Inflammatory and bone remodelling related biomarkers following periodontal transplantation of the tissue engineered biocomplex.

OBJECTIVES: To assess gingival crevicular fluid (GCF) levels of inflammatory and bone remodelling related biomarkers following transplantation of a tissue-engineered biocomplex into intrabony defects at several time-points over 12-months. MATERIALS AND METHODS: Group-A (n = 9) received the Minimal Access Flap (MAF) surgical technique combined with a biocomplex of autologous clinical-grade alveolar bone-marrow mesenchymal stem cells in collagen scaffolds enriched with an autologous fibrin/platelet lysate (aFPL). Group-B (n = 10) received the MAF surgery, with collagen scaffolds enriched with aFPL and Group-C (n = 8) received the MAF surgery alone. GCF was collected from the osseous defects of subjects via paper strips/30 sec at baseline, 6-weeks, 3-, 6-, 9-, 12-months post-surgery. Levels of inflammatory and bone remodelling-related biomarkers in GCF were determined by ELISA. RESULTS: Group-A demonstrated significantly higher GCF levels of BMP-7 at 6-9 months than baseline, with gradually decreasing levels of pro-inflammatory and pro-osteoclastogenic markers (TNF-α, RANKL) over the study-period; and an overall decrease in the RANKL/OPG ratio at 9-12 months than baseline (all p < 0.001). In comparison, only modest interim changes were observed in Groups-B and -C. CONCLUSIONS: At the protein level, the approach of MAF and biocomplex transplantation provided greater tissue regeneration potential as cell-based therapy appeared to modulate inflammation and bone remodelling in residual periodontal defects. CLINICAL RELEVANCE: Transplantation of a tissue engineered construct into periodontal intrabony defects demonstrated a biochemical pattern for inflammatory control and tissue regeneration over 12-months compared to the control treatments. Understanding the biological healing events of stem cell transplantation may facilitate the design of novel treatment strategies. CLINICAL DATABASE REGISTRATION: ClinicalTrials.gov ID: NCT02449005.

Expression of Toll-like receptor and cytokine mRNAs in feline odontoclastic resorptive lesion (FORL) and feline oral health.

Feline odontoclastic resorptive lesion (FORL) is a common chronic inflammatory condition whose aetiopathogenesis remains unclear. FORL affects 20-75% of cats and causes excruciating pain and tooth loss. The purpose of this study was to evaluate chronic inflammation in FORL by assessing differences in Toll-like receptor (TLR) and cytokine transcripts in gingival tissues between diseased and healthy cats. Gingival tissue samples were collected from 14 healthy cats with no known clinical signs of oral disease and 41 cats with FORL. Levels of mRNA encoding TLR2, TLR3, TLR4, TLR7, TLR9 and the cytokines interleukin-1ß (IL-1ß), IL-4, IL-6, IL-10, IL-12, interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) was evaluated using quantitative real-time PCR. Statistical significance of the results was assessed using non-parametric tests. Levels of TLR and cytokine transcripts were upregulated in gingival tissue from cats with FORL as compared with healthy gingival tissue: TLR2, TLR3 and TLR9, p ≤ 0.001; TLR4 and TLR7, p ≤ 0.01; IFN-γ, IL-4, IL-6, IL-10, IL-12, IL-1ß and TNF-α, p ≤ 0.001). In conclusion, expression of TLR and both pro- and anti-inflammatory cytokines were significantly increased, confirming an ongoing chronic inflammatory response to the microbiome in FORL. It is likely that dysbiosis of the oral microbiota in cats with FORL activates the innate immune response, leading to active inflammation that results in tooth resorption.

Effects of smoking on non-surgical periodontal therapy in patients with periodontitis Stage III or IV, and Grade C.

