bacterial front attenuates the secretion of inflammatory mediators demonstrating the protective effect of GCF flow. This proposed in vitro platform offers the potential to study complex host-microbe interactions in periodontal disease and the development of periodontal therapeutics under near-microphysiological conditions.

Suppression of host innate immune response by Burkholderia pseudomallei through the virulence factor TssM.

Burkholderia pseudomallei is a Gram-negative saprophyte that is the causative agent of melioidosis, a severe infectious disease endemic in Northern Australia and Southeast Asia. This organism has sparked much scientific interest in the West because of its classification as a potential bioterrorism agent by the U.S. Centers for Disease Control and Prevention. However, relatively little is known about its pathogenesis. We demonstrate that B. pseudomallei actively inhibits NF-kappaB and type I IFN pathway activation, thereby downregulating host inflammatory responses. We found the virulence factor TssM to be responsible for this activity. TssM interferes with the ubiquitination of critical signaling intermediates, including TNFR-associated factor-3, TNFR-associated factor-6, and IkappaBalpha. The expression but not secretion of TssM is regulated by the type III secretion system. We demonstrate that TssM is important for B. pseudomallei infection in vivo as inflammation in the tssM mutant-infected mice is more severe and corresponds to a more rapid death compared with wild-type bacteria-infected mice. Abs to TssM can be detected in the sera of melioidosis patients, indicating that TssM is functionally expressed in vivo and thus could contribute to bacterial pathogenesis in human melioidosis.

Oral streptococci subvert the host innate immune response through hydrogen peroxide.

In periodontal health, oral streptococci constitute up to 80% of the plaque biofilm. Yet, destructive inflammatory events of the periodontium are rare. This observation suggests that oral streptococci may possess mechanisms to co-exist with the host. However, the mechanisms employed by oral streptococci to modulate the innate immune response have not been well studied. One of the key virulence factors produced by oral streptococci is hydrogen peroxide (H2O2). In mammalian cells, H2O2 triggers the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a key pathway mediating antioxidant defence. This study aimed to determine (1) if H2O2 producing oral streptococci activated the Nrf2 pathway in macrophages, and (2) if the activation of Nrf2 influenced the innate immune response. We found that oral streptococci downregulated the innate immune response in a H2O2 dependent manner through the activation of the Nrf2. The activation of the Nrf2 signalling pathway led to the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NFĸB), the key transcription factor regulating pro-inflammatory response. This study showed for the first time that oral streptococci are unlikely passive bystanders but could play an active role in the maintenance of periodontal health by preventing overt inflammation.

Differential immune responses of 3D gingival and periodontal connective tissue equivalents to microbial colonization.

Gingival and periodontal ligament fibroblasts are functionally distinct cell types within the dento-gingival unit that participate in host immune response. Their microenvironment influences the behavior and immune response to microbial challenge. We developed three-dimensional gingival and periodontal connective tissue equivalents (CTEs) using human fibrin-based matrix. The CTEs were characterized, and the heterogeneity in their innate immune response was investigated. The CTEs demonstrated no to minimal response to planktonic Streptococcus mitis and Streptococcus oralis, while their biofilms elicited a moderate increase in IL-6 and IL-8 production. In contrast, Fusobacterium nucleatum provoked a substantial increase in IL-6 and IL-8 production. Interestingly, the gingival CTEs secreted significantly higher IL-6, while periodontal counterparts produced higher IL-8. In conclusion, the gingival and periodontal CTEs exhibited differential responses to various bacterial challenges. This gives insights into the contribution of tissue topography and fibroblast heterogeneity in rendering protective and specific immune responses toward early biofilm colonizers.

Lactobacillus reuteri DSM 17938 and ATCC PTA 5289 ameliorates chemotherapy-induced oral mucositis.

Oral mucositis (OM) is a common complication of cancer therapy, however OM management remains unsatisfactory. There is a growing interest in the therapeutic potential of probiotics in OM due to positive findings of its use in intestinal mucositis. This study aimed to determine the efficacy and safety of the probiotic combination Lactobacillus reuteri DSM 17938 and ATCC PTA 5289 strains in chemotherapy-induced OM. Mice were divided into 4 groups. PBS/water and PBS/LR groups comprised of mice injected with PBS intraperitoneally (i.p.), and were given water or the mixture of L. reuteri (LR) DSM 17938 and ATCC PTA 5289 in water respectively. The 5-FU/water and 5-FU/LR groups comprised of mice injected with 5-FU i.p., and were given water or L. reuteri DSM 17938 and ATCC PTA 5289 in water respectively. Histopathological analysis revealed that the oral epithelia of the 5-FU/water and 5-FU/LR groups were thinner compared to PBS/water and PBS/LR groups. However, epithelial damage was significantly reduced in the 5-FU/LR compared to 5-FU/water group. Additionally, the 5-FU/LR group showed reduced oxidative stress and inflammation in the oral mucosa. We further showed that L. reuteri reduced oxidative stress through the nuclear factor E2-related factor-2 (Nrf-2) signalling. There was no evidence of translocation of L. reuteri systemically. This study demonstrated for the first time that L. reuteri protected oral mucosa against damage induced by chemotherapy.

