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The assignment of functions to uncharacterized proteins discovered in genome projects requires easily accessible tools and computational resources for large-scale, user-friendly leveraging of the protein, genome, and metagenome databases by experimentalists. This article describes the web resource developed by the Enzyme Function Initiative (EFI; accessed at https://efi.igb.illinois.edu/ ) that provides "genomic enzymology" tools ("web tools") for (1) generating sequence similarity networks (SSNs) for protein families (EFI-EST); (2) analyzing and visualizing genome context of the proteins in clusters in SSNs (in genome neighborhood networks, GNNs, and genome neighborhood diagrams, GNDs) (EFI-GNT); and (3) prioritizing uncharacterized SSN clusters for functional assignment based on metagenome abundance (chemically guided functional profiling, CGFP) (EFI-CGFP). The SSNs generated by EFI-EST are used as the input for EFI-GNT and EFI-CGFP, enabling easy transfer of information among the tools. The networks are visualized and analyzed using Cytoscape, a widely used desktop application; GNDs and CGFP heatmaps summarizing metagenome abundance are viewed within the tools. We provide a detailed example of the integrated use of the tools with an analysis of glycyl radical enzyme superfamily (IPR004184) found in the human gut microbiome. This analysis demonstrates that (1) SwissProt annotations are not always correct, (2) large-scale genome context analyses allow the prediction of novel metabolic pathways, and (3) metagenome abundance can be used to identify/prioritize uncharacterized proteins for functional investigation.

Owing to its molecular stability in body fluids, soluble urokinase-type plasminogen activator receptor (suPAR) is used as a biomarker for the level of systemic inflammation. This study compares the suPAR levels in serum with those in the saliva of adolescents and evaluates their association with the periodontal conditions. Adolescents identified as screen positive (n = 87) or screen negative (n = 73) for periodontitis had saliva and serum samples taken, along with subgingival plaque samples. The concentrations of suPAR were determined in saliva and serum, and 18 microbial species and the immunoglobulin response to them was evaluated. Factor analyses were used to reduce the number of variables within each of the domains of clinical, microbiological, and immunological findings. The median salivary suPAR concentration was 13.18 [(interquartile range (IQR): 6.20-23.36] μg l -1 and was not associated with the serum suPAR levels (median 2.05; IQR: 1.62-2.46 μg l -1 ). Linear regression analysis showed that the log 10 (salivary suPAR concentration) was statistically significantly positively associated with the clinical phenotype 'Periodontitis Extent' (β = 0.28; 95% CI: 0.16-0.39) along with 'Putative periodontopathogens' (β = 0.65; 95% CI: 0.51-0.79). The study represents the first determination of salivary suPAR concentration in a larger well-defined adolescent population. Our results suggest the potential for clinical use of suPAR in saliva as an inflammatory risk indicator/biomarker of periodontitis., (© 2018 Eur J Oral Sci.)

Aims: The aims of the present study were to assess the prevalence of periodontal disease and Helicobacter pylori (H. pylori) infection and their associations within a predefined Indian population., Methods: A community-based cross-sectional study of 500 selected individuals using a questionnaire, oral examination, rapid urease testing of dental plaque, and serological examination for immunoglobulin G antibody to H. pylori was carried out., Results: Periodontal disease and H. pylori infection were prevalent in more than 50% of the population. Age, smoking, and diabetic status of the individuals were risk factors for periodontal disease after multivariate analysis, and a lack of proper sewage and waste disposal facilities were found to increase the risk of H. pylori infection. Although there was no association between periodontal disease and H. pylori seropositivity in the community, a highly-significant association was found between periodontal disease and colonization of H. pylori in dental plaque., Conclusions: Because periodontal disease is associated with the increased colonization of H. pylori, new treatment modalities, such as plaque control measures, should be employed for the complete management of H. pylori-associated gastric disease., (© 2014 Wiley Publishing Asia Pty Ltd.)

Objective: The aim of this paper was to study pH conditions between dental sites, taking account the presence of caries, calculus, and microbial composition and alkali production., Materials and Methods: One hundred 13-year-old Thai schoolchildren were recorded for caries experience (DMFT, DT), calculus, plaque, and gingivitis. Ex vivo urease activity was measured on 11, 26, 31, and 46 (distal aspect) with the rapid urease test and pH at baseline and after rinse with 0.25 % urea solution on mesial site in vivo. Interproximal plaque from contralateral teeth was microbiological analysed with the checkerboard technique., Results: Thirty-four children were caries free. Plaque and calculus were abundant; all children showed a high resting plaque pH and the mandibular incisor showed significantly (p < 0.01) higher pH at baseline, max pH and AOC7.0 after urea challenge, ex vivo urease activity and calculus but lower caries experience than other teeth. A significant inverse correlation (p < 0.02) was found between caries frequency and ex vivo urease activity for tooth 11. Anaerobes predominated over streptococci, but no significant differences between dental sites were found., Conclusions: The study group had a high baseline plaque pH, in vivo and ex vivo urease activity, and calculus but low caries experience, which was best reflected in the lower incisor region., Clinical Relevance: Urease activity and pH on site level may be important determinants for individuals at caries risk.

Objectives: This study investigated the relationship between urease and arginine deiminase system (ADS) activities and dental caries through a cross-sectional study., Material and Methods: Urease and ADS activities were measured in saliva and plaque samples from 10 caries-free subjects and 13 caries-active. Urease activity was obtained from the ammonia produced by incubation of plaque and saliva samples in urea. ADS activity was obtained from the ammonia generated by the arginine-HCl and Tris-maleate buffer. Specific activity was defined as micromoles of ammonia per minute per milligram of protein. Shapiro-Wilk statistical test was used to analyze the distribution of the data, and Mann-Whitney test was used to determine the significance of the data., Results: The specific urease activity in saliva and plaque was significantly higher in individuals with low DMFT scores. ADS activity in saliva (6.050 vs 1.350, p=0.0154) and plaque (8.830 vs 1.210, p=0.025) was also higher in individuals with low DMFT scores., Conclusions: Caries-free subjects had a higher ammonia generation activity by urease and arginine deiminase system for both saliva and plaque samples than low caries-active subjects. High levels of alkali production in oral environment were related to caries-free subjects.

Arginine metabolism by oral bacteria via the arginine deiminase system (ADS) increases the local pH, which can neutralize the effects of acidification from sugar metabolism and reduce the cariogenicity of oral biofilms. To explore the relationship between oral arginine metabolism and dental caries experience in children, we measured ADS activity in oral samples from 100 children and correlated it with their caries status and type of dentition. Supragingival dental plaque was collected from tooth surfaces that were caries-lesion-free (PF) and from dentinal (PD) and enamel (PE) caries lesions. Regardless of children's caries status or type of dentition, PF (378.6) had significantly higher ADS activity compared with PD (208.4; p < .001) and PE (194.8; p = .005). There was no significant difference in the salivary arginolytic activity among children with different caries status. Mixed-model analysis showed that plaque caries status is significantly associated with ADS activity despite children's age, caries status, and dentition (p < .001), with healthy plaque predicting higher ADS activity compared with diseased plaque. Plaque arginine metabolism varies greatly among children and tooth sites, which may affect their susceptibility to caries.

Alkali production by oral bacteria is believed to have a major impact on oral microbial ecology and to be inhibitory to the initiation and progression of dental caries. A substantial body of evidence is beginning to accumulate that indicates the modulation of the alkalinogenic potential of dental biofilms may be a promising strategy for caries control. This brief review highlights recent progress toward understanding molecular genetic and physiologic aspects of important alkali-generating pathways in oral bacteria, and the role of alkali production in the ecology of dental biofilms in health and disease.

