Engineering tumor-colonizing E. coli Nissle 1917 for detection and treatment of colorectal neoplasia.

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment. Here, first, we demonstrate selective colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition and orthotopic models of CRC. We next undertake an interventional, double-blind, dual-centre, prospective clinical trial, in which CRC patients take either placebo or EcN for two weeks prior to resection of neoplastic and adjacent normal colorectal tissue (ACTRN12619000210178). We detect enrichment of EcN in tumor samples over normal tissue from probiotic-treated patients (primary outcome of the trial). Next, we develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate. Oral delivery of this strain results in increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. To assess therapeutic potential, we engineer EcN to locally release a cytokine, GM-CSF, and blocking nanobodies against PD-L1 and CTLA-4 at the neoplastic site, and demonstrate that oral delivery of this strain reduces adenoma burden by ~50%. Together, these results support the use of EcN as an orally-deliverable platform to detect disease and treat CRC through the production of screening and therapeutic molecules.

Association Between RAS/BRAF Mutations and Complete Response Following Total Neoadjuvant Therapy in Patients with Rectal Cancer: A Prospective Multicentered Study.

BACKGROUND: The impact of RAS/BRAF mutation on primary response rates after total neoadjuvant therapy (TNT) in patients with advanced rectal cancer is unclear. The aim of this study was to assess complete response rates after TNT according to RAS/BRAF mutation status. METHODS: A prospective observational study was performed in patients with rectal cancer who underwent TNT with curative intent at three South Australian hospitals between 2019 and 2023. Patients were classified according to their mutation status: mutant RAS/BRAF (mutRAS) or wild-type RAS/BRAF (wtRAS). The primary endpoint was overall complete response (oCR) rate, defined as the proportion of patients who achieved clinical complete response (cCR) and/or pathological complete response (pCR). RESULTS: Of the 150 patients eligible for inclusion, 80 patients with RAS/BRAF status available were identified. Of these, 43 (53.8%) patients were classified as mutRAS and 37 (46.3%) patients as wtRAS. Patients with mutRAS had significantly lower cCR and oCR rates after TNT than patients with wtRAS (14% vs. 37.8%, p = 0.014; 11.6% vs. 43.2%, p = 0.001, respectively). There was no significant difference in pCR rate between the groups. Of the 80 rectal cancer patients tested, 35 (43.8%) had metastatic disease (M1). There was no significant difference in complete M1 response rates between the groups (17.6% vs. 38.9%, p = 0.254). CONCLUSION: RAS/BRAF mutations negatively impact primary tumor response rates after TNT in patients with advanced rectal cancer. Large-scale national studies are needed to determine whether RAS/BRAF status could be used to select optimal oncologic therapy in rectal cancer patients.

Colorectal cancer detection and treatment with engineered probiotics.

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment strategies. Here, we demonstrate the phenomenon of selective, long-term colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition. We show that, after oral administration, adenomas can be monitored over time by recovering EcN from stool. We also demonstrate specific colonization of EcN to solitary neoplastic lesions in an orthotopic murine model of CRC. We then exploit this neoplasia-homing property of EcN to develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate, and demonstrate that oral delivery of this strain results in significantly increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. We also assess EcN engineered to locally release immunotherapeutics at the neoplastic site. Oral delivery to mice bearing adenomas, reduced adenoma burden by ∻50%, with notable differences in the spatial distribution of T cell populations within diseased and healthy intestinal tissue, suggesting local induction of robust anti-tumor immunity. Together, these results support the use of EcN as an orally-delivered platform to detect disease and treat CRC through its production of screening and therapeutic molecules.

Personalized total neoadjuvant therapy (pTNT) for advanced rectal cancer with tailored treatment sequencing based on clinical stage at presentation.

