Publications by authors named "Shauna Culshaw"

27 Publications

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The Subgingival Plaque Microbiome, Systemic Antibodies Against Bacteria and Citrullinated Proteins Following Periodontal Therapy.

Pathogens 2021 Feb 10;10(2). Epub 2021 Feb 10.

Oral Sciences, College of Medical, Veterinary and Life Sciences, Dental School, University of Glasgow, Glasgow G12 8QQ, UK.

Periodontitis (PD) shows an association with rheumatoid arthritis (RA) and systemic inflammation. Periodontal pathogens, namely and , are proposed to be capable of inducing citrullination of peptides in the gingiva, inducing the formation of anti-citrullinated protein antibodies (ACPAs) within susceptible hosts. Here, we sought to investigate whether periodontal treatment influenced systemic inflammation and antibody titres to , , and ACPA in 42 systemically health patients with periodontal disease. Subgingival plaque and serum samples were collected from study participants before (baseline) and 90 days after treatment to analyse the abundance of specific bacteria and evaluate anti-bacterial antibodies, C-reactive protein (CRP), tumour necrosis factor α (TNF-α), interleukin 6 (IL-6) and ACPA in serum. Following treatment, all patients showed reduced periodontal inflammation. Despite observing a weak positive correlation between CRP and IL-6 with periodontal inflammation at baseline, we observed no significant reductions in any indicators of systemic inflammation 90 days after treatment. In contrast, anti- IgG significantly reduced post-treatment ( < 0.001, Wilcoxon signed rank test), although no changes were observed for other antibody titres. Patients who had detectable in subgingival plaques had significantly higher anti- IgG and ACPA titres, suggesting a potential association between colonisation and systemic antibody titres.
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http://dx.doi.org/10.3390/pathogens10020193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916579PMC
February 2021

The systemic inflammatory response following hand instrumentation versus ultrasonic instrumentation-A randomized controlled trial.

J Clin Periodontol 2020 09 27;47(9):1087-1097. Epub 2020 Jul 27.

Oral Sciences, Glasgow Dental Hospital and School, School of Medicine, Dentistry and Nursing, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.

Objective: This study sought to investigate whether the immediate systemic inflammatory response following full-mouth debridement differs following use of hand compared with ultrasonic instruments.

Methods: Thirty-nine periodontitis patients were randomized to treatment with full-mouth debridement using either hand or ultrasonic instrumentation completed within 24 hr. Serum and periodontal clinical parameters were collected at baseline, day 1, day 7 and day 90 post-treatment. Differences in systemic inflammatory markers were assessed using general linear models at each timepoint, corrected for age, gender, smoking status, body mass index and baseline levels of each marker.

Results: Across all patients, serum C-reactive protein increased at day 1, with no differences between hand and ultrasonic groups (p(adjusted) = .22). There was no difference between groups in interleukin-6 (p(adjusted) = .29) or tumour necrosis factor α (p(adjusted) = .53) at day 1. Inflammatory markers returned to baseline levels by day 7. Treatment resulted in equal and marked improvements in clinical parameters in both groups; however, total treatment time was on average shorter for ultrasonic instruments (p(adjusted) = .002).

Conclusions: Ultrasonic instrumentation resulted in shorter treatment time with comparable clinical outcomes. Levels of serum C-reactive protein at day 1 were similar following debridement with hand or ultrasonic instruments.
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http://dx.doi.org/10.1111/jcpe.13342DOI Listing
September 2020

Biofilm-stimulated epithelium modulates the inflammatory responses in co-cultured immune cells.

Sci Rep 2019 10 31;9(1):15779. Epub 2019 Oct 31.

Oral Sciences Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.

The gingival epithelium is a physical and immunological barrier to the microbiota of the oral cavity, which interact through soluble mediators with the immune cells that patrol the tissue at the gingival epithelium. We sought to develop a three-dimensional gingivae-biofilm interface model using a commercially available gingival epithelium to study the tissue inflammatory response to oral biofilms associated with "health", "gingivitis" and "periodontitis". These biofilms were developed by sequential addition of microorganisms to mimic the formation of supra- and sub-gingival plaque in vivo. Secondly, to mimic the interactions between gingival epithelium and immune cells in vivo, we integrated peripheral blood mononuclear cells and CD14 monocytes into our three-dimensional model and were able to assess the inflammatory response in the immune cells cultured with and without gingival epithelium. We describe a differential inflammatory response in immune cells cultured with epithelial tissue, and more so following incubation with epithelium stimulated by "gingivitis-associated" biofilm. These results suggest that gingival epithelium-derived soluble mediators may control the inflammatory status of immune cells in vitro, and therefore targeting of the epithelial response may offer novel therapies. This multi-cellular interface model, both of microbial and host origin, offers a robust in vitro platform to investigate host-pathogens at the epithelial surface.
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http://dx.doi.org/10.1038/s41598-019-52115-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823452PMC
October 2019

Polymicrobial oral biofilm models: simplifying the complex.