BACKGROUND: To evaluate possible effects of smoking on clinical, biochemical, and microbiological outcomes of non-surgical periodontal treatment in patients with periodontitis Stage III or IV and Grade C. METHODS: Conventional quadrant-wise non-surgical periodontal treatment was performed and whole-mouth periodontal measurements were recorded at baseline, 1, 3, and 6 months after completion of treatment. Saliva, gingival crevicular fluid, subgingival plaque, and blood samples were obtained at the same time points. Inflammatory cytokine levels, presence, and quantities of 11 different bacterial species were determined. Smoking status was validated by cotinine assay. RESULTS: Fourteen smoker and 13 non-smoker patients completed the study protocol and revealed similar clinical findings except for the higher plaque scores in the non-smokers at 6 months (P <0.01). Significant differences were found between the study groups in biofluid cytokine levels at 1 and 3 months (P <0.01). Gram-negative bacteria were more abundant in the smokers at baseline and so were Gram-positive bacteria in the non-smokers (P <0.01). Gram-negative bacteria repopulated in the smokers faster than in the non-smokers (P <0.01). CONCLUSIONS: The present findings suggest that smoker patients with periodontitis Stage III and IV, Grade C respond well to the non-surgical periodontal treatment during the 6-month follow-up. However, smokers exhibit faster repopulation of Gram-negative bacteria.

Plaque Accumulation and Inflammation Adjacent to Restorations of Amorphous Calcium Phosphate-containing Composite in Early Childhood Caries.

PURPOSE: To evaluate the clinical, biochemical, and microbiological reactions to nanocomposite containing amorphous calcium phosphate (ACP) in comparison to a traditional composite restorative material in early childhood caries. MATERIALS AND METHODS: Eighteen teeth were restored with the test material (ACP-containing resin) and 18 teeth were restored with the control material (traditional composite, TC) in fourteen paediatric patients using a split-mouth design. One caries- and restoration-free intact tooth in each patient was selected as the healthy control. Gingival crevicular fluid (GCF) and supragingival plaque samples were collected at baseline before the treatment and also on days 1, 7, 14 and 30 after treatment. Unstimulated whole saliva samples were obtained from each patient at baseline, and 1 and 6 months after restoration. GCF and saliva samples were assayed for IL-17A, IL-17F IL-17A/F, IL-17E, OPG and RANKL levels by ELISA, and plaque composition was assessed using RT-PCR. RESULTS: Clinical evaluation indicated no statistically significant differences between the two restorative materials according to the FDI criteria surface lustre, material retention and marginal adaptation properties. Pro-inflammatory IL-17 levels decreased statistically significantly at 6 months compared to baseline and 1-month values (p < 0.05). The baseline pro-inflammatory IL-17 cytokine levels in GCF samples around the carious teeth were higher than those obtained around the healthy teeth (p < 0.05), but similar in GCF from the ACP-test and TC teeth. Microbiological findings were similar in the ACP and T groups. CONCLUSION: It may be suggested that both ACP-containing and traditional resin composites show similar antimicrobial and biochemical effects in early childhood caries.

Clinical associations between acetylcholine levels and cholinesterase activity in saliva and gingival crevicular fluid and periodontal diseases.

AIM: The oral mucosa possesses a non-neuronal cholinergic system. This study aimed to determine clinical evidence for a role of cholinergic mechanisms in the pathogenesis of periodontal diseases. MATERIALS AND METHODS: Fifty healthy participants, 52 patients with gingivitis and 49 with periodontitis were recruited. Full periodontal parameters were recorded and saliva and gingival crevicular fluid (GCF) collected. Levels of acetylcholine and inflammatory mediators were quantified using commercially available assay kits. Acetylcholinesterase and butyrylcholinesterase activities were measured using a published biochemical assay. RESULTS: Acetylcholine levels are significantly elevated in saliva and GCF, whereas GCF levels of butyrylcholinesterase activity are significantly decreased, in patients with periodontal diseases. Acetylcholine levels in saliva and GCF correlated positively with clinical markers of disease severity and with increased levels of IL-17A and IL-17F. In contrast, butyrylcholinesterase activity levels in GCF showed significant negative correlations with clinical markers of disease severity and IL-17A and IL-17F levels. None of the findings were due to smoking. CONCLUSIONS: Elevated acetylcholine levels and reduced butyrylcholinesterase activity are clinically associated with periodontal diseases and elevated levels of IL-17A and IL-17F. Therefore, non-neuronal cholinergic mechanisms may influence IL-17 biology and the aetiopathogenesis of periodontal diseases and therefore are possible therapeutic targets.

Intraoral versus extraoral cementation of implant-supported single crowns: Clinical, biomarker, and microbiological comparisons.