BACKGROUND: This study aimed to assess short-term outcomes of a personalized total neoadjuvant treatment (pTNT) protocol, with treatment sequencing based on clinical stage at presentation. METHODS: A multidisciplinary pTNT protocol was implemented across two metropolitan hospitals. This consists of two-schema based on clinical stage: patients with distant failure risk were offered induction chemotherapy before chemoradiation (nCRT), and patients with locoregional failure risk received nCRT followed by consolidation chemotherapy. Patients underwent surgical resection unless a complete clinical response (cCR) was achieved, in which case non-operative management (NOM) was offered. A prospective cohort analysis of all patients with rectal cancer who underwent pTNT with curative intent between Jan 2019 and Aug 2022 was performed. RESULTS: Of 270 patients referred with rectal cancer, 102 received pTNT with curative intent and 79 have completed their treatment thus far. Thirty-three patients (41.8%) received induction chemotherapy and 46 (58.2%) received consolidation chemotherapy per protocol. The percentage of patients with EMVI, resectable M1 disease, cT4 disease, and positive lateral lymph nodes were 54.4%, 36.7%, 27.8% and 15.2%, respectively. Overall, 32 (40.5%) patients had cCR and 4 (5.1%) pCR, and 40 (50.6%) patients had non-operative management. Grade 3 toxicity was reported in 10.1% of patients and only three patients (3.8%) experienced Grade 4 chemotherapy-related toxicity, with no treatment related mortality. CONCLUSION: Early results with a defined two-schema pTNT protocol are encouraging and suggest that tailoring sequencing to disease risk at presentation may represent the optimal balance between local and distant disease control, as well as treatment toxicity.

Establishing a state-wide pelvic exenteration multidisciplinary team meeting in South Australia.

BACKGROUND: Pelvic exenteration surgery is complex, necessitating co-ordinated multidisciplinary input and improved referral pathways. A state-wide pelvic exenteration multidisciplinary team (MDT) meeting was established in SA and the outcomes of this were audited and compared with historical data. METHODS: All patients referred for discussion between August 2021 and July 2022 to the SA State-wide Pelvic Exenteration MDT were included in this study. MDT discussion centred around disease resectability, risk versus benefit of surgery, and need for local or interstate referral. Prospective data collection included patient demographics and MDT recommendations of surgery, palliation, or referral. Patients referred for surgery locally or interstate were compared with a retrospective patient cohort treated previously between January and December 2020. RESULTS: Over 12 months, 91 patients were discussed (including nine multiple times), by a mean of 18 meeting participants each month. Forty-eight patients (58.5%) had primary malignancy, 25 (30.5%) recurrent malignancy, and 9 (11.0%) had non-malignant disease. Colorectal cancer was the most common presentation (56.1%), followed by gynaecological (30.5%) and urological (6.1%) malignancy. Pelvic exenteration surgery was recommended to be performed locally in 53.7% of patients and the remainder for non-surgical treatment, palliation, or re-discussion. During this time, 44 patients underwent surgery locally (versus 34 in 2020) and only 4 referred interstate (versus 8 in 2020). CONCLUSION: The establishment of a dedicated state-wide pelvic exenteration MDT has resulted in better coordination of care for patients with locally advanced pelvic malignancy in SA, and significantly reduced the need for interstate referral.

Personalized total neoadjuvant therapy versus chemotherapy during the 'wait period' versus standard chemoradiotherapy for locally advanced rectal cancer.