J Med Microbiol 2019 Nov 13;68(11):1573-1584. Epub 2019 Sep 13.

Oral Sciences Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.

Over the past century, numerous studies have used oral biofilm models to investigate growth kinetics, biofilm formation, structure and composition, antimicrobial susceptibility and host-pathogen interactions. animal models provide useful models of some oral diseases; however, these are expensive and carry vast ethical implications. Oral biofilms grown or maintained offer a useful platform for certain studies and have the advantages of being inexpensive to establish and easy to reproduce and manipulate. In addition, a wide range of variables can be monitored and adjusted to mimic the dynamic environmental changes at different sites in the oral cavity, such as pH, temperature, salivary and gingival crevicular fluid flow rates, or microbial composition. This review provides a detailed insight for early-career oral science researchers into how the biofilm models used in oral research have progressed and improved over the years, their advantages and disadvantages, and how such systems have contributed to our current understanding of oral disease pathogenesis and aetiology.
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http://dx.doi.org/10.1099/jmm.0.001063DOI Listing
November 2019

Impact of frequency of denture cleaning on microbial and clinical parameters - a bench to chairside approach.

J Oral Microbiol 2019 29;11(1):1538437. Epub 2018 Oct 29.

School of Medicine, Dentistry and Nursing, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.

: Robust scientific and clinical evidence of how to appropriately manage denture plaque is lacking. This two-part study (i) developed an model of denture plaque removal, and (ii) assessed effectiveness of these approaches in a randomised clinical trial. : (i) a complex denture plaque model was developed using the dominant microbial genera from a recent microbiome analyses. Biofilms formed on polymethylmethacrylate were brushed daily with a wet toothbrush, then either treated daily for 5 days or only on Days 1 and 5 with Polident® denture cleanser tablets (3 min soaking). Quantitative and qualitative microbiological assessments were performed. (ii), an examiner-blind, randomised, crossover study of complete maxillary denture wearers was performed (n = 19). Either once-daily for 7 days or on Day 7 only, participants soaked dentures for 15 min using Corega® denture cleansing tables, then brushed. Denture plaque microbiological assessment used sterilized filter paper discs. : The model showed daily cleaning with denture cleanser plus brushing significantly reduced microbial numbers compared to intermittent denture cleaning with daily brushing (p < 0.001). The clinical component of the study showed a statistically significant reduction in denture plaque microbial numbers in favour of daily versus weekly treatment (aerobic bacteria p = 0.0144). Both and studies showed that denture plaque biofilm composition were affected by different treatment arms. : This study demonstrated that daily denture cleansing regimens are superior to intermittent denture cleansing, and that cleansing regimens can induce denture plaque compositional changes. Clinicaltrials.gov registration: NCT02780661.
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http://dx.doi.org/10.1080/20002297.2018.1538437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6225516PMC
October 2018

Periodontal Disease: Its Impact on Restorative Dentistry.

Authors:
Shauna Culshaw

Prim Dent J 2017 Feb;6(1):25-31

A healthy periodontium provides stable gingival margins and stable tooth position prior to tooth preparations for indirect restorations. Good periodontal health allows easier tissue handling during tooth preparation, impression taking and restoration fitting. Periodontal health is integral to successful restorative care. This report documents common clinical scenarios in which periodontal problems cause aesthetic concern.
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http://dx.doi.org/10.1177/205016841700600103DOI Listing
February 2017

In Vitro Effect of Porphyromonas gingivalis Methionine Gamma Lyase on Biofilm Composition and Oral Inflammatory Response.

PLoS One 2016 29;11(12):e0169157. Epub 2016 Dec 29.

Research Centre for Clinical & Diagnostic Oral Sciences, Blizard Institute, Queen Mary University of London, London, United Kingdom.