Pterostilbene complexed with cyclodextrin exerts antimicrobial and anti-inflammatory effects.

Resveratrol (RES) is a natural polyphenol with potential as an adjunctive therapeutic modality for periodontitis. However, its inferior pharmacokinetics and toxicity concerns about its commonly used solvent dimethyl sulfoxide (DMSO) hinder translation to clinical applicability. Our study aimed to investigate the comparative antimicrobial properties of RES and its analogues (pterostilbene [PTS], oxyresveratrol [OXY] and piceatannol [PIC]), utilizing 2-hydroxypropyl-ß-cyclodextrin (HPßCD) as a solubiliser, which has a well-documented safety profile and FDA approval. These properties were investigated against Fusobacterium nucleatum, a key periodontal pathogen. PTS demonstrated the most potent antibacterial effects in HPßCD, with MIC > 60-fold lower than that of RES, OXY and PIC. In addition, PTS inhibited F. nucleatum biofilm formation. PTS exerted antimicrobial effects by eliciting leakage of cellular contents, leading to loss of bacterial cell viability. PTS also conferred immunomodulatory effects on F. nucleatum-challenged macrophages via upregulation of antioxidant pathways and inhibition of NF-κB activation. Given the superior antimicrobial potency of PTS against F. nucleatum compared to RES and other analogues, and coupled with its immunomodulatory properties, PTS complexed with HPßCD holds promise as a candidate nutraceutical for the adjunctive treatment of periodontitis.

Biotransformation of Piceatannol, a Dietary Resveratrol Derivative: Promises to Human Health.

SCOPE: To evaluate the health-promoting potentials of piceatannol (PIC), a dietary resveratrol derivative, its biotransformation is examined. METHODS AND RESULTS: The biotransformation is tested in human/rat hepatic microsomes and cytosols; its pharmacokinetic profiles are assessed in rats. Although limited phase I metabolism exists in microsomes, PIC is rapidly converted to two pharmacologically active metabolites, namely rhapontigenin (RHA) and isorhapontigenin (ISO) in cytosols. Such biotransformation is completely blocked by entacapone, a well-known catechol-O-methyltransferase (COMT) inhibitor, demonstrating that the O-methylation is mediated by COMT. Moreover, PIC is identified as a substrate inhibitor of COMT, suggesting its potential benefits in Alzheimer's disease. Due to extensive phase II metabolism including glucuronidation, sulfation, and O-methylation, PIC displays rapid clearance and at least 4.02% ± 0.61% and 17.70% ± 0.91% of PIC is converted to RHA and ISO, respectively, in rats after intravenous administration. Similarly, PIC serves as an effective precursor of ISO upon oral administration. CONCLUSION: Since PIC and its metabolites possess pleiotropic health-promoting activities, it has emerged as a promising nutraceutical candidate for further development. This study also reinforces the importance of in vivo testing in nutritional researches as the active metabolite(s) may be absent from the in vitro system.

Antibiotics Used in Regenerative Endodontics Modify Immune Response of Macrophages to Bacterial Infection.

INTRODUCTION: Ciprofloxacin, amoxicillin, and metronidazole are antibiotics used in regenerative endodontic therapy (RET). Although their antimicrobial properties are well-documented, there is a lack of information on the effects of these antibiotics on the immune response by host macrophages and periapical healing. Thus, this study had 2 objectives: (1) to determine the immune response of macrophages to bacterial infection in response to the combination of ciprofloxacin or amoxicillin and metronidazole and (2) using conditioned media produced by these macrophages to simulate the periapical microenvironment, to determine the impact on the expression of extracellular matrix (ECM) components by periodontal fibroblasts. METHODS: Macrophages were treated with ciprofloxacin and metronidazole or amoxicillin and metronidazole at 10-1000 µg/mL. The treated macrophages were exposed to lipopolysaccharide, and the pro- and anti-inflammatory cytokines produced were quantified with enzyme-linked immunosorbent assay. Periodontal fibroblasts were treated with conditioned media from these treated macrophages, and the expression of ECM genes was determined by quantitative polymerase chain reaction. RESULTS: Lipopolysaccharides elicited the production of proinflammatory cytokines interleukin 1 beta and tumor necrosis factor alpha by macrophages, but this was suppressed by ciprofloxacin and metronidazole. Moreover, only conditioned media from macrophages treated with ciprofloxacin and metronidazole rescued microbial-induced down-regulation of ECM genes by periodontal fibroblasts. Specifically, ciprofloxacin was the antibiotic responsible for these observations. In contrast, these effects were not observed with amoxicillin and metronidazole. CONCLUSIONS: Apart from disinfection of the root canal system, the combination of ciprofloxacin and metronidazole also exerts an immunomodulatory effect, which may aid in periapical healing.

