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BACKGROUND AND OBJECTIVE Cigarette smoking is a major risk factor for periodontitis, and smoking perturbs neutrophil reactive oxygen species production. This study tested the hypothesis that cigarette smoke extract (CSE) and its components/metabolites nicotine, cotinine and thiocyanate (SCN-), may influence neutrophil functions. MATERIAL AND METHODS Chemotaxis was assessed in neutrophils pre-treated with CSE using real-time video microscopy. Neutrophil extracellular trap (NET) release in response to CSE, nicotine, cotinine, SCN- as well as to phorbol 12-myristate-13-acetate and hypochlorous acid following pre-treatment with CSE, nicotine, cotinine or SCN- was assessed using fluorescence-based assays. The impact of CSE and SCN- treatment on neutrophil respiratory burst- and inflammation-related gene expression (NFKBIE, DNAJB1, CXCL8, NCF1, NCF2, CYBB) was determined by real-time polymerase chain reaction. RESULTS Both CSE and SCN- pre-treatment inhibited phorbol 12-myristate-13-acetate-stimulated NET release. Additionally, SCN- inhibited hypochlorous acid-stimulated NET formation, while SCN- alone stimulated NET release. Overall, neutrophils pre-treated with CSE exhibited reduced speed, velocity and directionality relative to untreated neutrophils. Although CSE and SCN- promoted DNAJB1 expression, increased redox-related gene expression was only detected in response to SCN-. CONCLUSION These results suggest that CSE can alter ex vivo neutrophil activation by mechanisms independent of SCN- and nicotine, and SCN- may contribute to the perturbed innate immune responses observed in smokers.

Palmer LJ, Chapple ILC, Wright HJ, Roberts A, Cooper PR. Extracellular deoxyribonuclease production by periodontal bacteria. J Periodont Res 2012; 47: 439–445. © 2011 John Wiley & Sons A/S Background and Objective: Whilst certain bacteria have long been known to secrete extracellular deoxyribonuclease (DNase), the purpose in microbial physiology was unclear. Recently, however, this enzyme has been demonstrated to confer enhanced virulence, enabling bacteria to evade the host’s immune defence of extruded DNA/chromatin filaments, termed neutrophil extracellular traps (NETs). As NETs have recently been identified in infected periodontal tissue, the aim of this study was to screen periodontal bacteria for extracellular DNase activity. Material and Methods: To determine whether DNase activity was membrane bound or secreted, 34 periodontal bacteria were cultured in broth and on agar plates. Pelleted bacteria and supernatants from broth cultures were analysed for their ability to degrade DNA, with relative activity levels determined using an agarose gel electrophoresis assay. Following culture on DNA-supplemented agar, expression was determined by the presence of a zone of hydrolysis and DNase activity related to colony size. Results: Twenty-seven bacteria, including red and orange complex members Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, Parvimonas micra, Prevotella intermedia, Streptococcus constellatus, Campylobacter rectus and Prevotella nigrescens, were observed to express extracellular DNase activity. Differences in DNase activity were noted, however, when bacteria were assayed in different culture states. Analysis of the activity of secreted DNase from bacterial broth cultures confirmed their ability to degrade NETs. Conclusion: The present study demonstrates, for the first time, that DNase activity is a relatively common property of bacteria associated with advanced periodontal disease. Further work is required to determine the importance of this bacterial DNase activity in the pathogenesis of periodontitis.

The search for markers of periodontal disease activity and progression has accelerated over the last decade, in an effort to replace existing subjective clinical measures of periodontal health status. Research is being aimed at establishing more objective and quantitative methodology, capable of rapid diagnosis prior to the appearance of clinical signs of destructive disease. Such tests need to be sensitive enough to evaluate individual periodontal sites in health as well as disease states. We report the development of a new chemiluminescent assay for the enzyme alkaline phosphatase, that is capable of quantifying the enzyme in sub-microliter volumes of gingival crevicular fluid and serum. The technique will measure alkaline phosphatase (ALP) whilst immobilised on paper strips, without the need for an elution stage. It is simple, versatile and amenable to chair-side use. We discuss in detail the assay procedure and have examined levels of ALP in 11 adult volunteers with clinically healthy periodontal tissues. The mean ALP concentration was 2135 IU/L for GCF and 183 IU/L for serum, a 12-fold difference. There also appeared to be an "oral pattern" of enzyme distribution in healthy periodontal sites, with levels being higher in the anterior region of the mouth and highest in the lower anterior region.

This paper reports the detailed calibration of the new Periotron 8000 with different fluids and uses the method of least squares to derive polynomial regression equations up to the 6th order, to investigate the most accurate descriptor of the resulting calibration lines. The use of a 4th order polynomial regression equation (recommended by the manufacturer) provided better coefficients of determination (R2: 0.999) and root mean square errors (RMSE = 1.6) than either linear regression (R2: 0.986, RMSE = 10.9) or quadratic models (R2: 0.998, RMSE = 3.2). Data derived using the manufacturer's MLCONVERT software program lacked accuracy and incurred large errors for volumes > 0.5 microliter. Calibrations performed on one day could be used with accuracy to derive volumes > 0.1 microliter collected on subsequent days, when using the same machine (s.d. for residuals plot = 2.49 Periotron units), but this was not the case for different machines (s.d. = 9.57 Periotron units). Varying serum protein concentration by up to 500% had a negligible effect on calculated volumes above 0.1 microliter. We conclude that the Periotron 8000 is at least as reliable a machine as the Periotron 6000, and that the calibration lines for both machines are best described using 4th order polynomial regression equations and "look-up" tables, rather than quadratic (Periotron 6000) or the manufacturer's software (Periotron 8000). Serum seems to be an acceptable GCF substitute for calibrations, which can be performed 1 day, and used on subsequent days for a given machine and for volumes above 0.1 microliter.

The Periotron 6000 fluid analyser has become widely used as a diagnostic tool for a variety of oral diseases and recent work has questioned its reliability. This paper investigates for the first time, the detailed calibration curves of 2 Periotron 6000 machines across a range of 23 different fluid volumes. Within and between machine reliability is analyzed and the shape of the calibration line determined. The measurement errors incurred by using a single fluid sample, as opposed to mean values of triplicate samples are also determined. We conclude that there are 3 sections to the calibration line, 2 linear and a quadrilateral zone, and that 2 separate regression equations should be used; from 0-0.1 microliter and from 0.1-1.0 microliter. Within machine calibration errors were only 3.2 +/- 7.5%, but values for volumes below 0.2 microliter were as high as 18.7%. Using a single fluid sample rather than mean values of multiple samples, incurred a further 4 +/- 4% error, which was as high as 7% for volumes lower than 0.12 microliter. Whilst significant differences in volume reading existed between different machines (p < 0.0004) and between the same volumes of different fluids (p < 0.00001), individual Periotron calibrations were extremely reproducible and reliable. We conclude that the Periotron 6000 is a reliable and convenient instrument for measuring fluid volumes greater than 0.2 microliter. For volumes lower than 0.2 microliter errors in measurement may be too high for some investigations, but this is likely to be due to problems with evaporation and with measurement technique, rather than errors directly due to the Periotron itself. Finally, for optimum accuracy, the digital display should be re-set to zero after each sample is measured.