Unlabelled: Bacterial urease activity in dental plaque and in saliva generates ammonia, which can increase the plaque pH and can protect acid-sensitive oral bacteria. Recent cross-sectional studies suggest that reduced ability to generate ammonia from urea in dental plaque can be an important caries risk factor. In spite of this proposed important clinical role, there is currently no information available regarding important clinical aspects of oral ureolysis in children., Objective: The objective of this study was to evaluate the distribution and pattern of urease activity in the dental plaque and in the saliva of children during a three-year period, and to examine the relationship of urease with some important caries risk factors., Methods: A longitudinal study was conducted with repeated measures over a three-year period on a panel of 80 children, aged 3-6 years at recruitment. The dynamics of change in urease activity were described and associated with clinical, biological, and behavioural caries risk factors., Results: Urease activity in plaque showed a trend to remain stable during the study period and was negatively associated with sugar consumption (P<0.05). Urease activity in unstimulated saliva increased with age, and it was positively associated with the levels of mutans streptococci in saliva and with the educational level of the parents (P<0.05)., Conclusions: The results of this study reveal interesting and complex interactions between oral urease activity and some important caries risk factors. Urease activity in saliva could be an indicator of mutans infection in children., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Infection is a risk factor for preterm birth. This study was conducted in the field and addressed the link between periodontal pathogens measured with the benzoyl-DL-arginine-naphthylamide (BANA) test and preterm birth., Methods: This prospective study was performed in Changhua, Taiwan. Periodontal examinations included the plaque index, papillary bleeding scores, and measurement of the BANA enzyme in plaque samples at the second and third trimesters. Independent variables included maternal demographic characteristics, previous pregnancy histories, risk factors, plaque and gingivitis scores, and current pregnancy outcomes., Results: There were 19 (7%) preterm deliveries among the 268 subjects. A history of a previous preterm birth and low birth weight, frequency of prenatal visits, preterm uterine contractions, antepartum hemorrhages, placenta previae, and preterm premature rupture of membranes were significantly related to preterm birth (P = 0.035, 0.027, <0.001, 0.025, 0.006, 0.014, and <0.001, respectively). Maternal weight gain was higher with a normal term delivery (P = 0.003). Multivariable logistic regression analyses showed that the number of BANA-infected sites in the third trimester (odds ratio [OR]: 5.89; 95% confidence interval [CI]: 1.5 to 31.6), maternal weight gain (OR: 0.78; 95% CI: 0.65 to 0.91), antepartum hemorrhages (OR: 10.0; 95% CI: 2.2 to 46.9), and preterm premature rupture of membranes (OR: 12.6; 95% CI: 3.97 to 42.71) had significant influences on preterm-birth outcomes., Conclusions: BANA-positive plaque in the third trimester was associated with preterm births after controlling for other risk factors. The BANA test can be used to screen pregnant women at chairside and/or bedside to apply suitable intervention tactics.

Background: Microbial denitrification is not considered important in human-associated microbial communities. Accordingly, metabolic investigations of the microbial biofilm communities of human dental plaque have focused on aerobic respiration and acid fermentation of carbohydrates, even though it is known that the oral habitat is constantly exposed to nitrate (NO3-) concentrations in the millimolar range and that dental plaque houses bacteria that can reduce this NO3- to nitrite (NO2-)., Results: We show that dental plaque mediates denitrification of NO3- to nitric oxide (NO), nitrous oxide (N2O), and dinitrogen (N2) using microsensor measurements, 15N isotopic labelling and molecular detection of denitrification genes. In vivo N2O accumulation rates in the mouth depended on the presence of dental plaque and on salivary NO3- concentrations. NO and N2O production by denitrification occurred under aerobic conditions and was regulated by plaque pH., Conclusions: Increases of NO concentrations were in the range of effective concentrations for NO signalling to human host cells and, thus, may locally affect blood flow, signalling between nerves and inflammatory processes in the gum. This is specifically significant for the understanding of periodontal diseases, where NO has been shown to play a key role, but where gingival cells are believed to be the only source of NO. More generally, this study establishes denitrification by human-associated microbial communities as a significant metabolic pathway which, due to concurrent NO formation, provides a basis for symbiotic interactions.

Objective: Dietary sugar exposures induce an immediate drop of the plaque pH. Based on in vitro observations, it was hypothesized that oral bacteria may rapidly respond to this environmental change by increasing the activity or expression of alkali-generating pathways, such as the urease pathway. The objective of this exploratory in vivo study was to determine the short-term effect of a brief sucrose exposure on plaque and saliva urease activity and expression, and to relate this effect to caries experience., Methods: Urease activity levels were measured in plaque and saliva samples collected from 20 children during fasting conditions and 30 min after rinsing with a sucrose solution. Streptococcus salivarius ureC-specific mRNA in saliva was quantified using real-time RT-PCR. The impact of host-related factors, such as age, gender, sugar consumption, salivary mutans streptococci levels and caries status on urease activity was evaluated., Results: Plaque urease activity under fasting conditions was higher in subjects with low caries and mutans streptococci levels. This difference was not observed after the sucrose exposure. The response of urease to sucrose in vivo did not depend on caries experience or salivary mutans levels. Significant increase in urease activity of plaque and saliva after exposure to sucrose was observed only in the subjects who had low urease levels at baseline., Conclusions: The findings of this exploratory study suggest that plaque urease activity may have an important long-term influence in caries development but not during a cariogenic challenge., (Copyright 2010 Elsevier Ltd. All rights reserved.)

This study investigated the relationship of arginine deiminase (ADS) and urease activities with dental caries through a case-control study. ADS and urease activities were measured in dental smooth-surface supragingival plaque and whole saliva samples from 93 subjects, who were in three different groups: caries-free (n = 31), caries-active (n = 30), and caries-experienced (n = 32). ADS activity was measured by quantification of the ammonia generated from the incubation of plaque and saliva samples in a mixture containing 50 mM arginine-HCl and 50 mM Tris-maleate buffer, pH 6.0. ADS-specific activity was defined as nanomoles of ammonia generated per minute per milligram of protein. Urease activity was determined by quantification of ammonia produced from 50 mM urea. For bacterial identification and enumeration real-time qPCR analysis was used. Groups were compared using Kruskal-Wallis tests. Spearman correlations were used to analyze plaque metabolic activity and bacterial relationships. The results revealed significantly higher ammonia production from arginine in saliva (1.06 vs. 0.18; p < 0.0001) and plaque samples (1.74 vs. 0.58; p < 0.0001) from caries-free subjects compared to caries-active subjects. Urease levels were about 3-fold higher in the plaque of caries-free subjects (p < 0.0001). Although higher urease activity in saliva of caries-experienced and caries-free subjects was evident, no significant difference was found between the groups., (Copyright © 2010 S. Karger AG, Basel.)

The degradation of complex substrates, like salivary mucins, requires an arsenal of glycosidases and proteases to sequentially degrade the oligosaccharides and polypeptide backbone. The mucin MUC5B is a complex oligomeric glycoprotein, heterogeneous in molecular mass (14-40 x 10(6) Da), with a diverse repertoire of oligosaccharides, differing in composition and charge. The aim of this study was to investigate whether proteolytic degradation of the mucin polypeptide backbone could be identified and if cooperation of dental biofilm bacteria was required. Cooperative bacteria-mediated proteolysis of MUC5B was determined by comparing individual species and mixed consortia of strains isolated from supragingival plaque, and freshly harvested supragingival plaque. Proteolytic activity was analysed using fluorescent labelled substrate and by visualizing mucin degradation by SDS-PAGE. Dental plaque degraded the polypeptide backbone of the salivary MUC5B mucin. The mucin was also degraded by a specific consortium of isolated species from supragingival plaque, although individual species and other consortia did not. Certain bacteria in supragingival dental plaque therefore cooperate as a consortium to proteolyse human salivary MUC5B and hydrolyse glycosides.