OBJECTIVES: Implant supported single metal-ceramic crowns cemented either extraorally or intraorally were comparatively evaluated by clinical, radiologic, biomarker, and microbiological parameters. MATERIALS AND METHODS: Twelve patients with bilateral single tooth gap in the maxillary posterior region received two locking-taper implants; 4.5 mm width, 8 mm length. Selection of intraoral (IOC) or extraoral cementation (EOC) using screwless titanium abutments was done randomly. Peri-implant crevicular fluid (PICF), gingival crevicular fluid (GCF) samples were collected from the implants, adjacent teeth, and bleeding on probing, soft tissue thickness, keratinized tissue width were recorded before starting the prosthetic procedures (baseline) and 3, 6 months after implant loading. Crestal bone loss was measured on radiographs taken immediately and 6 months after cementation. Cytokine levels, amounts of bacteria were determined in PICF/GCF samples. Data were tested by appropriate statistical analyses. RESULTS: Clinical findings were similar in the crowns cemented extraorally or intraorally at all times (P < .05). PICF and GCF data were similar. At 3 month, interleukin-17E and osteoprotegerin levels were lower in the intraorally cemented crowns. CONCLUSION: Extraorally and intraorally cemented crowns exhibited similar crestal bone loss after loading. Higher amount of osteoprotegerin at 3 month at the EOC than the IOC sites might bode well for good osseointegration.

The Anti-Adhesive Effect of Curcumin on Candida albicans Biofilms on Denture Materials.

The use of natural compounds as an alternative source of antimicrobials has become a necessity given the growing concern over global antimicrobial resistance. Polyphenols, found in various edible plants, offers one potential solution to this. We aimed to investigate the possibility of using curcumin within the context of oral health as a way of inhibiting and preventing the harmful development of Candida albicans biofilms. We undertook a series of adsorption experiments with varying concentrations of curcumin, showing that 50 µg/ml could prevent adhesion. This effect could be further synergized by the curcumin pre-treatment of yeast cells to obtain significantly greater inhibition (>90%, p < 0.001). Investigation of the biological impact of curcumin showed that it preferentially affected immature morphological forms (yeast and germlings), and actively promoted aggregation of the cells. Transcriptional analyses showed that key adhesins were down-regulated (ALS1 and ALS3), whereas aggregation related genes (ALS5 and AAF1) were up-regulated. Collectively, these data demonstrated that curcumin elicits anti-adhesive effects and that induces transcription of genes integrally involved in the processes related to biofilm formation. Curcumin and associated polyphenols therefore have the capacity to be developed for use in oral healthcare to augment existing preventative strategies for candidal biofilms on the denture surface.

Biomarkers and Bacteria Around Implants and Natural Teeth in the Same Individuals.

BACKGROUND: This cross-sectional study assesses cytokine levels in peri-implant crevicular fluid (PICF)/gingival crevicular fluid (GCF) and a selection of subgingival/submucosal plaque bacteria from clinically healthy or diseased sites in the same individuals. METHODS: Samples from 97 implants/teeth (58 implants [19 healthy, 20 mucositis, 19 peri-implantitis] and 39 natural teeth [19 healthy, 12 gingivitis, eight periodontitis] in 15 systemically healthy patients were investigated by immunoassay and real-time polymerase chain reaction. Samples were obtained first, with probing depth, clinical attachment level, bleeding on probing, plaque index scores, and keratinized tissue width then recorded. Data were analyzed by Wilcoxon, Mann-Whitney U, and permutation tests on dependent, independent, and mixed dependent and independent samples and Spearman correlation. RESULTS: Interleukin (IL)-1ß levels were significantly higher in PICF samples of healthy implants than in GCF samples of healthy teeth (P = 0.003), and soluble receptor activator of nuclear factor-κB ligand (sRANKL) concentrations were significantly higher in the gingivitis than the mucositis group (P = 0.004). Biomarker levels were similar in peri-implantitis and periodontitis groups (P >0.05). Actinomyces naeslundi and Streptococcus oralis levels were significantly higher in the healthy implant group than in healthy teeth (P <0.05). Prevotella intermedia and Treponema denticola (Td) levels were lower in the mucositis group than the gingivitis group (P <0.05). Prevotella oralis and S. oralis levels were significantly higher in the periodontitis group (P <0.05), and Td levels were significantly higher in the peri-implantitis group (P <0.05). CONCLUSION: There were many similarities but, crucially, some differences in biomarker levels (IL-1ß and sRANKL) and bacterial species between peri-implant and periodontal sites in the same individuals, suggesting similar pathogenic mechanisms.