BACKGROUND: This study aimed to compare current treatment response rates with personalized Total Neoadjuvant Therapy (pTNT), against extended chemotherapy in the 'wait period' (xCRT) and standard chemoradiotherapy (sCRT) with adjuvant chemotherapy for rectal cancer. METHODS: This was a multicentre retrospective cohort analysis. Consecutive patients with rectal cancer treated with pTNT over a 3.9-year period were compared to a historical cohort of patients treated with xCRT or sCRT as part of the published WAIT Trial. pTNT patients received 8 cycles mFOLFOX6 or 6 cycles CAPOX in the neoadjuvant setting (no adjuvant treatment). Patients in the WAIT Trial received either 3 cycles 5-FU/LV during the 10-week wait period after chemoradiotherapy or standard chemoradiotherapy, followed by adjuvant chemotherapy. The primary endpoint was overall complete response (oCR) rate defined as the proportion of patients who achieved either complete clinical response (cCR) or pathological complete response (pCR). RESULTS: Of 284 patients diagnosed with rectal cancer during the 3.9-year period, 107 received pTNT. Forty of these were matched with 49 patients from the WAIT Trial (25 received xCRT and 24 received sCRT). There was a significant difference in oCR between the groups (pTNT n = 21, xCRT n = 6, sCRT n = 7, P = 0.043). Of the patients that underwent surgery, pCR occurred in 13 patients with no significant difference between groups (P = 0.415). There were no significant differences in 2-year disease-free survival or overall survival. CONCLUSION: Compared with sCRT and xCRT, pTNT results in a significantly higher complete response rate which may facilitate organ preservation.

Assessing the impact of the 2018 National Health and Medical Research Council polyp surveillance guidelines on compliance with surveillance intervals at two public hospitals.

BACKGROUND: Several studies have highlighted poor compliance with surveillance colonoscopy guidelines. The National Health and Medical Research Council (NHMRC) guidelines were revised in 2018 and were more complex than the previous iteration (2011). The aim of this study was to determine the impact of 2018 NHMRC polyp surveillance guidelines on compliance with colonoscopy surveillance intervals. METHODS: A multicentre retrospective clinical audit was conducted between January 2020 and February 2021. Patients awaiting a colonoscopy for polyp surveillance at two public tertiary care hospitals in South Australia were included. Compliance rates of recommended polyp surveillance colonoscopy intervals after implementation of 2018 NHMRC guidelines were compared with 2011 NHMRC guidelines. The projected impact on colonoscopy bookings of the change in guideline intervals was modelled to 5 and 10 years, factoring in differences in compliance. RESULTS: Of 3996 patients awaiting colonoscopy services at two public hospitals in South Australia, 1984 patients (60% male, median age 61 years) were waitlisted for polyp surveillance. Overall compliance with surveillance guidelines was >60%. Implementation of the 2018 NHMRC guidelines significantly reduced compliance from 65.8% (2011 guidelines) to 50.8% (2018) (χ2 <0.001, OR 0.5). Modelling projections to 5 and 10 years demonstrated that application of the 2018 guidelines significantly increases the projected number of colonoscopy bookings per year. CONCLUSION: The revised 2018 NHMRC guidelines have resulted in significantly poorer compliance post-implementation, possibly due to their increased complexity. This has potential to increase the surveillance colonoscopy waiting list burden.

A snapshot of intraoperative conditions to predict prolonged postoperative ileus after colorectal surgery.

BACKGROUND: The cause of prolonged postoperative ileus (PPOI) is multifactorial. The influence of preoperative factors on PPOI has been well documented, but little is known about the impact of intraoperative conditions. The aim of this study was to investigate the influence of intraoperative factors on PPOI in patients undergoing colorectal surgery. METHODS: The LekCheck study database of the Colorectal Unit at the Royal Adelaide Hospital was analysed. Per patient, over 60 data points were prospectively collected between March 2018 and July 2020. Intraoperative data were collected in theatre during a one-off snapshot measure. Univariate and multivariable logistic regression analyses were performed. RESULTS: Data of 336 patients were included. The median age was 66 years and 58.3% were male. Ninety-three patients (27.7%) developed PPOI. Univariate analysis identified the following intraoperative variables as risk-factors of PPOI: greater volumes of intraoperative IV fluid administration (464 versus 415 mL/h for those without PPOI; p = 0.04), side-to-side anastomosis orientation (53.8 versus 41.2%; p = 0.04) and increased perioperative opioid use (6.73 versus 4.11 mg/kg morphine equivalents for patients with and without PPOI, respectively; p = 0.02). Upon multivariable analysis, increased perioperative opioid use remained significant (p = 0.05), as well as the preoperative factors anticoagulation use (p = 0.04) and higher levels of serum total protein (p = 0.02). CONCLUSION: This study suggests that intraoperative factors may also contribute to the development of PPOI, but this could not be confirmed in the multivariate analysis. Further studies including larger patient numbers will be required to determine the impact of intraoperative conditions on the development of PPOI.