Introduction: The arginine deiminase system (ADS) of oral bacteria is a major generator of alkali (ammonia) in dental plaque and is considered to have anticaries effects. However, many of the antimicrobial agents used in oral care products may reduce alkali production by the ADS. The objective of our work was to assess the sensitivity of the ADS of oral streptococci to commonly used antimicrobials, fluoride, triclosan and organic weak acids., Methods: Streptococcus sanguinis NCTC 10904 and Streptococcus ratti FA-1 were grown in suspension cultures and mono-organism biofilms. ADS activity at pH values of 4, 5 and 6 was assessed, and the actions of the agents was determined in terms of reduced production of alkali from arginine, inhibition of ADS enzymes and changes in uptake of arginine., Results: ADS activity was not greatly affected by pH changes between 4 and 6 and was greater per unit of biomass for cell suspensions than for biofilms. NaF was a poor inhibitor, while triclosan was highly effective with a 50% inhibitory dose for the two organisms between 0.03 and 0.05 and between 0.10 and 0.15 mm-h for suspension cells and biofilms, respectively. The weak acid indomethacin was nearly as potent at pH 4.0 as triclosan, while capric and lauric acids were less potent, especially for biofilms. The methyl ester of lauric acid was slightly stimulatory. The major targets for the inhibitors appeared to be transport systems for arginine uptake, although carbamate kinase was a secondary target., Conclusion: Triclosan, indomethacin, caprate and laurate can reduce ADS activity in dental plaque.

Background/aim: Alkali generation by oral bacteria plays a key role in plaque pH homeostasis and may be a major impediment to the development of dental caries. To determine if the capacity of oral samples to produce ammonia from arginine or urea was related to caries experience, the arginine deiminase system (ADS) and urease activity in saliva and dental plaque samples were measured in 45 adult subjects., Methods: The subjects were divided into three groups according to caries status; 13 caries-free (CF) individuals (decayed, missing, and filled teeth = 0); 21 caries-active (CA) individuals (decayed teeth >or= 4); and 11 caries-experienced (CE) individuals (decayed teeth = 0; missing and filled teeth > 0). Real-time polymerase chain reaction was used to quantify the proportion of certain acid- or alkali-producing organisms in the samples., Results: The amount of ammonia generated from the test substrates by plaque samples was generally higher than that produced by salivary samples in all groups. Significantly higher levels of salivary ADS activity and plaque urease activity were observed in CF subjects compared to CA subjects (P = 0.0004 and P = 0.014, respectively). The proportions of Streptococcus mutans from saliva and dental plaque of CA subjects were significantly higher than those from the CF group (P = 0.0153 and P = 0.0009, respectively). In the CA group, there was an inverse relationship between urease activity and the levels of S. mutans (P < 0.0001)., Conclusion: This study supports the theory that increased caries risk is associated with reduced alkali-generating capacity of the bacteria colonizing the oral cavity; providing compelling evidence to further our understanding of oral alkali-generation in health and disease.

The innate host response system is comprised of various mechanisms for orchestrating host response to microbial infection of the oral cavity. The heterogeneity of the oral cavity and the associated microenvironments that are produced give rise to different chemistries that affect the innate defense system. One focus of this review is on how these spatial differences influence the two major defensive peroxidases of the oral cavity, salivary peroxidase (SPO) and myeloperoxidase (MPO). With hydrogen peroxide (H(2)O(2)) as an oxidant, the defensive peroxidases use inorganic ions to produce antimicrobials that are generally more effective than H(2)O(2) itself. The concentrations of the inorganic substrates are different in saliva vs. gingival crevicular fluid (GCF). Thus, in the supragingival regime, SPO and MPO work in unison for the exclusive production of hypothiocyanite (OSCN(-), a reactive inorganic species), which constantly bathes nascent plaques. In contrast, MPO is introduced to the GCF during inflammatory response, and in that environment it is capable of producing hypochlorite (OCl(-)), a chemically more powerful oxidant that is implicated in host tissue damage. A second focus of this review is on inter-person variation that may contribute to different peroxidase function. Many of these differences are attributed to dietary or smoking practices that alter the concentrations of relevant inorganic species in the oral cavity (e.g.: fluoride, F(-); cyanide, CN(-); cyanate, OCN(-); thiocyanate, SCN(-); and nitrate, NO(3)(-)). Because of the complexity of the host and microflora biology and the associated chemistry, it is difficult to establish the significance of the human peroxidase systems during the pathogenesis of oral diseases. The problem is particularly complex with respect to the gingival sulcus and periodontal pockets (where the very different defensive stratagems of GCF and saliva co-mingle). Despite this complexity, intriguing in vitro and in vivo studies are reviewed here that reveal the interplay between peroxidase function and associated inorganic chemistry.

Introduction: Model systems with oral bacteria from dental plaque have demonstrated that the utilization of complex glycoproteins as a food source cannot be undertaken by single species but requires concerted degradation by a multi-species consortium, with each member contributing one or a few hydrolytic enzymes. Unlike previous studies, the aim of the present investigation was to explore the ability of fresh dental plaque to degrade salivary mucin, MUC5B, isolated by methods designed to retain intact the natural polymeric structure and physiological conformation, in an attempt to mimic the naturally occurring interaction between the oral microbiota and salivary mucins., Methods: Human salivary MUC5B was isolated from whole saliva by density-gradient centrifugation and incubated with freshly isolated supragingival dental plaque with samples subjected to fluorescent staining for viability and metabolic activity. In addition, the degradation of MUC5B oligosaccharide side chains was studied using a lectin assay, recognizing three different carbohydrate epitopes commonly found on mucin oligosaccharide side chains., Results: The addition of purified salivary MUC5B elicited a strong metabolic response from the biofilm cells, whereas individual strains of Streptococcus oralis and Streptococcus gordonii isolated from the same plaque were not able to utilize the MUC5B. The degradation of terminal saccharide moieties on the MUC5B was demonstrated by a marked decrease in both sialic acid and fucose reactivity., Conclusion: These results have shown that dental plaque is capable of utilizing human salivary MUC5B as a nutrient source, a process possibly requiring the synergistic degradation of the molecule by a consortium of oral bacteria in the plaque community.

Aim: To investigate the effect of the collagenase gene (prtC) product of Porphyromonas gingivalis on inducing host cells to secrete inflammatory cytokines, and to discuss the correlation between the PrtC level in subgingival plaque samples and clinical parameters., Methods: A prokaryotic expression system pET32a-prtC-Escheria coli BL21DE3 was constructed. Antigenicity and immunoreactivity of the recombinant PrtC protein (rPrtC) was identified by Western blotting. ELISA was applied to detect interleukin (IL)-1alpha, IL-8, and TNF-alpha levels in supernatants from rPrtC-induced human umbilical vein endothelial cells (HUVEC) originated ECV304 cells. Clinical parameters recorded at baseline and after treatment included bleeding on probing (BOP), probing depth (PD), and attachment loss (AL). ELISA was established to measure the PrtC level in 196 subgingival plaque samples from 49 patients with chronic periodontitis., Results: After coincubation with 1 microg/mL rPrtC for 24 h and with 5 or 10 microg/mL rPrtC for 12 h, the levels of IL-1 alpha, IL-8, and TNF-alpha secreted by the ECV304 cells increased significantly (P<0.05). The PrtC level in the BOP-positive or the > or =5 mm AL or > or = 6 mm PD sites was higher than that in the BOP-negative or the < or =2 mm AL or < or =6 mm PD sites (P<0.05), respectively. Compared with baseline, the PrtC levels in different AL sites or in the < or =6 mm PD pockets decreased remarkably after treatment (P<0.01), but in the BOP-positive or in the > 6 mm PD sites, the PrtC levels changed insignificantly (P>0.05)., Conclusion: rPrtC is able to directly induce host cells to synthesize and secrete IL-1 alpha, IL-8, and TNF-alpha. The PrtC level in subgingival samples is correlated with BOP, AL, and PD.