Smoking, Porphyromonas gingivalis and the immune response to citrullinated autoantigens before the clinical onset of rheumatoid arthritis in a Southern European nested case-control study.

BACKGROUND: Antibodies to citrullinated proteins (ACPA) occur years before RA diagnosis. Porphyromonas gingivalis expresses its own peptidylarginine deiminase (PPAD), and is a proposed aetiological factor for the ACPA response. Smoking is a risk factor for both ACPA-positive RA and periodontitis. We aimed to study the relation of these factors to the risk of RA in a prospective cohort. METHODS: We performed a nested case-control study by identifying pre-RA cases in four populations from the European Prospective Investigation into Cancer and nutrition, matched with three controls. Data on smoking and other covariates were obtained from baseline questionnaires. Antibodies to CCP2 and citrullinated peptides from α-enolase, fibrinogen, vimentin and PPAD were measured. Antibodies to arginine gingipain (RgpB) were used as a marker for P.gingivalis infection and validated in a separate cohort of healthy controls and subjects with periodontitis. RESULTS: We studied 103 pre-RA cases. RA development was associated with several ACPA specificities, but not with antibodies to citrullinated PPAD peptides. Antibody levels to RgpB and PPAD peptides were higher in smokers but were not associated with risk of RA or with pre-RA autoimmunity. Former but not current smoking was associated with antibodies to α-enolase (OR 4.06; 95 % CI 1.02, 16.2 versus 0.54; 0.09-3.73) and fibrinogen peptides (OR 4.24; 95 % CI 1.2-14.96 versus 0.58; 0.13-2.70), and later development of RA (OR 2.48; 95 % CI 1.27-4.84 versus 1.57; 0.85-2.93), independent of smoking intensity. CONCLUSIONS: Smoking remains a risk factor for RA well before the clinical onset of disease. In this cohort, P.gingivalis is not associated with pre-RA autoimmunity or risk of RA in an early phase before disease-onset. Antibodies to PPAD peptides are not an early feature of ACPA ontogeny.

The Influence of Glycated Hemoglobin on the Cross Susceptibility Between Type 1 Diabetes Mellitus and Periodontal Disease.

BACKGROUND: Periodontal disease is a major complication of type 1 diabetes mellitus (T1DM). The aim of the present study is to investigate the relationship between glycated hemoglobin and circulating levels of interleukin (IL)-6, IL-8, and C-X-C motif chemokine ligand 5 (CXCL5) in non-smoking patients suffering from T1DM, with and without periodontitis. In addition, to determine the effect of advanced glycation end products (AGE) in the presence and absence of Porphyromonas gingivalis lipopolysaccharide (LPS) on IL-6, IL-8, and CXCL5 expression by THP-1 monocytes and OKF6/TERT-2 cells. METHODS: There were 104 participants in the study: 19 healthy volunteers, 23 patients with periodontitis, 28 patients with T1DM, and 34 patients with T1DM and periodontitis. Levels of blood glucose/glycated hemoglobin (International Federation of Clinical Chemistry [IFCC]) were determined by high-performance liquid chromatography. Levels of IL-6, IL-8, and CXCL5 in plasma were determined by enzyme-linked immunosorbent assay (ELISA). In vitro stimulation of OKF6/TERT-2 cells and THP-1 monocytes was performed with combinations of AGE and P. gingivalis LPS. Changes in expression of IL-6, IL-8, and CXCL5 were monitored by ELISA and real-time polymerase chain reaction. RESULTS: Patients with diabetes and periodontitis had higher plasma levels of IL-8 than patients with periodontitis alone. Plasma levels of IL-8 correlated significantly with IFCC units, clinical probing depth, and attachment loss. AGE and LPS, alone or in combination, stimulated IL-6, IL-8, and CXCL5 expression in both OKF6/TERT-2 cells and THP-1 monocytes. CONCLUSIONS: Elevated plasma levels of IL-8 potentially contribute to the cross-susceptibility between periodontitis and T1DM. P. gingivalis LPS and AGE in combination caused significantly greater expression of IL-6, IL-8, and CXCL5 from THP-1 monocytes and OKF6/TERT-2 cells than LPS alone.