Beta2 adrenergic receptor activation stimulates pro-inflammatory cytokine production in macrophages via PKA- and NF-kappaB-independent mechanisms.

Activation of the beta(2) adrenergic receptor (beta(2)AR) located on macrophages has been reported to possess anti-inflammatory properties, inhibiting nuclear factor kappaB (NF-kappaB) activation and cytokine production induced by pro-inflammatory stimuli. Here, we show that activation of the beta(2)AR in the absence of pro-inflammatory stimuli produced up to an 80- and 8-fold increase in IL-1beta and IL-6 transcripts, respectively, in the RAW 264.7 murine macrophage cell line. This increase in mRNA expression was accompanied by a significant increase in IL-1beta and IL-6 protein production. Pre-treatment of RAW cells with pharmacological inhibitors of protein kinase A (PKA) or NF-kappaB pathway failed to block this cytokine increase. Instead, the beta(2)AR-mediated increase in cytokines required activation of both the B-raf-ERK1/2 and p38 pathways. Treatment of RAW cells with the exchange protein directly activated by cAMP (EPAC) agonist also resulted in the up-regulation of IL-1beta and IL-6 transcripts. Examination of the main transcription factors downstream of the ERK1/2 and p38 signaling revealed that beta(2)AR activation resulted in the stimulation of CRE-, but not C/EBPbeta-, ETS-, or NF-kappaB-dependent transcription. Western blot analysis further showed that among the transcription factors which recognize the CRE-binding site, ATF-1 and ATF-2 but not CREB proteins were phosphorylated in an ERK1/2- and p38-dependent manner. Collectively, these results demonstrate that beta(2)ARs possess pro-inflammatory properties and that their activation leads to IL-1beta and IL-6 production through ERK1/2- and p38-dependent activation of ATF-1 and ATF-2 transcription factors.

Himar1 Transposon for Efficient Random Mutagenesis in Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is the primary etiological agent of aggressive periodontal disease. Identification of novel virulence factors at the genome-wide level is hindered by lack of efficient genetic tools to perform mutagenesis in this organism. The Himar1 mariner transposon is known to yield a random distribution of insertions in an organism's genome with requirement for only a TA dinucleotide target and is independent of host-specific factors. However, the utility of this system in A. actinomycetemcomitans is unknown. In this study, we found that Himar1 transposon mutagenesis occurs at a high frequency (×10-4), and can be universally applied to wild-type A. actinomycetemcomitans strains of serotypes a, b, and c. The Himar1 transposon inserts were stably inherited in A. actinomycetemcomitans transconjugants in the absence of antibiotics. A library of 16,000 mutant colonies of A. actinomycetemcomitans was screened for reduced biofilm formation. Mutants with transposon inserts in genes encoding pilus, putative ion transporters, multidrug resistant proteins, transcription regulators and enzymes involved in the synthesis of extracellular polymeric substance, bacterial metabolism and stress response were discovered in this screen. Our results demonstrated the utility of the Himar1 mutagenesis system as a novel genetic tool for functional genomic analysis in A. actinomycetemcomitans.

Effects of root debridement and adjunctive photodynamic therapy in residual pockets of patients on supportive periodontal therapy: A randomized split-mouth trial.

BACKGROUND: The study aims to compare the effects of adjunctive photodynamic therapy (PDT) with scaling and root debridement alone on periodontal parameters and inflammatory cytokines in residual pockets of patients undergoing maintenance therapy. METHODS: 27 subjects, each with at least 2 residual pockets ≥5mm, were recruited for this randomized, split-mouth controlled trial, providing total of 72 sites. Probing pocket depth (PPD), recession, clinical attachment loss (CAL), plaque and bleeding on probing of all sites were examined at baseline, 3 and 6 months. Gingival crevicular fluids (GCFs) were collected to determine levels of IL-1ß, IL-6, IL-8, TNF-α and MMP-8 via enzyme-linked immunosorbent assay. At baseline, all sites received subgingival instrumentation and polishing. In addition, test sites received a single application of PDT using Fotosan® with toluidine blue O solution photosensitizer. At 3 and 6 months, site level analysis was performed for changes in clinical parameters and cytokine level. RESULTS: Based on mixed model analysis, at 3 months, test sites showed significant reduction in CAL (p=0.016) and PPD (p=0.027) (from 6.14±0.28mm to 5.49±0.20mm and 5.42±0.16mm to 4.65±0.18mm respectively) compared to control sites (from 6.32±0.24mm to 6.08±0.17mm and 5.32±0.13mm to 5.15±0.15mm respectively). At 6 months, these differences were no longer significant (p=0.510). Adjunctive PDT did not offer additional reduction in levels of GCF cytokines. CONCLUSIONS: A single application of PDT to residual pockets provided a modest improvement of CAL and PPD over 3 months. Application of adjunctive PDT may lead to faster resolution of residual pockets and may be recommended for periodontal patients with slower healing capacity.