Background: The aim of this clinical trial was to compare the outcome of non-surgical treatment of interproximal and non-interproximal Class II furcation involvements., Methods: Thirty-eight patients presenting at least one Class II furcation involvement that bled on probing with a probing depth (PD) > or = 5 mm were recruited. Furcation involvements were grouped as either buccal and lingual furcation involvements (BLFI) or interproximal furcation involvements (IFI). The following clinical outcomes were evaluated: visible plaque index, bleeding on probing (BOP), position of the gingival margin, relative attachment level (RAL), PD, and relative horizontal attachment level (RHAL). N-benzoyl-l-arginine-p-nitroanilide (BAPNA) testing was used to analyze trypsin-like activity in dental biofilm. All parameters were evaluated at baseline and 1, 3, and 6 months after non-surgical subgingival instrumentation., Results: Six months after treatment, both groups had similar means of RAL and RHAL gain (P >0.05). These variables were 1.22 and 1.07 mm in the IFI group and 1.38 and 1.20 mm in the BLFI group, respectively. The PD reduction was significantly greater in the BLFI group than in the IFI group (2.59 and 2.11 mm, respectively; P <0.05). The BLFI group presented fewer sites with PD > or = 5 mm than the IFI group at all post-treatment periods. At 6 months, the BAPNA test showed that only the BLFI group had values significantly different from baseline. This means that the BLFI group had significantly lower BAPNA values compared to the IFI group at 6 months., Conclusion: Buccal and lingual Class II furcation involvements respond better to non-surgical therapy compared to interproximal Class II furcation involvements.

Introduction: Ammonia production from the metabolism of urea by urease enzymes of oral bacteria moderates plaque acidification and may inhibit dental caries, as suggested by in vitro studies and indirect clinical observations. The objective of this study was to examine the relationship of urease activity with dental caries at the clinical level., Methods: Urease activity was measured in dental plaque and saliva samples from 25 caries-free subjects (CF) and in eight subjects with six or more open caries lesions (CA). Plaque and saliva collection was repeated for each subject 1 week later using identical procedures., Results: Urease-specific activity in the dental plaque of CF subjects was significantly higher compared to that in the subjects with caries. The association of low plaque urease levels with increased caries was further supported by odds ratio analysis using different plaque urease cut-off points. Using a receiver operating characteristic curve it was estimated that there was an approximately 85% probability of correctly classifying the subjects as CA or CF based on the relative ordering of their plaque urease activity levels. No statistically significant differences were observed in salivary urease activity., Conclusion: This study suggests that loss of alkali-generating potential of tooth biofilms via the urease pathway has a positive relationship to dental caries.

Helicobacter pylori infection is common in people. However, the existence of extra gastric reservoirs and transmission routes remain controversial in the field. Because the oral cavity has been proposed as a reservoir for H. pylori, a study was carried out to determine the presence of H. pylori in dental plaque and saliva. The results were asociated with those obtained in the gastric biopsy. Ninety-seven dyspeptic and fifty asymptomatic patients were studied and samples taken for biopsy, dental plaque and saliva. The gastric biopsies were evaluated using microbiology and histology methods. Cultures and urease tests were carried out on the oral cavity samples and included pretreatment methods using urea and HCl. The frequency of H. pylori for all the patients evaluated was 75.5%. H. pylori was not isolated in saliva or dental plaque in any of the two groups studied with or without sample pretreatment. The urease test in dental plaque was positive in 99.3% of the patients and 89.8% in saliva. There was no statistically significant difference between the infection prevalence by H. pylori in dyspeptic or not dyspeptic patients. The obtained results suggest that the methodology used for the detection of H. pylori is not sufficiently sensitive for the determination of the microorganism in the oral cavity.

Background: The aim of the present study was to evaluate the clinical effects of one-stage periodontal debridement with an ultrasonic instrument, associated with 0.5% povidone (pvp)-iodine irrigation in patients with chronic periodontitis., Methods: Forty-five patients were randomly assigned into three groups: the control group (CG) received quadrant root planing at 1-week intervals over four consecutive sessions; the periodontal debridement plus pvp-iodine group (PD-PIG) received a 45-minute full-mouth debridement with an ultrasonic instrument, associated with 0.5% pvp-iodine irrigation; and the periodontal debridement group (PDG) received a 45-minute full-mouth periodontal debridement with an ultrasonic instrument, associated with NaCl irrigation., Results: At the 3-month evaluation, the mean probing depth (PD) reduction in CG was 2.51+/-0.52 mm, 2.53+/-0.50 mm in PD-PIG, and 2.58+/-0.60 mm in PDG (P<0.05). The clinical attachment level (CAL) analysis showed a statistically significant gain in all groups compared to baseline (1.87+/-0.56 mm [CG], 1.94+/-0.70 mm [PD-PIG], and 1.99+/-0.92 mm [PDG]). Intergroup analysis of PD and CAL at 1 and 3 months showed no differences (P>0.05). The N-benzoyl-L-arginine-p-nitroanilide (BAPNA) test showed a significant reduction in trypsin activity only during the first month (P<0.05); at 3 months there were no differences compared to baseline (P=0.80)., Conclusion: This study provides no evidence that pvp-iodine is effective as an adjunct for one-stage periodontal debridement.

Purpose: The aim of this investigation was to determine the presence of aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in crevicular fluid collected from implants with and without clinical and radiographic signs of peri-implantitis., Materials and Methods: There were 17 implants with symptoms of peri-implantitis in 12 subjects, including 4 females and 8 males, compared to 17 implants in 13 subjects, including 5 females and 8 males, with healthy peri-implant tissues. Filter paper strips were used to collect peri-implant crevicular fluid for 30 seconds in the base of the crevice/pocket. SPSS statistical software (SPSS, Inc., Chicago, IL) was used to determine AST and ALP activity., Results: The results showed that there was a significant difference in the activity of AST and ALP between the 2 study groups (P < 0.0001). AST activity was significantly associated with the amount of bleeding on probing (P = 0.02), but no statistical correlation was found between ALP activity and increased amount of bleeding on probing (P = 0.05)., Conclusions: Within the limits of this study, our results may suggest that peri-implant crevicular fluid analysis could be further investigated in longitudinal studies as a suitable diagnostic strategy in the evaluation of dental implants.

Objective: Carbonic anhydrase (CA) VI is a unique secreted isozyme of CA, which catalyzes the reversible reaction CO2 +H2O<-->H+ +HCO3-. CA VI has been thought to provide a greater buffering capacity to fluids into which it is secreted. This study was performed to confirm this in saliva., Design: Nine healthy subjects participated in the study. The pH of the dental plaque from each subject was monitored after a mouth rinse with 10% sucrose with or without 10(-5)M acetazolamide, a specific inhibitor of CA. Also CA was examined in plaque by enzyme histochemistry, immunohistochemistry and Western blot analysis., Results: Though sucrose and sucrose plus inhibitor yielded Stephan curves with a similar temporal pattern, the pH values of the latter were significantly lower than those of the former. Plaque exhibited CA activity by enzyme histochemistry. Immunohistochemistry and Western analysis demonstrated that the activity was due to CA VI but not to CA I or CA II., Conclusions: The results indicate that CA VI in saliva penetrates plaque and facilitates acid neutralization by salivary bicarbonate. Therefore, CA VI may be considered an anti-caries protein in saliva.