Aspergillus fumigatus enhances elastase production in Pseudomonas aeruginosa co-cultures.

In the cystic fibrosis (CF) lung the presence of bacteria and fungi in the airways promotes an inflammatory response causing progressive lung damage, ultimately leading to high rates of morbidity and mortality. We hypothesized that polymicrobial interactions play an important role in promoting airway pathogenesis. We therefore examined the interplay between the most commonly isolated bacterial CF pathogen, Pseudomonas aeruginosa, and the most prevalent filamentous fungi, Aspergillus fumigatus, to test this. Co-culture experiments showed that in the presence of A. fumigatus the production of P. aeruginosa elastase was enhanced. This was confirmed by the presence of zones of clearance on Elastin-Congo Red (ECR) agar, which was identified as elastase by mass spectrometry. When P. aeruginosa were grown in a co-culture model with mature A. fumigatus biofilms, 60% of isolates produced significantly more elastase in the presence of the filamentous fungi than in its absence (P < .05). The expression of lasB also increased when P. aeruginosa isolates PA01 and PA14 were grown in co-culture with A. fumigatus. Supernatants from co-culture experiments were also significantly toxic to a human lung epithelial cell line (19-38% cell cytotoxicity) in comparison to supernatants from P. aeruginosa only cultures (P < .0001). Here we report that P. aeruginosa cytotoxic elastase is enhanced in the presence of the filamentous fungi A. fumigatus, suggesting that this may have a role to play in the damaging pathology associated with the lung tissue in this disease. This indicates that patients who have a co-colonisation with these two organisms may have a poorer prognosis.

The Association Between Thalassemia Major and Periodontal Health.

BACKGROUND: The aim of this cross-sectional study is to compare the local and systemic levels of soluble receptor activator of nuclear factor-κB ligand (sRANKL), osteoprotegerin (OPG), a proliferation-inducing ligand (APRIL), B-cell activating factor (BAFF), interleukin (IL)-6, and IL-8 in biofluids of patients with thalassemia major (TM) with or without gingivitis. METHODS: Seventy-seven patients are included in this study (TM, n = 29; systemically healthy, n = 48). Gingival crevicular fluid (GCF), saliva, and serum levels of IL-6, IL-8, sRANKL, OPG, BAFF, and APRIL were determined by enzyme-linked immunosorbent assay. Data were analyzed by appropriate non-parametric or parametric statistical tests. RESULTS: Median GCF, serum, and saliva levels for BAFF (P <0.001) and IL-6 and IL-8 (P <0.005) were higher in TM gingivitis than in systemically healthy gingivitis (P <0.001). GCF, serum, and saliva levels for APRIL, sRANKL, IL-6, and IL-8 were higher in TM than in systemically and periodontally healthy comparison groups (P <0.05). Positive correlations were found between bleeding on probing (BOP), plaque index (PI) scores, and GCF APRIL, serum sRANKL, serum OPG, and sRANKL concentrations in TM groups (P <0.05). Several significant positive correlations were found between BOP, PI scores, and biofluid parameters also in systemically healthy groups. CONCLUSION: TM may have a role in the underlying systemic hematologic condition and potentially affect gingival inflammation via dysregulation of lymphocytes and increased activation of osteoclasts.

Evaluation of biochemical parameters and local and systemic levels of osteoactive and B-cell stimulatory factors in gestational diabetes in the presence or absence of gingivitis.