Methanethiol (methyl mercaptan) is an important contributor to oral malodour and periodontal tissue destruction. Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum are key oral microbial species that produce methanethiol via methionine gamma lyase (mgl) activity. The aim of this study was to compare an mgl knockout strain of P. gingivalis with its wild type using a 10-species biofilm co-culture model with oral keratinocytes and its effect on biofilm composition and inflammatory cytokine production. A P. gingivalis mgl knockout strain was constructed using insertion mutagenesis from wild type W50 with gas chromatographic head space analysis confirming lack of methanethiol production. 10-species biofilms consisting of Streptococcus mitis, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp polymorphum, Fusobacterium nucleatum ssp vincentii, Veillonella dispar, Actinomyces naeslundii, Prevotella intermedia and Aggregatibacter actinomycetemcomitans with either the wild type or mutant P. gingivalis were grown on Thermanox cover slips and used to stimulate oral keratinocytes (OKF6-TERT2), under anaerobic conditions for 4 and 24 hours. Biofilms were analysed by quantitative PCR with SYBR Green for changes in microbial ecology. Keratinocyte culture supernatants were analysed using a multiplex bead immunoassay for cytokines. Significant population differences were observed between mutant and wild type biofilms; V. dispar proportions increased (p<0.001), whilst A. naeslundii (p<0.01) and Streptococcus spp. (p<0.05) decreased in mutant biofilms. Keratinocytes produced less IL-8, IL-6 and IL-1α when stimulated with the mutant biofilms compared to wild type. Lack of mgl in P. gingivalis has been shown to affect microbial ecology in vitro, giving rise to a markedly different biofilm composition, with a more pro-inflammatory cytokine response from the keratinocytes observed. A possible role for methanethiol in biofilm formation and cytokine response with subsequent effects on oral malodor and periodontitis is suggested.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169157PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5199072PMC
July 2017

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.

BMC Musculoskelet Disord 2015 Nov 4;16:331. Epub 2015 Nov 4.

Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK.

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.
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http://dx.doi.org/10.1186/s12891-015-0792-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4634856PMC
November 2015

Revealing microbial recognition by specific antibodies.

BMC Microbiol 2015 Jul 2;15:132. Epub 2015 Jul 2.

Department of Health and Genomics, FISABIO Foundation, Center for Advanced Research in Public Health, Avda. Cataluña 21, 46020, Valencia, Spain.

Background: Recognition of microorganisms by antibodies is a vital component of the human immune response. However, there is currently very limited understanding of immune recognition of 50 % of the human microbiome which is made up of as yet un-culturable bacteria. We have combined the use of flow cytometry and pyrosequencing to describe the microbial composition of human samples, and its interaction with the immune system.

Results: We show the power of the technique in human faecal, saliva, oral biofilm and breast milk samples, labeled with fluorescent anti-IgG or anti-IgA antibodies. Using Fluorescence-Activated Cell Sorting (FACS), bacterial cells were separated depending on whether they are coated with IgA or IgG antibodies. Each bacterial population was PCR-amplified and pyrosequenced, characterizing the microorganisms which evade the immune system and those which were recognized by each immunoglobulin.

Conclusions: The application of the technique to healthy and diseased individuals may unravel the contribution of the immune response to microbial infections and polymicrobial diseases.
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http://dx.doi.org/10.1186/s12866-015-0456-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4489363PMC
July 2015

Selected dietary (poly)phenols inhibit periodontal pathogen growth and biofilm formation.

Food Funct 2015 Mar;6(3):719-29

Human Nutrition, School of Medicine, Glasgow Royal Infirmary, College of Medical, Veterinary and Life Sciences, The University of Glasgow, Glasgow G31 2ER, UK.

Periodontitis (PD) is a chronic infectious disease mediated by bacteria in the oral cavity. (Poly)phenols (PPs), ubiquitous in plant foods, possess antimicrobial activities and may be useful in the prevention and management of periodontitis. The objective of this study was to test the antibacterial effects of selected PPs on periodontal pathogens, on both planktonic and biofilm modes of growth. Selected PPs (n = 48) were screened against Streptococcus mitis (S. mitis), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), Fusobacterium nucleatum (F. nucleatum) and Porphyromonas gingivalis (P. gingivalis). The antibacterial potential of each compound was evaluated in terms of planktonic minimum inhibitory concentration (PMIC) and planktonic minimum bactericidal concentration (PMBC) using standardized broth microdilution assays. The most active PPs were further tested for their effect on mono-species and multi-species biofilms using a colorimetric resazurin-based viability assay and scanning electron microscopy. Of the 48 PPs tested, 43 showed effective inhibition of planktonic growth of one or more test strains, of which curcumin was the most potent (PMIC range = 7.8-62.5 μg mL(-1)), followed by pyrogallol (PMIC range = 2.4-2500 μg mL(-1)), pyrocatechol (MIC range = 4.9-312.5 μg mL(-1)) and quercetin (PMIC range = 31.2-500 μg mL(-1)). At this concentration, adhesion of curcumin and quercetin to the substrate also inhibited adhesion of S. mitis, and biofilm formation and maturation. While both curcumin and quercetin were able to alter architecture of mature multi-species biofilms, only curcumin-treated biofilms displayed a significantly reduced metabolic activity. Overall, PPs possess antibacterial activities against periodontopathic bacteria in both planktonic and biofilm modes of growth. Further cellular and in vivo studies are necessary to confirm their beneficial activities and potential use in the prevention and or treatment of periodontal diseases.
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http://dx.doi.org/10.1039/c4fo01087fDOI Listing
March 2015