Disruption of Enterococcus Faecalis biofilms using individual and plasma polymer encapsulated D-amino acids.

OBJECTIVE: Our aim was to assess the anti-biofilm ability of previously unverified individual D-amino acids (DAAs), to produce plasma polymer encapsulated DAAs (PPEDAAs), to measure the shell thickness and subsequent release of DAAs, and to assess the effects of PPEDAAs on Enterococcus faecalis biofilms. MATERIALS AND METHODS: Microtitre tray assays were used to evaluate the effect of individual DAAs (D-leucine, D-methionine, D-tryptophan, and D-tyrosine) on E. faecalis biofilms of different maturity. A mixture and individual DAAs were encapsulated with a plasma polymer for 10, 20, 40, and 60 min. The shell thickness of PPEDAAs was analyzed by ultra-high-resolution scanning electron microscopy. The release of DAAs from the PPEDAAs encapsulated for 60 min was measured over 7 days using high-performance liquid chromatography. Static biofilms were used to assess the effect of PPEDAAs on E. faecalis biofilms. RESULTS: Individual DAAs reduced biofilm formation to various degrees, according to the DAA and the experimental times. The shell thicknesses of the PPEDAAs ranged between 31 and 76 nm and increased with encapsulation time. Diffusion of DAAs from the PPEDAAs occurred over 60 min for encapsulated D-leucine, D-methionine, and D-tyrosine and up to 7 days for D-tryptophan. PPEDAAs disrupted biofilms at every experimental time. CONCLUSIONS: PPEDAAs of various shell thickness can be produced with the proposed methodology, DAAs are subsequently released, and the anti-biofilm activity remains unaltered. CLINICAL RELEVANCE: Individual DAAs and PPEDAAs have anti-biofilm ability and can be considered as part of a biological strategy in endodontics.

Histone deacetylases 1 and 2 inhibition suppresses cytokine production and osteoclast bone resorption in vitro.

The regulation of epigenetic factors is an emerging therapeutic target of immune function in a variety of osteolytic pathologies. Histone deacetylases (HDAC) modify core histone proteins and transcriptional processes, in addition to nonhistone protein activity. The activated immune response in rheumatoid arthritis, periodontitis, and prosthetic implant particle release stimulates the catabolic activity of osteoclasts. In this study, we investigated the effects of novel therapeutic agents targeting HDAC isozymes (HDAC 1, 2, and 5), previously shown to be upregulated in inflammatory bone disorders, in cytokine-stimulated human monocytes and osteoclasts in vitro. Inhibiting HDAC 1 and 2 significantly reduced gene expression of IL-1ß, TNF, MCP-1, and MIP-1α in TNF-stimulated monocytes, while suppressing secretions of IL-1ß, IL-10, INF-γ, and MCP-1 (P < .05). Osteoclast formation and bone resorption were also significantly diminished with HDAC 1 and 2 inhibition, through reduced NFATc1 expression and osteoclast specific target genes, TRAF6, CTR, TRAP, and Cathepsin K (P < .05). Similar trends were observed when inhibiting HDAC 1 and to a lesser extent, HDAC 2, in isolation. However, their combined inhibition had the greatest anti-inflammatory and antiosteoclastic effects. Targeting HDAC 5 had minimal effects on these processes investigated in this study, whereas a broad acting HDACi, 1179.4b, had widespread suppressive outcomes. This study demonstrates that targeting HDACs is a potent and effective way of regulating the inflammatory and catabolic processes in human monocytes and osteoclasts. It also demonstrates the importance of targeting individual HDACs with an overall aim to improve efficiency and reduce any potential off target effects.