BACKGROUND: Gestational diabetes mellitus (GDM) is defined as varying glucose intolerance, with first onset or recognition in pregnancy. This study evaluates clinical and biochemical parameters in a possible association between GDM and gingivitis. METHODS: A total of 167 pregnant females was included in the study. There were 101 females with GDM and 66 females without GDM. Subgroups were created according to the presence or absence of gingival inflammation. Plaque index, bleeding on probing, and probing depth were recorded at four sites per tooth. Serum, saliva, and gingival crevicular fluid (GCF) levels of interleukin (IL)-6, IL-8, soluble receptor activator of nuclear factor-kappa B ligand (sRANKL), osteoprotegerin (OPG), B-cell activating factor (BAFF), and a proliferation-inducing ligand (APRIL) were determined by enzyme-linked immunosorbent assay. Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests and Spearman correlation analysis. RESULTS: Age and anthropometric indices were higher in the GDM than non-GDM group (P <0.0001). Clinical periodontal recordings, serum BAFF, IL-8, and saliva sRANKL levels were higher in the GDM group (P <0.05). Saliva IL-6 level was higher in the GDM with gingivitis group than non-GDM with gingivitis group (P = 0.044). Serum and GCF BAFF (P <0.0001), serum, saliva, and GCF APRIL (P <0.0001; P <0.0001; P = 0.032, respectively), GCF OPG (P = 0.036), and serum and saliva sRANKL (P <0.0001) were higher in the GDM with gingivitis group than GDM without gingivitis group. CONCLUSIONS: The inflammatory response seems to be more pronounced in females with GDM. The observed increase in both local and systemic levels of inflammatory cytokines may suggest an interaction between gingivitis and GDM.

Development of an in vitro periodontal biofilm model for assessing antimicrobial and host modulatory effects of bioactive molecules.

BACKGROUND: Inflammation within the oral cavity occurs due to dysregulation between microbial biofilms and the host response. Understanding how different oral hygiene products influence inflammatory properties is important for the development of new products. Therefore, creation of a robust host-pathogen biofilm platform capable of evaluating novel oral healthcare compounds is an attractive option. We therefore devised a multi-species biofilm co-culture model to evaluate the naturally derived polyphenol resveratrol (RSV) and gold standard chlorhexidine (CHX) with respect to anti-biofilm and anti-inflammatory properties. METHODS: An in vitro multi-species biofilm containing S. mitis, F. nucleatum, P. gingivalis and A. actinomycetemcomitans was created to represent a disease-associated biofilm and the oral epithelial cell in OKF6-TERT2. Cytotoxicity studies were performed using RSV and CHX. Multi-species biofilms were either treated with either molecule, or alternatively epithelial cells were treated with these prior to biofilm co-culture. Biofilm composition was evaluated and inflammatory responses quantified at a transcriptional and protein level. RESULTS: CHX was toxic to epithelial cells and multi-species biofilms at concentrations ranging from 0.01-0.2%. RSV did not effect multi-species biofilm composition, but was toxic to epithelial cells at concentrations greater than 0.01%. In co-culture, CHX-treated biofilms resulted in down regulation of the inflammatory chemokine IL-8 at both mRNA and protein level. RSV-treated epithelial cells in co-culture were down-regulated in the release of IL-8 protein, but not mRNA. CONCLUSIONS: CHX possesses potent bactericidal properties, which may impact downstream inflammatory mediators. RSV does not appear to have bactericidal properties against multi-species biofilms, however it did appear to supress epithelial cells from releasing inflammatory mediators. This study demonstrates the potential to understand the mechanisms by which different oral hygiene products may influence gingival inflammation, thereby validating the use of a biofilm co-culture model.

Bacteria in the adventitia of cardiovascular disease patients with and without rheumatoid arthritis.

The incidence of atherosclerosis is significantly increased in rheumatoid arthritis (RA). Infection is one factor that may be involved in the pathogenesis of both diseases. The cause of RA and atherosclerosis is unknown, and infection is one of the factors that may be involved in the pathogenesis of both diseases. The aims of this study were to identify bacteria in the aortic adventitia of patients with cardiovascular disease (CVD) in the presence and absence of RA, and to determine the effect of identified candidate pathogens on Toll-like receptor (TLR)-dependent signalling and the proinflammatory response. The aortic adventitia of 11 CVD patients with RA (RA+CVD) and 11 CVD patients without RA (CVD) were collected during coronary artery bypass graft surgery. Bacteria were detected in four samples from CVD patients and three samples from RA+CVD patients and identified by 16S rRNA gene sequencing. Methylobacterium oryzae was identified in all three RA+CVD samples, representing 44.1% of the bacterial flora. The effect of M. oryzae on TLR-dependent signalling was determined by transfection of HEK-293 cells. Although mild TLR2 signalling was observed, TLR4 was insensitive to M. oryzae. Human primary macrophages were infected with M. oryzae, and a TLDA qPCR array targeting 90 genes involved in inflammation and immune regulation was used to profile the transcriptional response. A significant proinflammatory response was observed, with many of the up-regulated genes encoding proinflammatory cytokines (IL-1α, IL-1ß, IL-6, TNF-α) and chemokines (CCR7, IL-8). The aortic adventitia of CVD patients contains a wide range of bacterial species, and the bacterial flora is significantly less diverse in RA+CVD than CVD patients. M. oryzae may stimulate an proinflammatory response that may aggravate and perpetuate the pathological processes underlying atherosclerosis in RA patients.