Erratum to: Clinical associations between IL-17 family cytokines and periodontitis and potential differential roles for IL-17A and IL-17E in periodontal immunity.

Inflamm Res 2015 Jan;64(1):83

Infection and Immunity Research Group, Immunology, Level 9, Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, 378 Sauchiehall Street, Glasgow, G2 3JZ, UK.

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http://dx.doi.org/10.1007/s00011-014-0788-3DOI Listing
January 2015

Clinical associations between IL-17 family cytokines and periodontitis and potential differential roles for IL-17A and IL-17E in periodontal immunity.

Inflamm Res 2014 Dec 5;63(12):1001-12. Epub 2014 Nov 5.

Infection and Immunity Research Group, Immunology, Level 9, Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, 378 Sauchiehall Street, Glasgow, G2 3JZ, UK.

Objective: IL-17A is implicated in periodontitis pathogenesis. The roles of IL-17B-IL-17F and IL-17A/F are unknown. This study aimed to determine clinical associations between IL-17 family cytokines and periodontitis and to investigate the biological roles of IL-17A and IL-17E using in vitro model systems.

Materials And Methods: Samples from 97 patients with periodontitis and 77 healthy volunteers were used in the study. Serum, saliva and gingival crevicular fluid (GCF) levels of IL-17 family cytokines were measured by ELISA. Oral keratinocytes were stimulated with a P. gingivalis biofilm, or IL-17A, in the presence and absence of IL-17E and the expression of IL-8 and CXCL5 were investigated by ELISA and real-time-PCR. NF-κB phosphorylation in similar experiments was also measured using a cell-based ELISA.

Results: Serum, saliva and GCF IL-17A levels were higher in periodontitis patients and correlated positively with clinical parameters of attachment loss, pocket depth and bleeding on probing. Serum IL-17E levels were lower in periodontitis patients and the serum IL-17A:IL-17E ratio correlated positively with clinical parameters. In vitro, IL-17E inhibited Porphyromonas gingivalis and IL-17A induced expression of chemokines by reducing phosphorylation of the NF-κB p65 subunit.

Conclusions: Serum IL-17A:IL-17E may be a marker of disease severity. IL-17E may have opposing roles to IL-17A in periodontitis pathogenesis. IL-17E can negatively regulate IL-17A and periodontal pathogen induced expression of chemokines by oral keratinocytes.
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http://dx.doi.org/10.1007/s00011-014-0776-7DOI Listing
December 2014

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

BMC Oral Health 2014 Jun 28;14:80. Epub 2014 Jun 28.

Infection and Immunity Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, 378 Sauchiehall Street, Glasgow G2 3JZ, UK.

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.
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http://dx.doi.org/10.1186/1472-6831-14-80DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4080992PMC
June 2014

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

Inflamm Res 2014 Jul 8;63(7):557-68. Epub 2014 Mar 8.

Infection and Immunity Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Level 9, 378 Sauchiehall Street, Glasgow, G2 3JZ, UK.

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.
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http://dx.doi.org/10.1007/s00011-014-0725-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050294PMC
July 2014

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

BMC Oral Health 2013 Sep 24;13:47. Epub 2013 Sep 24.

Infection and Immunity Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, 378 Sauchiehall Street, Glasgow G2 3JZ, UK.

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.
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http://dx.doi.org/10.1186/1472-6831-13-47DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3849008PMC
September 2013

Treatment of peri-implant diseases: a review of the literature and protocol proposal.

Dent Update 2013 Jul-Aug;40(6):472-4, 476-8, 480

Glasgow Dental Hospital and School, 378 Sauchiehall Street, Glasgow G2 3JZ, UK.

Unlabelled: Over 100,000 implants were placed in the UK in 2010. As the numbers of patients with implant-retained prostheses increases, operators are encountering an increasing number of biological implant complications, most commonly peri-implant mucositis and peri-implantitis. The effective management of these complications is crucial to maintain patients' oral health. In particular, in contrast to common periodontal infections, some peri-implant infections may benefit from surgical intervention as a first line approach.