Efficacy of laser and ultrasonic-activated irrigation on eradicating a mixed-species biofilm in human mesial roots.

This study investigated the efficacy of Er,Cr:YSGG laser and ultrasonic activated irrigation on eradicating a mixed-species biofilm grown in root canals with complex anatomy. The biofilm was grown over 4-weeks in the root canals of decoronated human mandibular molar teeth. Control roots received no further treatment. The remaining roots were chemomechanically prepared using different irrigating protocols: 4% NaOCl and 15% EDTAC with ultrasonic activated irrigation and laser activated irrigation using power settings of 0.5 W and 0.75 W. Cellular viability was determined using serial plating. One tooth from each group was subjected to qualitative SEM analysis. Quantification by culturing revealed significant differences between control group and all other treatment groups. This study demonstrated that chemomechanical irrigation with laser and ultrasonic activated irrigation significantly reduced the bacterial load from complex root canal systems; however, there were no significant differences found between the experimental groups.

Probiotic Lactobacillus rhamnosus GG prevents alveolar bone loss in a mouse model of experimental periodontitis.

AIM: This study investigated the role of Lactobacillus rhamnosus GG (LGG) on bone loss and local and systemic inflammation in an in vivo mouse model of experimental periodontitis (PD). MATERIALS AND METHODS: Experimental PD was induced in mice by oral inoculation with Porphyromonas gingivalis and Fusobacterium nucleatum over a period of 44 days. The probiotic LGG was administered via oral inoculation or oral gavage prior to, and during disease induction. The antimicrobial activity of LGG on the inoculum was also tested. Alveolar bone levels and gingival tissue changes were assessed using in vivo microcomputed tomography and histological analysis. Serum levels of mouse homologues for IL-8 were measured using multiplex assays. RESULTS: Pre-treatment with probiotics either via oral gavage or via oral inoculation significantly reduced bone loss (p < .0001) and gingival inflammation (p < .0001) when compared with PD group. Oral gavage treatment group had significantly less tartrate-resistant acid phosphatase positive cells (p < .02) then PD group. LGG showed no antimicrobial activity against P. gingivalis and F. nucleatum. CONCLUSIONS: Lactobacillus rhamnosus GG effectively suppresses bone loss in a mouse model of induced PD irrespective of the mode of administration.

Compromised inflammatory cytokine response to P. gingivalis LPS by fibroblasts from inflamed human gingiva.

OBJECTIVES: The aims of this study were to compare the in vitro cytokine response of gingival fibroblasts (GF's) from healthy and inflamed human gingival tissues and to assess whether GF's from inflamed gingivae are capable of mounting a secondary inflammatory response after exposure to P. gingivalis LPS. MATERIALS AND METHODS: GF's were obtained from healthy donors and periodontitis patients and cultured in vitro. Cells were exposed to P. gingivalis LPS for 24h before measurement of MCP-1, GRO, IL-6, IL-8 and VEGF using a bead-based multiplex assay. Statistical comparisons were made between LPS-exposed GF's and unstimulated cells as well as the two patient groups by two-way ANOVA. RESULTS: GF's exposed to P. gingivalis LPS significantly increased their production of MCP-1, GRO, IL-6, IL-8 and VEGF compared to unstimulated cells. GF's isolated from inflamed tissue from periodontitis patients demonstrated consistently less cytokine production after exposure to P. gingivalis LPS, most notably for GRO and IL-6. CONCLUSIONS: The current study demonstrates that GF's play an active role in the inflammatory response in periodontal disease by producing a number of chemokines and cytokines. Furthermore, inflamed GF's may be compromised in their ability to mount an adequate secondary immune response in relation to chemokine/cytokine production. CLINICAL RELEVANCE: The compromised inflammatory cytokine response of inflamed human gingival fibroblasts to P. gingivalis LPS may impact on their ability to recruit and activate inflammatory cells while maintaining persistent inflammation, a key feature of periodontal disease.