This study uses a cross-sectional design to examine the endothelial and inducible nitric oxide synthase (eNOS and iNOS, respectively) levels of gingival tissue. Fifteen subjects, 10 female and 5 male individuals (aged 14.6-21.2 years; mean 17.4 +/- 1.8 years), who needed extraction of the four first premolars for orthodontic reasons and who had indications for a gingivectomy were enrolled in the study. In each patient, two maxillary/mandibular premolars were extracted, and two months later an orthodontic appliance was placed in the same arch. A canine undergoing treatment for distal movement served as the test tooth (TT), whereas its contralateral canine was used as the control tooth (CT). The CT was included in the orthodontic appliance but was not subjected to the orthodontic force. Two weeks after the orthodontic appliance placement, clinical data consisting of the presence of supragingival plaque, bleeding on probing, and probing depth were collected from each experimental tooth. Immediately after, gingival tissue was collected from the distal aspect of each TT and CT for immunohistochemistry, messenger RNA reverse transcription by polymerase chain reaction, and Western blot analysis for both eNOS and iNOS. The results showed that no differences in clinical conditions occurred between the experimental teeth. On the contrary, both the eNOS and iNOS levels and the expression of the TTs were significantly greater than those of the CTs (all comparisons significant to P < .01). Our results indicate a role for gingival eNOS and iNOS during the early phases of orthodontic treatment in humans.

Background: The objective of this study was to evaluate the effect of three commercial anti-plaque dentifrices containing 0.3% triclosan + 2% pvm/ma (Colgate Total), 0.3% triclosan + 0.75% Zn (Signal Global) and 0.3% triclosan + 5% PPi (Crest Complete) in comparison with an experimental dentifrice (0.3% triclosan + 2% pvm/ma + 0.75% Zn + 4% PPi) and a control dentifrice without anti-plaque agents on trypsin-like activity in dental plaque (detected by the hydrolysis of [Na-Benzoyl-DL-Anginine p-Nitroanilide (BAPNA)] and volatile sulphur compounds (VSCs) in mouth air during experimental gingivitis development., Method: A 5-step double blind, crossover experimental gingivitis study was conducted on 19 volunteers during a 21-day period. The volunteers refrained from brushing an experimental quadrant of teeth. The dentifrices were applied to those teeth via toothshield three times per day; simultaneously they brushed the other teeth with the same dentifrice. After each period, VSCs in mouth air and BAPNA hydrolysis by dental plaque accumulated in the experimental quadrant were determined., Results: There was an increase (p < 0.05) in VSCs in mouth air when experimental gingivitis was induced in only one quadrant of teeth. None of the dentifrices was able to avoid the increase of VSCs during the experimental gingivitis development. The majority of the antiplaque dentifrices evaluated reduced the increase of VSC formation in comparison with the control (p < 0.05). There was no relationship between the ability of the dentifrices in reducing VSC formation and the inhibition of trypsin-like activity in dental plaque., Conclusions: Anti-plaque dentifrices reduce the increase of VSCs that occurs during the development of experimental gingivitis.

The growth and pathogenic properties of dental plaque result from interactions between the microbiota and the oral environment and have been studied in laboratory experimental systems ranging from single or a few species (such as in chemostats) to dental plaque microcosms. Microcosm plaque is an in vitro version of natural plaque and has been explored as a microflora model because it is sited a more manipulable and controllable environment. It is obtained as microcosm biofilms in an 'artificial mouth' plaque culture system by culturing the bacteria in natural plaque-enriched saliva (i.e. salivary bacteria where a whole-saliva donor has abstained from oral hygiene for 24 h to increase the plaque bacteria in the saliva). The aim here was to examine whether a new, chemically defined analogue of saliva (defined medium mucin, DMM) could substitute for a previously used, chemically undefined medium (basal medium mucin, BMM) as an analogue of saliva for large-scale biofilm culturing. DMM contains various ions, mucin, amino acids, vitamins and growth factors at concentrations generally similar to those in saliva, whereas BMM contains yeast extract, peptones and mucin. To model the nutrient functions of salivary proteins, amino acids equivalent to 5 g/l casein were also included in DMM. In earlier studies, BMM-grown plaques were similar to natural plaques in structure, composition, growth rate and pH response to substrates. Their doubling-time patterns over a 20-day period were similar, except that the DMM-grown plaques showed biphasic growth patterns that were more pronounced than with BMM. Variation in enzyme profiles between BMM- and DMM-grown plaque, measured using the API-ZYM technique, provided evidence of nutritional effects on plaque composition. It was concluded that realistic growth rates and patterns are generated in microcosm plaque biofilms by supplying both DMM and BMM. However, the use of DMM enables specific modifications to be made to nutrient conditions during large-scale culture in our 'artificial mouth' biofilm system.

Fluorescence polarization (FP) was examined as a rapid quantitative method to assay the proteases in subgingival plaque. Protease activity was measured by a decrease in FP at 0.5-min intervals over 5 min, using BODIPY-alpha-casein, a protein substrate. To quantitate activity, the least absolute deviation (LAD) slope for each assay was determined. Protease activity increased with the quantity of plaque (r=0.416, P<0.001). Of the 208 subgingival plaque samples, 87 contained detectable protease activity, with a mean of about 4 microg trypsin equivalents above a general background of 1 microg per site. The mean plaque protease activity of 89 paired samples from 15 individuals had decreased by 1.1 microg trypsin equivalents per site when measured at 8 months after tooth scaling and root planing (P<0.01). Most isolates of Porphyromonas gingivalis, Treponema denticola, Prevotella nigrescens, and Prevotella intermedia implicated in the pathogenesis of adult periodontitis exhibited high activity in the FP assay. The assay is rapid, quantitative and requires only one-tenth of the plaque sampled using a single pass with a Gracey curette at a single tooth site.

Background: The goal of this study was to see whether Helicobacter pylori (H. pylori) in the oral cavity might adversely affect the outcome of eradication therapy for gastric H. pylori., Materials and Methods: Forty-seven patients (36 males, 11 females) with gastric H. pylori infection were enrolled in this study. Gastric H. pylori infection was confirmed by both immunohistological staining with anti-H. pylori antibody and bacterial culture of biopsy specimens. The therapeutic regimen consisted of 30 mg/day lansoprazole, 750 mg/day metronidazole, and 400 mg/day clarithromycin administered for 2 weeks. A fragment of the H. pylori urease gene was amplified by nested PCR for DNA extracted from saliva and dental plaque from the same patients. We examined the correlation between the gastric eradication success rate and the prevalence of H. pylori in the oral cavity as determined by PCR before and after the eradication therapy., Results: The eradication success rate was significantly lower in the oral H. pylori-positive cases (12/23, 52.1%) than in the negative cases (22/24, 91.6%) at 4 weeks after the therapy (p =. 0028). Two years later, only 16 of the 23 (69.5%) oral H. pylori-positive cases were disease-free, as compared to 23 of the 24 (95.8%) oral H. pylori-negative cases (p =.018)., Conclusions: H. pylori in the oral cavity affected the outcome of eradication therapy and was associated with a recurrence of gastric infection. We recommend that oral H. pylori should be examined by nested PCR and, if positive, should be considered a causal factor in refractory or recurrent cases.