Clinical Relevance: This article reviews the literature on the treatment options for peri-implant mucositis and peri-implantitis and proposes a protocol for their treatment.
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http://dx.doi.org/10.12968/denu.2013.40.6.472DOI Listing
September 2013

Influence of periodontal disease, Porphyromonas gingivalis and cigarette smoking on systemic anti-citrullinated peptide antibody titres.

J Clin Periodontol 2013 Oct 1;40(10):907-15. Epub 2013 Aug 1.

Infection and Immunity, University of Glasgow Dental School, School of Medicine, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow, UK.

Background: Anti-citrullinated protein antibody (ACPA) responses may precede clinical onset of rheumatoid arthritis. Porphyromonas gingivalis peptidylarginine deiminase can citrullinate proteins possibly inducing autoimmunity in susceptible individuals.

Aim: To determine whether periodontitis, carriage of P. gingivalis, smoking and periodontal therapy influence ACPA titres.

Methods: Serum and plaque samples were collected from 39 periodontitis patients before and after non-surgical periodontal treatment, and from 36 healthy subjects. Carriage of P. gingivalis was determined by PCR of plaque DNA. ACPA was determined by anti-cyclic citrullinated peptide (CCP) enzyme-linked immunosorbent assay (ELISA). Anti-P. gingivalis titres were determined by ELISA.

Results: Untreated periodontitis patients had higher anti-CCP antibody titres than healthy controls [three patients (8%) greater than manufacturer suggested assay diagnostic threshold (5 Assay Units/AU) versus none (0%); mean ± SEM: 1.37 ± 0.23 versus 0.40 ± 0.10 AU, p < 0.0001]. Periodontitis patients who smoked demonstrated lower anti-P. gingivalis (15956 ± 4385 versus 2512 ± 1290 Units/ml, p < 0.05), but similar anti-CCP than non-smoking periodontitis patients (smokers: 1.31 ± 0.35; non-smokers: 1.41 ± 0.32 AU). Healthy smokers demonstrated elevated anti-CCP titres (0.75 ± 0.19 AU), at levels between healthy non-smokers (0.15 ± 0.05 AU) and non-smoker periodontitis patients. Six months after periodontal treatment, there were significant reductions in anti-CCP (non-smokers p < 0.05) and anti-P. gingivalis (all participants p < 0.01).

Conclusion: In subjects with periodontitis, P. gingivalis infection may be responsible for inducing autoimmune responses that characterize rheumatoid arthritis.
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http://dx.doi.org/10.1111/jcpe.12138DOI Listing
October 2013

Heightened immune response to autocitrullinated Porphyromonas gingivalis peptidylarginine deiminase: a potential mechanism for breaching immunologic tolerance in rheumatoid arthritis.

Ann Rheum Dis 2014 Jan 5;73(1):263-9. Epub 2013 Mar 5.

Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, , London, UK.

Background: Rheumatoid arthritis (RA) is characterised by autoimmunity to citrullinated proteins, and there is increasing epidemiologic evidence linking Porphyromonas gingivalis to RA. P gingivalis is apparently unique among periodontal pathogens in possessing a citrullinating enzyme, peptidylarginine deiminase (PPAD) with the potential to generate antigens driving the autoimmune response.

Objectives: To examine the immune response to PPAD in patients with RA, individuals with periodontitis (PD) and controls (without arthritis), confirm PPAD autocitrullination and identify the modified arginine residues.

Methods: PPAD and an inactivated mutant (C351A) were cloned and expressed and autocitrullination of both examined by immunoblotting and mass spectrometry. ELISAs using PPAD, C351A and another P gingivalis protein arginine gingipain (RgpB) were developed and antibody reactivities examined in patients with RA (n=80), individuals with PD (n=44) and controls (n=82).

Results: Recombinant PPAD was a potent citrullinating enzyme. Antibodies to PPAD, but not to Rgp, were elevated in the RA sera (median 122 U/ml) compared with controls (median 70 U/ml; p<0.05) and PD (median 60 U/ml; p<0.01). Specificity of the anti-peptidyl citrullinated PPAD response was confirmed by the reaction of RA sera with multiple epitopes tested with synthetic citrullinated peptides spanning the PPAD molecule. The elevated antibody response to PPAD was abolished in RA sera if the C351A mutant was used on ELISA.