Abnormal pregnancy outcomes in mice using an induced periodontitis model and the haematogenous migration of Fusobacterium nucleatum sub-species to the murine placenta.

OBJECTIVES: To investigate if there is subspecies specific migration to the placenta by Fusobacterium nucleatum (Fn) and to determine whether experimentally induced periodontitis results in adverse pregnancy outcomes (APO) in mice. METHODS: Periodontitis was induced in pregnant mice using an inoculum of Fn and Porphyromonas gingivalis. In parallel, four sub-species of Fn were individually injected into the circulatory system. At day 18 of gestation, the placenta, liver, spleen and blood were harvested and litter size, number of viable fetuses and resorptions, maternal, fetal and placenta weights were recorded. For the direct inoculation group, some mice were allowed to deliver for assessment of length of gestation, litter size, maternal, placental and pup weight. The presence of Fn was assessed by PCR and inflammatory mediators were measured by ELISA or multiplex analysis. RESULTS: Mice with alveolar bone loss, a marker of periodontitis, demonstrated significantly higher fetal weights (p = 0.015) and fetal/placental weight ratios (p = 0.030). PCR analysis of maternal organs did not identify Fn in any extracted tissues. In mice that received direct injection of Fn subspecies, varying degrees of APO were observed including preterm birth, intrauterine growth restriction, and fetal loss. Haematogenous spread of only Fn subsp. nucleatum to the placenta was confirmed. Litter size was significantly smaller (p = 0.023) and the number of resorptions was higher in inoculated versus control groups. Mice injected with subsp. nucleatum had significantly increased circulating CRP levels (p = 0.020) compared to controls while the mice with induced periodontitis had increased levels of IL-6 (p = 0.047) and IL-8 (p = 0.105). CONCLUSIONS: Periodontitis in mice elevated fetal weight and the fetal weight/placental weight ratio. This study found that subsp. nucleatum migrated haematogenously to the placenta, leading to APO in mice. The study supports the potential role of Fn in the association between periodontitis and APO.

C-reactive protein in gingival crevicular fluid may be indicative of systemic inflammation.

BACKGROUND AND AIM: Periodontitis is associated with elevated C-reactive protein (CRP) in both serum and gingival crevicular fluid (GCF). Although the liver is the primary source of CRP, extra-hepatic production of CRP has been reported. This study aimed to determine whether CRP in GCF is produced locally in the gingivae. MATERIALS AND METHODS: Gingivae and GCF were collected from non-periodontitis and periodontitis sites. Presence of CRP in gingivae was assessed by immunohistochemistry. CRP in GCF was measured using ELISA. Gene expression for CRP in gingivae was determined using real-time polymerase chain reaction. RESULTS: CRP was found in both the gingivae and GCF. No gingivae had detectable amounts of CRP mRNA. Not all patients with periodontitis had detectable levels of CRP in the GCF. Some non-periodontitis patients had detectable levels of CRP in the GCF. CONCLUSION: CRP in the GCF appears to be of systemic origin, and therefore may be indicative of systemic inflammation from either a periodontal infection or inflammatory disease elsewhere. The correlation between levels of CRP in GCF and serum requires validation in future studies.

Local and systemic biomarkers in gingival crevicular fluid increase odds of periodontitis.