This study was intended to explore the relationship between the proteolytic enzymes in dental plaque and the patient's age and sex. Fluorometry and spectrometry were used to separately determine the activities of leucine amino peptidase, dipeptidyl peptidase IV and trypsin-like proteinase in children(10 boys and 10 girls), adults(n = 20) and elderly people(n = 10). The results showed that sex almost had no influence on the activities of the three enzymes in dental plaque in the children(P > 0.05), and with the increase of age, the activities of the three enzymes apparently increased (P < 0.01). These findings imply that the proteolytic enzymes may only play important roles in root lesions.

Objective: The purpose of this paper was to study the role of proteolytic enzymes in dental plaque in deciduous caries occurrence., Methods: Fluorometry and spectrometry were used separately to determine the activities of leucine amino peptidase, dipeptidyl peptidase IV and trypsin-like proteinase in caries-positive and caries-free children., Results: The activities of the three enzymes in dental plaque in different children were same (P > 0.05)., Conclusion: The three enzymes may be not related to deciduous caries.

Thirty-one strains of 23 gram-negative oral bacterial species were examined for dextran-degrading activity on agar plates containing blue dextran. One strain each of Capnocytophaga ochracea, Capnocytophaga sputigena, Prevotella loescheii, Prevotella melaninogenica and Prevotella oralis had detectable dextranase activity. The culture supernatants of P. melaninogenica and P. oralis cells contained dextranases of multiple sizes, but those of the other three species had a single size of enzyme. A 56-kDa dextranase was purified from the culture supernatant of P. oralis and the antiserum against the enzyme was prepared with a rabbit. The Ouchterlony test showed that the antibody reacted with the supernatants of both P. melaninogenica and P. oralis but not with the others. Dot-blot hybridization using the dextranase gene of Streptococcus mutans as a probe revealed that there was no significantly homologous sequence in the chromosomal DNA of the five species.

The purpose of this study was to investigate the types and activities of proteolytic enzymes in human root surface plaque. Sephadex G-200 gel filtration, fluorometry and spectrometry were used to isolate, purify and demonstrate the activities of proteolytic enzymes. The results indicated that three enzymes were present in the root surface plaque, namely leucine amino peptidase, dipeptidyl peptidase i.v. and trypsin-like proteinase, and their activities were different.

Analysis of beta-glucuronidase (betaG) in the gingival crevicular fluid (GCF) provides an indication of neutrophil influx into the crevicular environment. The aim of this study was to test the hypotheses that: (1) betaG is significantly elevated in individuals with early-onset periodontitis (EOP) and that betaG activity correlates with disease severity; and (2) betaG level may reflect the local bacterial challenge in the gingival crevice. The study subjects consisted of a sub-sample of individuals examined in the National Survey of Oral Health of United States Children, which was undertaken during the 1986/87 school year. A total of 249 individuals were selected based on presence or absence of clinical attachment loss at baseline. The individuals were examined a second time 6 years later and the clinical attachment loss was assessed, and subgingival plaque and GCF were collected. The subjects were classified into 3 types of EOP and a control group. BetaG activity in the GCF and the levels of 7 putative micro-organisms in the pocket were assessed. The generalized EOP group had the highest betaG activity, followed by the localized and incidental EOP groups, and the controls, respectively. There was a significant increase in betaG activity with the increase in probing depth. Also, sites with bleeding on probing had a significantly higher betaG activity than sites without bleeding. However, the effect of gingival inflammation on betaG activity was more evident in the generalized and localized EOP groups. Sites harboring high levels of one or more of the micro-organisms tended to have high betaG activity. There were moderate differences between the organisms with respect to their effect on betaG activity, but sites with high numbers of Porphyromonas gingivalis, Prevotella intermedia, or Treponema denticola also had the highest betaG activity. The present findings suggest that betaG activity in GCF from patients with EOP can be of value in the early identification of individuals at higher risk of developing EOP The findings also suggest that host mechanisms leading to higher betaG activity in EOP represent systemic responses and are only partly related to the presence of local factors at the site-level.

The potential of bacterial markers for the detection of periodontal disease activity is discussed. Chronic periodontitis has been associated with subgingival bacteria. Techniques are available to detect and distinguish different bacteria. In subgingival plaque, bacterial proteases can be detected. Commercial diagnostic tests based on bacterial factors are described.

Plaque samples were collected from tooth surface by the sampling method in 28 volunteers (two groups, 14 young people, ages ranged from 19-26 years, mean: 22.29 years old; 14 old people, aged between 60-70 years, mean: 64.35 years old, DMFT > 3). The activity for three enzymes including amylases, LDH, GTF was tested by micro-chemistry enzyme analysis method. The results showed that there were no significant difference for the activity of three enzymes in two groups (P > 0.05). The finding suggests that dental plaque is a specific microecologic environment and cariogenic factor, age does not affect the activity of enzymes, plaque in different ages has similar cariogenicity.

In 23 untreated adult periodontitis patients, the occurrence of beta-lactamase producing periodontal bacteria was determined. In addition to non-selective isolation media, selective isolation and growth of beta-lactamase positive subgingival bacterial species was carried out on blood agar plates supplemented with amoxicillin and plates with amoxicillin+clavulanic acid. Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, Peptostreptococcus micros, Fusobacterium nucleatum, Bacteroides forsythus and Campylobacter rectus isolates from the non-selective medium were tested for beta-lactamase activity by a nitrocefin disk method (DrySlide) and by a laboratory chromogenic nitrocefin-based test. Isolates from the amoxicillin plates that were absent on the amoxicillin/clavulanic acid plates were identified and tested for beta-lactamase production. Based on the non-selective plates, six of 23 P. intermedia isolates, 2 of 19 B. forsythus isolates and 3 of 23 F. nucleatum isolates were beta-lactamase positive. The beta-lactamase positive species Prevotella loescheii, Prevotella buccae, Prevotella buccalis and Actinomyces spp were recovered from the selective amoxicillin plates. beta-Lactamase positive subgingival species were recovered from 17 of 23 patients (74%) but usually comprised low proportions of the subgingival microbiota (range < 0.01-15%). Comparison of the DrySlide test and the nitrocefin-based laboratory test revealed full agreement of test results. beta-Lactamase activity in whole subgingival plaque was detected in 12 patient samples (52%). It was concluded that beta-lactamase activity in subgingival bacteria in adult periodontitis is a common feature. However, since the majority of the samples showed only low-level enzymatic activity, the clinical relevance of this observation with regard to therapy with unprotected enzyme-susceptible beta-lactams is uncertain, though failure on the other hand, is difficult to rule out when a mechanism of resistance is present. The majority of beta-lactamase positive strains was found among species of the Prevotella genus.

Xylitol reduces plaque but the reduction mechanism is largely unknown. The main aim of the present study was to determine whether the xylitol-induced reduction in the amount of plaque and the number of mutans streptococci could be demonstrated in subjects with (presumably) high levels of xylitol-resistant (XR; not inhibited by xylitol) mutans streptococci acquired following previous xylitol consumptions. 37 healthy dental students participated in the double-blind study. All subjects had been uncontrolled, habitual consumers of xylitol-containing products for at least 1 yr before the study. A 1-month washout period was followed by a 2-week test period during which either xylitol, xylitol-sorbitol or unsweetened chewing gum base was chewed 3-5 x a day. Plaque and saliva samples were collected at baseline and at the 2-week point for determination of the amount of plaque, microbiological variables, and hydrolytic enzymes. Mixtures of xylitol and sorbitol seemed to perform equally well with respect to reduction in the amount of plaque but not the number of mutans streptococci. Thus, polyols were the active ingredients of chewing gums able to modulate the amount of plaque and its microbial composition. Xylitol reduced plaque with a mechanism which appeared not to be associated with the study-induced changes in the proportion (%) of mutans streptococci in plaque, the number of salivary mutans streptococci, the proportion of XR strains in plaque or saliva, or the hydrolytic enzyme activities of plaque.