Conclusions: The peptidyl citrulline-specific immune response to PPAD supports the hypothesis that, as a bacterial protein, it might break tolerance in RA, and could be a target for therapy.
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http://dx.doi.org/10.1136/annrheumdis-2012-202726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3888615PMC
January 2014

Antifungal, cytotoxic, and immunomodulatory properties of tea tree oil and its derivative components: potential role in management of oral candidosis in cancer patients.

Front Microbiol 2012 18;3:220. Epub 2012 Jun 18.

Infection and Immunity Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow Glasgow, UK.

Candida albicans forms oral biofilms that cause disease and are difficult to treat with conventional antifungal agents. Tea tree oil (TTO) is a natural compound with reported antimicrobial and immunomodulatory activities. The aims of the study were to evaluate the antifungal efficacy of TTO and key derivatives against C. albicans biofilms, to assess the toxicological effects of TTO on a clinically relevant oral cell line, and to investigate its impact on inflammation. TTO and its derivatives were examined against 100 clinical strains of C. albicans. Planktonic minimum inhibitory concentrations (MICs) were determined using the CLSI M-27A broth microdilution method. Sessile MICs were determined using an XTT reduction assay. Inhibition, time-kill, and mode of action studies were performed. OKF6-TERT2 epithelial cells were used for cytotoxicity and cytokine expression assays. Planktonic C. albicans isolates were susceptible to TTO, terpinen-4-ol (T-4-ol), and α-terpineol, with an MIC(50) of 0.5, 0.25, and 0.25%, respectively. These three compounds also displayed potent activity against the 69 biofilm-forming strains, of which T-4-ol and α-terpineol displayed rapid kill kinetics. For all three compounds, 1 × MIC(50) effectively inhibited biofilm growth when C. albicans were treated at 0, 1, and 2 h post adhesion. By scanning electron microscopy analysis and PI uptake, TTO and derivative components were shown to be cell membrane active. TTO and T-4-ol were cytotoxic at 1 × MIC(50), whereas at 0.5 × MIC(50) T-4-ol displayed no significant toxicity. Transcript and protein analysis showed a reduction of IL-8 when treated with TTO and T-4-ol. These data provide further in vitro evidence that TTO and its derivative components, specifically T-4-ol, exhibit strong antimicrobial properties against fungal biofilms. T-4-ol has safety advantages over the complete essential oil and may be suitable for prophylaxis and treatment of established oropharyngeal candidosis. A clinical trial of T-4-ol is worthy of consideration.
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http://dx.doi.org/10.3389/fmicb.2012.00220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3376416PMC
October 2012

What can the periodontal community learn from the pathophysiology of rheumatoid arthritis?

J Clin Periodontol 2011 Mar;38 Suppl 11:106-13

Glasgow Dental Hospital and School, University of Glasgow, Glasgow, UK.

Aim: The aim of this paper is to provide a narrative review of the aetiopathogeneis and treatments of rheumatoid arthritis (RA), focusing on aspects that may share commonality with periodontitis.

Results: A myriad of cell types, cytokines and pathways have been investigated in both periodontitis and RA. Chronic inflammatory diseases, including RA, psoriatic arthritis, ankylosing spondylitis and periodontitis are likely to share pathogenic mechanisms of inflammation-mediated solid tissue destruction. The aetiopathogenesis of these diseases has been extensively researched over the last several decades and advances in understanding have revolutionized arthritis therapeutics.

Conclusion: The rational, targeted inhibition of mediators in RA has provided clinically useful therapeutics and shed light on mechanisms underpinning disease pathogenesis. RA should be considered a prototypic disease revealing how understanding disease pathogenesis may transform therapeutic options and patient outcomes.
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http://dx.doi.org/10.1111/j.1600-051X.2010.01669.xDOI Listing
March 2011

Are we any closer to beating the biofilm: novel methods of biofilm control.

Curr Opin Infect Dis 2010 Dec;23(6):560-6

Glasgow Dental School and Hospital, Faculty of Medicine, University of Glasgow, UK.

Purpose Of Review: A multidisciplinary approach to the treatment and management of biofilms has resulted from the growing appreciation of the role that biofilms play in modern medicine. Conventional antimicrobial agents are generally ineffective against biofilms, and as a result novel laboratory-based and clinical strategies have emerged. The purpose of this review is to analyse the recent literature relating to novel treatment strategies targeting the growing spectrum of clinically relevant biofilms.

Recent Findings: Microscopy and molecular techniques have provided greater insights into identifying the key bacterial and fungal biofilm pathogens. Knowledge of these microorganisms has provided a foundation for the development of specific molecules, often microbial derived, with antimicrobial and/or biofilm disruptive properties, augmenting conventional antibiotics treatments. The validity of some such rationally designed therapeutics has been explored in clinical trials.