Bacteria and Toll-like receptor and cytokine mRNA expression profiles associated with canine arthritis.

The major forms of inflammatory canine arthritis are immune-mediated arthritis (IMA) and septic arthritis (SA), although some cases of cruciate disease (CD) are associated with significant levels of synovitis. In this study, the bacteria associated with canine arthritis were identified and mRNA expression levels of Toll-like receptors (TLRs) and pro-inflammatory cytokines determined. Of the 40 synovial fluid samples analysed, bacteria were isolated from 12 samples by culture (2 CD, 10 SA) and detected in 4 samples (3 CD, 1 SA) using culture-independent methods. Statistically significant increases in TLR2, tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-12 mRNA expression were seen in all disease groups compared to normal controls. All disease groups had decreased mRNA expression of other TLRs compared to normal controls, but this did not reach statistical significance. Synovial fluid cell counts revealed that the highest number and proportion of mononuclear cells and neutrophils were found in the IMA and SA samples, respectively. Age had an effect on the TLR and cytokine mRNA expression profiles: TNF-α (p=0.043) and IL-12 (p=0.025) mRNA expression was increased and TLR4 mRNA expression was reduced (p=0.033) in dogs up to 4 years of age compared to older animals. In the 10 SA samples from which bacteria were isolated, statistically significant increases in TLR2, TLR7, TNF-α and IL-6 mRNA expression were observed. It is concluded that canine arthritis is associated with increased mRNA levels of pro-inflammatory cytokines, which could in some cases be mediated by bacteria through activation of TLR2.

The alpha 7 nicotinic receptor agonist PHA-543613 hydrochloride inhibits Porphyromonas gingivalis-induced expression of interleukin-8 by oral keratinocytes.

OBJECTIVE: The alpha 7 nicotinic receptor (α7nAChR) is expressed by oral keratinocytes. α7nAChR activation mediates anti-inflammatory responses. The objective of this study was to determine if α7nAChR activation inhibited pathogen-induced interleukin-8 (IL-8) expression by oral keratinocytes. MATERIALS AND METHODS: Periodontal tissue expression of α7nAChR was determined by real-time PCR. OKF6/TERT-2 oral keratinocytes were exposed to Porphyromonas gingivalis in the presence and absence of a α7nAChR agonist (PHA-543613 hydrochloride) alone or after pre-exposure to a specific α7nAChR antagonist (α-bungarotoxin). Interleukin-8 (IL-8) expression was measured by ELISA and real-time PCR. Phosphorylation of the NF-κB p65 subunit was determined using an NF-κB p65 profiler assay and STAT-3 activation by STAT-3 in-cell ELISA. The release of ACh from oral keratinocytes in response to P. gingivalis lipopolysaccharide was determined using a GeneBLAzer M3 CHO-K1-bla cell reporter assay. RESULTS: Expression of α7nAChR mRNA was elevated in diseased periodontal tissue. PHA-543613 hydrochloride inhibited P. gingivalis-induced expression of IL-8 at the transcriptional level. This effect was abolished when cells were pre-exposed to a specific α7nAChR antagonist, α-bungarotoxin. PHA-543613 hydrochloride downregulated NF-κB signalling through reduced phosphorylation of the NF-κB p65-subunit. In addition, PHA-543613 hydrochloride promoted STAT-3 signalling by maintenance of phosphorylation. Furthermore, oral keratinocytes upregulated ACh release in response to P. gingivalis lipopolysaccharide. CONCLUSION: These data suggest that α7nAChR plays a role in regulating the innate immune responses of oral keratinocytes.