The enzymatic degradation of sodium monofluorophosphate by whole saliva in patients with differing oral health status was compared Ten patients each with good or poor oral health were selected. Assessment of oral health status included indices of the amount of dental plaque, caries experience and counts of Streptococcus mutans and lactobacilli. Whole-saliva samples were collected under standardized conditions and monofluorophosphatase (MFPase) activity was measured in 2-ml saliva samples during 2 h of incubation at 37 C with 1 ml of monofluorophosphate solution (equivalent 31.5 mmol/1 F). MFPase activity was found in all the saliva samples. The values ranged from 0.6 to 17.8 nmol F/ml saliva per min. High MFPase activities correlated well with large amounts of plaque with the D component of the DMFT index and with high counts of salivary S. mutans and lactobacilli. The results show a wide range of MFPase activity in individuals and a statistically significant correlation (p < 0.05) between high MFPase activity and poor oral health status.

Perioscan requires a plaque sample to detect the presence of enzymes capable of degrading N-benzoyl-DL-arginine-2-naphthylamide (BANA) from relatively few anaerobic periodontal pathogens. Periocheck assays the presence of neutral proteases in crevicular fluid. The aim of this study was to compare these test kits with traditional clinical methods of detecting periodontal disease and to monitor the ability of the kits to reflect the response to initial therapy. 19 patients with moderately severe chronic periodontitis were seen before and after a course of oral hygiene and root instrumentation consisting of 4 appointments. Clinical measurements and test assays were collected at 5 diseased sites and 2 healthy sites in each subject. Complete data from 125 sites were available for statistical analysis. At baseline Periocheck had a sensitivity of 88% and a specificity of 61% whereas Perioscan had a sensitivity of 99% and a specificity of 55%, when related to the clinical diagnosis. A composite clinical assessment, based on improvement or deterioration of one whole unit change of the subjective clinical indices and 2 mm changes or greater in probing depth or probing attachment level, revealed 75 sites which improved following treatment, whereas 45 sites did not change and 5 sites deteriorated. The probability that the tests agreed with the clinical outcome after treatment, was calculated as 50.4% for Periocheck and 52% for Perioscan. The diagnostic kits did not reliably reflect the clinical assessment of periodontal disease in the cross sectional study, or the outcome following treatment.

Triclosan monophosphate is a phosphorylated derivative of the antimicrobial agent, triclosan. In comparison with triclosan, it is highly soluble in aqueous solutions. It is hypothesized that, within the oral environment, triclosan monophosphate (which may be devoid of antimicrobial activity) will be hydrolyzed into triclosan by the action of microbial phosphatases. The liberated triclosan may then exert antimicrobial activity. To test this hypothesis, we designed experiments to measure the phosphatase activity of plaque and selected species of oral micro-organisms and to demonstrate hydrolysis of triclosan monophosphate. Tests comparing the minimal inhibitory concentration and minimal bactericidal concentration of triclosan and triclosan monophosphate were also undertaken. Dental plaque and the majority of the bacterial strains tested showed phosphatase activity against p-nitrophenyl phosphate which peaked below neutral pH (acid phosphatases) or above neutral pH (alkaline phosphatases). Dental plaque showed the highest levels of alkaline phosphatase (optimum at pH 9.0) and relatively high levels of acid phosphatase (optimum at pH 6.0 to 6.5). Dental plaque and selected species of micro-organisms were all capable of hydrolyzing triclosan monophosphate, albeit at different rates. The minimal inhibitory concentration and minimal bactericidal concentration values for triclosan monophosphate against eight bacterial strains were always considerably higher than the corresponding values for triclosan. Addition of triclosan monophosphate to an established culture (ca. 10(9) cfu/mL) of Capnocytophaga gingivalis growing continuously showed that triclosan monophosphate was rapidly hydrolyzed into triclosan with concomitant loss of total bacterial viability. It is therefore likely that triclosan monophosphate will be broken down into triclosan within the oral environment with concomitant antimicrobial activity.

The purpose of this study was to investigate how levels of gingival crevicular fluid (GCF) alkaline phosphatase (ALP) change in relation to levels of plaque and gingival inflammation in 20 adults during a 21 day period of experimental gingivitis. The source of ALP within GCF was also investigated using a repeat sampling protocol; by determining enzyme levels derived from 30 putative periodontal pathogenic and non-pathogenic species; and by examining inhibition profiles from a variety of host and bacterial ALP isoenzymes. Total 30-s GCF ALP levels increased significantly (p < 0.002) during experimental gingivitis and preceded an increase in gingival index (GI) by approximately 7 days. Enzyme levels correlated with GCF volume (R = 0.7; p < 0.0001), but repeat sampling indicated that entry of ALP into the gingival crevice was independent of the rate of fluid flow. Only 5 of the bacterial species investigated produced clearly detectable levels of ALP in culture supernatants, these were P. gingivalis (381), P. intermedia (581), P. nigrescens (8944), Dentin P. gingivalis (TW 471: clinical isolate) and C. ochracea (25). Levamisole inhibition and studies on suspensions of washed plaque demonstrated that host-derived ALP contributed to > 80% of the enzyme in GCF. We conclude that elevated 30-s GCF ALP levels measured using the chemiluminescent assay reported, are detectable before increases in gingival indices and appear to be a better marker of gingival inflammation than ALP concentrations. The major source of ALP within GCF is host derived and in early inflammatory disease is likely to be of polymophonuclear leukocyte origin.

The presence, localization and activities of cathepsin G in gingival tissue specimens and crevicular fluid (GCF) from 9 adult periodontitis patients and 6 controls with clinically healthy periodontium were studied by use of avidinbiotin-peroxidase complex method, Western and dot blotting, and spectrophotometric activity assay. In contrast to healthy gingival tissue specimens, gingival tissue specimens collected from adult periodontitis patients contained inflammatory cells in lamina propria, beneath the oral sulcular epithelium, 10-50% of which were cathepsin G positive polymorphonuclear neutrophilic leukocytes (PMNs) and monocyte/macrophage-like cells. Cathepsin G activities were increased in adult periodontitis GCF when compared to periodontally healthy controls' GCF (p < 0.05). In adult periodontitis GCF, Western blotting disclosed free cathepsin G but also clear complexes of cathepsin G with its predominant endogenous inhibitor alpha 1-antichymotrypsin (alpha 1-ACT). The present results demonstrate that part of the cathepsin G, despite the presence of increased concentrations of alpha 1-ACT, was in an uncomplexed, free and functionally active form. Our results suggest that GCF cathepsin G reflects the disease process in adjacent inflamed gingiva and also increased host response to microbiota and/or dental plaque in the periodontitis lesions. Cathepsin G may contribute to periodontal tissue destruction directly and indirectly, via proteolytic activation of latent neutrophil procollagenase (promatrix metalloproteinase-8 [proMMP-8]).