Summary: Biofilms are inherently difficult to treat, and mechanical disruption is the mainstay of clinical management. With scientific progress in molecular microbiology, there is an abundance of newly discovered molecules and pathways, providing novel therapeutic and prophylactic targets.
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http://dx.doi.org/10.1097/QCO.0b013e32833e5850DOI Listing
December 2010

Peptidylarginine deiminase from Porphyromonas gingivalis citrullinates human fibrinogen and α-enolase: implications for autoimmunity in rheumatoid arthritis.

Arthritis Rheum 2010 Sep;62(9):2662-72

Imperial College London, London, UK.

Objective: To investigate protein citrullination by the periodontal pathogen Porphyromonas gingivalis as a potential mechanism for breaking tolerance to citrullinated proteins in rheumatoid arthritis (RA).

Methods: The expression of endogenous citrullinated proteins was analyzed by immunoblotting of cell extracts from P gingivalis and 10 other oral bacteria. P gingivalis-knockout strains lacking the bacterial peptidylarginine deiminases (PADs) or gingipains were created to assess the role of these enzymes in citrullination. Citrullination of human fibrinogen and α-enolase by P gingivalis was studied by incubating live wild-type and knockout strains with the proteins and analyzing the products by immunoblotting and mass spectrometry.

Results: Endogenous protein citrullination was abundant in P gingivalis but lacking in the other oral bacteria. Deletion of the bacterial PAD gene resulted in complete abrogation of protein citrullination. Inactivation of arginine gingipains, but not lysine gingipains, led to decreased citrullination. Incubation of wild-type P gingivalis with fibrinogen or α-enolase caused degradation of the proteins and citrullination of the resulting peptides at carboxy-terminal arginine residues, which were identified by mass spectrometry.

Conclusion: Our findings demonstrate that among the oral bacterial pathogens tested, P gingivalis is unique in its ability to citrullinate proteins. We further show that P gingivalis rapidly generates citrullinated host peptides by proteolytic cleavage at Arg-X peptide bonds by arginine gingipains, followed by citrullination of carboxy-terminal arginines by bacterial PAD. Our results suggest a novel model where P gingivalis-mediated citrullination of bacterial and host proteins provides a molecular mechanism for generating antigens that drive the autoimmune response in RA.
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http://dx.doi.org/10.1002/art.27552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2941529PMC
September 2010

Murine neutrophils present Class II restricted antigen.

Immunol Lett 2008 Jun 19;118(1):49-54. Epub 2008 Mar 19.

Centre for Rheumatic Diseases, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK.

Neutrophils were originally described as short lived, terminally differentiated phagocytes that contribute only to the innate immune response. Recent evidence of neutrophil cytokine production and expression of numerous cell surface proteins has suggested that neutrophils are likely to influence adaptive responses and may satisfy the criteria of antigen presenting cells. Under certain inflammatory conditions human neutrophils express major histocompatibilty complex (MHC) Class II and the costimulatory molecules CD80 and CD86. We have employed a murine T cell hybridoma with a transgenic T cell receptor specific for ovalbumin peptide 323-339 (OVA(323-339)), and a green fluorescent reporter of T cell receptor ligation, to directly investigate neutrophil-T cell interactions. These cells provide an ideal model system, allowing precise identification of antigen specificity and a clear readout of T cell activation. Additionally, whilst murine neutrophils have previously been shown to stimulate MHC Class I-dependent CD8(+) T cell activation, CD4(+) T cells stimulation via MHC Class II-expressing neutrophils has not been investigated. We addressed this by isolating murine neutrophils, loading with OVA(323-339) and co-culturing with T cells specific for the OVA(323-339)/MHC Class II complex, and this resulted in T cell activation, as determined by expression of the green-fluorescent protein reporter. Antigen-pulsed neutrophils were also able to prime naïve OVA-specific CD4(+) T cells in a contact-dependent manner, as shown by proliferation and cytokine production. Activation of lymphocytes was not due to contaminating macrophages. These studies demonstrate that murine neutrophils present MHC Class II-restricted peptides and induce T cell proliferation, confirming findings in human neutrophils, and demonstrate a novel pro inflammatory effect of murine neutrophils.
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http://dx.doi.org/10.1016/j.imlet.2008.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430030PMC
June 2008

Prior elevation of IL-18 promotes rapid early IFN-gamma production during staphylococcal infection.

Eur J Immunol 2005 May;35(5):1438-44

Department of Immunology and Bacteriology, Western Infirmary, Glasgow, UK.