AIM: To determine the independent and combined associations of interleukin-1beta (IL-1beta) and C-reactive protein (CRP) in gingival crevicular fluid (GCF) on periodontitis case status in the Australian population. MATERIALS AND METHODS: GCF was collected from 939 subjects selected from the 2004-2006 Australian National Survey of Adult Oral Health: 430 cases had examiner-diagnosed periodontitis, and 509 controls did not. IL-1beta and CRP in GCF were detected by enzyme-linked immunosorbent assays. Odds ratios (OR) and 95% confidence intervals (CIs) were calculated in bivariate and stratified analysis and fully adjusted ORs were estimated using multivariate logistic regression. RESULTS: Greater odds of having periodontitis was associated with higher amounts of IL-1beta (OR=2.4, 95% CI=1.7-3.4 for highest tertile of IL-1beta relative to lowest tertile) and CRP (OR=1.9, 95% CI=1.5-2.5 for detectable CRP relative to undetectable CRP). In stratified analysis, there was no significant interaction between biomarkers (p=0.68). In the multivariate analyses that controlled for conventional periodontal risk factors, these relationships remained (IL-1beta OR=1.8, 95% CI=1.1-2.6; CRP OR=1.7, 95% CI=1.3-2.3). CONCLUSIONS: Elevated odds of clinical periodontitis was associated independently with each biomarker. This suggests that people with elevated biomarkers indicative of either local (IL-1beta) or systemic (CRP) inflammation are more likely to suffer from periodontal disease.

Effect of smoking on concentrations of receptor activator of nuclear factor kappa B ligand and osteoprotegerin in human gingival crevicular fluid.

AIM: To compare the levels of the soluble receptor activator of nuclear factor kappa B ligand (sRANKL), osteoprotegerin (OPG) and their relative ratio in gingival crevicular fluid (GCF) among periodontitis patients with varying smoking histories. MATERIAL AND METHODS: GCF samples were collected from 149 periodontitis patients who were never smokers (n=58), former smokers (n=39) and current smokers (n=52). sRANKL and OPG concentrations in GCF were measured by enzyme-linked immunosorbent assays. RESULTS: sRANKL, OPG and their relative ratio were not statistically significant among the never smokers, former smokers and current smokers. However, OPG was significantly reduced and subsequently the sRANKL:OPG ratio was significantly increased in the high pack-years group as compared with never smokers. The positive correlation between pack-years and the sRANKL:OPG ratio remained statistically significant after adjusting for age and current smoking status. CONCLUSION: Increased lifetime exposure to cigarette smoking above a minimum threshold suppresses OPG production and leads to increased sRANKL:OPG. This may partially explain increased bone loss in smoking-related periodontitis.

Physical activity, inflammatory biomarkers in gingival crevicular fluid and periodontitis.

AIMS: To examine the associations of physical activity with interleukin 1-beta (IL-1beta), C-reactive protein (CRP) and periodontitis and to investigate whether any relationship between physical activity and inflammatory mediators differs between periodontitis cases and non-cases. MATERIAL AND METHODS: In this population-based case control study of Australians aged 18+ years, dentists conducted oral epidemiologic examinations identifying cases with moderate or severe periodontitis and periodontally healthy controls. Gingival crevicular fluid samples collected during examinations were analysed for inflammatory biomarkers. Subject-completed questionnaires assessed leisure-time physical activity. Exposure odds ratios (ORs) were estimated in multivariable logistic regression models adjusting for periodontitis risk indicators. RESULTS: Of 751 subjects (359 cases, 392 controls), those meeting a prescribed threshold for leisure-time physical activity had lower adjusted odds of elevated IL-1beta: OR=0.69, (95% CI=0.50-0.94) and detectable CRP: OR=0.70 (0.50-0.98) than less active adults. Physical activity was not associated with periodontitis: OR=1.14 (0.80-1.62). Periodontitis modified the association between levels of physical activity and detectable CRP. Increasing quartiles of physically activity were associated with decreasing probability of detectable CRP, but the effect was limited to periodontitis cases and was not apparent among non-cases. CONCLUSION: Leisure-time physical activity may protect against an excessive inflammatory response in periodontitis.