Saliva and serum levels of B-cell activating factors and tumor necrosis factor-α in patients with periodontitis.

BACKGROUND: B-lymphocytes play a central and critical role in the adaptive immune response against invading pathogens. This study evaluates saliva and serum levels of APRIL (a proliferation-inducing ligand), B-cell activating factor (BAFF), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 in patients with chronic periodontitis (CP) or aggressive periodontitis (AgP) and periodontally healthy individuals. METHODS: Twenty-five patients with AgP, 20 patients with CP, and 20 periodontally healthy individuals were included. Smoking status was recorded, and all individuals were divided into non-smokers and smokers. Saliva and serum samples were collected before clinical periodontal measurements. APRIL, BAFF, TNF-α, IL-6, and IL-10 levels in serum and saliva samples were determined by enzyme-linked immunosorbent assay. Statistical analysis was performed using multivariate analysis of variance and bivariate correlation. RESULTS: Serum and saliva levels of TNF-α, APRIL, BAFF, IL-6, and IL-10 were similar in CP and AgP groups. Serum levels of TNF-α, APRIL, and BAFF and saliva levels of BAFF were significantly higher in periodontitis groups than healthy controls (P <0.05). Non-smokers with CP or AgP had lower levels of saliva TNF-α and APRIL and serum APRIL and IL-6 than smokers with CP or AgP (P <0.05). Saliva APRIL and serum TNF-α and IL-6 levels were significantly higher in healthy smokers than healthy non-smokers (P <0.05). Clinical periodontal parameters correlated positively with TNF-family cytokines and negatively with IL-10 (P <0.05). CONCLUSIONS: Within the limits of this study, it may be suggested that elevated salivary and serum TNF-α, APRIL, and BAFF in patients with periodontitis may contribute to the dominance of B cells in periodontitis lesions. Moreover, higher levels in healthy smokers than non-smoking counterparts may play a role in detrimental effects of smoking on periodontal tissues.

Investigating the biological properties of carbohydrate derived fulvic acid (CHD-FA) as a potential novel therapy for the management of oral biofilm infections.

BACKGROUND: A number of oral diseases, including periodontitis, derive from microbial biofilms and are associated with increased antimicrobial resistance. Despite the widespread use of mouthwashes being used as adjunctive measures to control these biofilms, their prolonged use is not recommended due to various side effects. Therefore, alternative broad-spectrum antimicrobials that minimise these effects are highly sought after. Carbohydrate derived fulvic acid (CHD-FA) is an organic acid which has previously demonstrated to be microbiocidal against Candida albicans biofilms, therefore, the aims of this study were to evaluate the antibacterial activity of CHD-FA against orally derived biofilms and to investigate adjunctive biological effects. METHODS: Minimum inhibitory concentrations were evaluated for CHD-FA and chlorhexidine (CHX) against a range of oral bacteria using standardised microdilution testing for planktonic and sessile. Scanning electron microscopy was also employed to visualise changes in oral biofilms after antimicrobial treatment. Cytotoxicity of these compounds was assessed against oral epithelial cells, and the effect of CHD-FA on host inflammatory markers was assessed by measuring mRNA and protein expression. RESULTS: CHD-FA was highly active against all of the oral bacteria tested, including Porphyromonas gingivalis, with a sessile minimum inhibitory concentration of 0.5%. This concentration was shown to kill multi-species biofilms by approximately 90%, levels comparable to that of chlorhexidine (CHX). In a mammalian cell culture model, pretreatment of epithelial cells with buffered CHD-FA was shown to significantly down-regulate key inflammatory mediators, including interleukin-8 (IL-8), after stimulation with a multi-species biofilm. CONCLUSIONS: Overall, CHD-FA was shown to possess broad-spectrum antibacterial activity, with a supplementary function of being able to down-regulate inflammation. These properties offer an attractive spectrum of function from a naturally derived compound, which could be used as an alternative topical treatment strategy for oral biofilm diseases. Further studies in vitro and in vivo are required to determine the precise mechanism by which CHD-FA modulates the host immune response.