Systemic Staphylococcus aureus infection is associated with significant morbidity and mortality arising from both bacterial and host immune factors. IL-18 is a pro-inflammatory cytokine of the IL-1 superfamily that exhibits broad functional effects in innate and acquired immune responses and which has been found in high levels in several chronic inflammatory and autoimmune diseases. Over-expression of IL-18 may promote early resolution of infection or could promote a detrimental exaggerated immune response. This was explored in a model of S. aureus infection. We report increased mortality in Swiss mice that were given recombinant IL-18 prior to inoculation with S. aureus LS-1. IL-18 administration prior to infection induced preferentially enhanced IFN-gamma mRNA expression in peripheral blood leukocytes and spleen, especially splenic NK cells. This correlated with increased IFN-gamma protein detection in serum, and leukocyte and spleen cultures at subsequent discrete time points. These data suggest that increased mortality following gram-positive infection in autoimmune diseases could in part reflect the impact of high levels of pleiotropic pro-inflammatory cytokines such as IL-18 present prior to the onset of infection.
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http://dx.doi.org/10.1002/eji.200425661DOI Listing
May 2005

A novel therapy of murine collagen-induced arthritis with soluble T1/ST2.

J Immunol 2004 Jul;173(1):145-50

Division of Immunology, Infection, and Inflammation and Centre for Rheumatic Diseases, Royal Infirmary, University of Glasgow, Glasgow G11 6NT, Scotland, UK.

Rheumatoid arthritis is characterized by chronic inflammatory infiltration of the synovium, leading to eventual cartilage and bone destruction. Previously, we have reported that soluble T1/ST2 (sST2), a member of the IL-1R gene family, inhibits LPS-induced macrophage proinflammatory cytokine production. In this study, we report the therapeutic effect of sST2-Fc in the murine model of collagen-induced arthritis. A short term administration of sST2-Fc fusion protein significantly attenuated disease severity compared with controls treated with normal IgG. Histological examination revealed that while control IgG-treated mice developed severe cellular infiltration in the joints, synovial hyperplasia, and joint erosion, this pathology was profoundly reduced in sST2-Fc-treated animals. Treatment of sST2-Fc also down-regulated serum levels of IL-6, IL-12, and TNF-alpha. Spleen cells from the sST2-Fc-treated mice produced significantly less IFN-gamma, TNF-alpha, IL-6, and IL-12 compared with cells from the control mice when cultured with collagen in vitro. Finally, pretreatment with ST2-Fc markedly inhibited the ability of human monocytic THP1 cells to release TNF-alpha when cocultured with peripheral blood T cells from rheumatoid patients. Together these results demonstrate that sST2-Fc may provide a novel approach in treating chronic autoimmune conditions by inhibiting the release of proinflammatory cytokines.
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http://dx.doi.org/10.4049/jimmunol.173.1.145DOI Listing
July 2004

IL-18 enhances collagen-induced arthritis by recruiting neutrophils via TNF-alpha and leukotriene B4.

J Immunol 2003 Jul;171(2):1009-15

Department of Pharmacology, School of Medicine Ribeirao Preto, University of São Paulo, São Paulo, Brazil.

IL-18 expression and functional activity have been associated with a range of autoimmune diseases. However, the precise mechanism by which IL-18 induces such pathology remains unclear. In this study we provide direct evidence that IL-18 activates neutrophils via TNF-alpha induction, which drives the production of leukotriene B(4) (LTB(4)), which in turn leads to neutrophil accumulation and subsequent local inflammation. rIL-18 administered i.p. resulted in the local synthesis of LTB(4) and a rapid influx of neutrophils into the peritoneal cavity, which could be effectively blocked by the LTB(4) synthesis inhibitor MK-886 (MK) or its receptor antagonist CP-105,696. IL-18-induced neutrophils recruitment and LTB(4) production could also be blocked by a neutralizing anti-TNF-alpha Ab. In addition, IL-18 failed to induce neutrophil accumulation in vivo in TNFRp55(-/-) mice. In an IL-18-dependent murine collagen-induced arthritis model, administration of MK significantly inhibited disease severity and reduced articular inflammation and joint destruction. Furthermore, MK-886-treated mice also displayed suppressed proinflammatory cytokine production in response to type II collagen in vitro. Finally, we showed that IL-18-activated human peripheral blood neutrophils produced significant amounts of LTB(4) that were effectively blocked by the MK. Together, these findings provide a novel mechanism whereby IL-18 can promote inflammatory diseases.
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http://dx.doi.org/10.4049/jimmunol.171.2.1009DOI Listing
July 2003
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