A simple and reproducible method of determining the quality and quantity of neutral lipids in human saliva was tested. Parotid, submandibular and whole stimulated saliva were collected from 10 healthy adults. The lipids were extracted by the Folch method. A special method for extraction of glycolipids was also tested but gave no additional recovery. Thin-layer chromatography was used for separating the different lipid classes. The concentrations of total lipids in parotid, submandibular and whole stimulated saliva were 0.2, 0.9 and 1.3 mg/dl, respectively. Cholesteryl esters, cholesterol, triglycerides, diglycerides, monoglycerides and free fatty acids accounted for 96-99 percent of the total salivary lipids. Thus, polar lipids such as phospholipids contributed only a minor fraction, indicating that the lipids are not primarily of membrane origin. Ultracentrifugation of saliva samples at d = 1.21 g ml(-1) showed that the salivary lipids did not float like blood plasma lipoproteins. Therefore, they must be in a different state of aggregation from lipids in blood or lymph. No significant lipase activity of the type that acts on plasma lipoproteins was found in parotid or submandibular saliva. The content of free fatty acids and partial glycerides was high.

Dental plaque is the major aetiological factor in periodontal diseases and contains several proteolytic enzymes. The origin of these proteinases is, however, poorly studied. This study was undertaken to characterize collagenase present in dental plaque of adult periodontitis patients. Vertebrate-type rather than bacterial-derived collagenase activity was detected in extracts of both supra- and subgingival dental plaque extracts of adult periodontitis patients. Dental plaque collagenase was found to exist predominantly in autoactive form. Dental plaque collagenase from periodontally healthy individuals existed in latent from. Latent dental plaque collagenase from periodontitis lesions could be activated by a 95 kD chymotrypsin-like proteinase from Treponema denticola and human leukocyte cathepsin G but not by human plasmin. Incubation of purified latent leukocyte collagenase with whole cells of Fusobacterium nucleatum, Eubacterium saburreum, Prevotella buccae and Porphyromonas gingivalis, however, did not result to the activation of the enzyme. Doxycycline in vitro inhibited dental plaque collagenase with an IC50-value of 20 microM. Dental plaque collagenase degraded more efficiently type I and II collagens than type III collagen. Western-blot analysis with specific anti-human neutrophil collagenase-antibody revealed that both in supra- and subgingival dental plaque extracts dental plaque collagenase had undergone proteolytic conversion from an 80 kD proform to a 58 kD active form which is associated with catalytic autoactivity as measured by functional collagenase assay. This reflects proteolytic activation of leukocyte collagenase in dental plaque probably by other proteases derived from potent periodontopathogenic bacteria such as T. denticola or other PMN proteases such as cathepsin G.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective: Trypsin-like protease (TLPase) enzyme produced by Porphyromonas gingivalis has been implicated as a virulence factor in the pathogenesis of periodontal disease. The aims of this study were to investigate the relationship between cultivable P. gingivalis, TLPase enzyme activity (BANA hydrolysis) and serum antibody levels against cell sonicate and a purified TLPase antigen from P. gingivalis W50., Materials and Methods: Sub-gingival plaque samples were cultured for levels of P. gingivalis together with a chairside analysis of TLPase enzyme activity (Perioscan) from periodontitis and gingivitis sites of adult periodontitis patients. A TLPase from P. gingivalis was purified by gel filtration and ion exchange chromatography from the vesicle fraction for use as a test antigen., Results: Elevated levels of P. gingivalis were found at periodontitis sites, however, there was no correlation with sub-gingival plaque TLPase enzyme activity. Adult periodontitis patients had higher levels of IgG and IgA against cell sonicate and TLPase antigens than did controls. Those patients who were P. gingivalis culture-positive demonstrated an elevated immune response against both cell sonicate and TLPase when compared to P. gingivalis culture-negative patients. Treatment resulted in an improvement of clinical indices and no cultivable P. gingivalis could be recovered from the treated sites and there was a concomitant decrease in IgG levels against the TLPase. There was no significant difference in BANA hydrolysis at gingivitis sites or periodontitis sites after treatment., Conclusions: Further longitudinal studies are suggested to investigate the role of the TLPase in the response to treatment of chronic adult periodontitis patients.

Although the ability of dental plaque to hydrolyze sodium monofluorophosphate (MFP) has been known for some time, its effect on the F- concentration at the plaque-enamel interface is undefined. We have determined enzyme kinetic values for MFP hydrolysis and diffusion coefficients so that the penetration and degradation of MFP in plaque can be modeled by computer. The KM and Vmax values for natural human plaque were 1.77 mmol/L and 41.4 nmol/min/mg protein, respectively, at pH 8.0. At pH 6.0, the Vmax was lower, 15.6 nmol/min/mg, but KM was not significantly different. Competitive inhibition by orthophosphate gave a Ki of 4.55 mmol/L. The diffusion coefficient for MFP in artificial plaque was 1.91 x 10(-6) cm2/sec. When these data were used for mathematical modeling of the effects of rinsing with MFP and F- solutions, compared with an equivalent NaF application, the concentration of F- from MFP was lower at the inner surface of plaque, and the peak occurred later. Both pH and plaque thickness had a marked effect on the amount of MFP that could penetrate: At pH 8.0, almost none reached the inner surface of a 1-mm-thick plaque intact. At pH 6.0, however, more MFP was able to penetrate, due to lower MFPase activity. While MFP diffusion is inherently slower than that of F-, enzymic degradation increases the gradient for inward diffusion. If the conventional view that MFP in toothpaste acts as a source of F- is true, then MFP toothpaste should be formulated to optimize MFPase activity in dental plaque.

The effect of sucrose rinses supplemented with L-LDH and NAD was investigated. When LDH or NAD were added to the sucrose rinse, the pH fall was less marked than with rinsing with sucrose alone. Significant differences (p < 0.05) in mean minimum pH and cH areas were observed when sucrose rinses were supplemented with LDH, NAD or with a combination of LDH and NAD, when compared with sucrose alone. Plaque fluid concentrations of lactate and acetate significantly decreased with all rinses when compared with sucrose alone. Phosphate levels also decreased, although not significantly, when NAD was the sole supplement. No significant decreases were observed in succinate, formate or propionate concentrations. The results confirm the beneficial effect of LDH in reducing the accumulation of lactate in plaque following a sucrose rinse. The inclusion of NAD in the rinse demonstrated the role of extracellular hydrogen acceptors in the reaction. Further work is required to identify endogenous hydrogen receptors and to clarify the fate of pyruvate resulting from the oxidation of lactate before the potential role of LDH as a cariostatic agent can be fully understood.

Salivary amylase is known to bind specifically to several species of oral streptococci. To assess the importance of this interaction in bacterial colonization of the oral cavity, we determined the proportion and identity of amylase-binding bacteria (ABB) in dental plaque of humans and various salivary amylase-secreting and non-secreting mammalian species. The numbers of ABB in undisturbed plaque collected over time from tooth surfaces of six human volunteers or from 14 other mammalian species were determined by means of a replicating assay. The mean proportion of ABB cultured aerobically from human teeth at 2 h was 10.5% (SD 10), at 8 h 7.9% (8), at 24 h 13% (11), and at 48 h 12% (9). The mean proportion of anaerobically cultured ABB found at 2 h was 3% (SD 4), at 8 h 5% (5), at 24 h 12% (9), and at 48 h 16% (12). Amylase-binding bacteria cultured from these samples resembled Streptococcus mitis, Streptococcus gordonii, Streptococcus salivarius, Streptococcus crista, or unidentified streptococci. In addition, only animals exhibiting salivary amylase activity in their saliva harbored ABB (ranging from 2 to 31% of the total flora), with the exception of the pig, where no ABB were found to colonize, despite considerable amylase activity in saliva. Only strains resembling S. mitis and S. salivarius and unspeciated strains were isolated from these mammals. These results suggest that amylase-binding streptococci are the predominant ABB in human plaque, and their numbers generally increase as plaque develops. Since ABB colonized only the oral cavities of hosts demonstrating salivary amylase activity, the ability to bind amylase may play an important role in oral colonization by these bacteria.