Characterization of protease activities in Capnocytophaga spp., Porphyromonas gingivalis, Prevotella spp., Treponema denticola and Actinobacillus actinomycetemcomitans.

Protease activities in cell sonicates of defined bacterial strains were examined using peptide substrates and class-specific inhibitors. Capnocytophaga spp. all produced serine dipeptidyl peptidase activity and arginine/lysine, elastase- and chymotrypsin-like enzymes with some metalloprotease characteristics. The elastase-like activity was strongest in Capnocytophaga sputigena, but the others were greatest in Capnocytophaga gingivalis. The latter also had a separate arginine-specific enzyme which appeared not to be present in the other two species. Porphyromonas gingivalis showed serine dipeptidyl peptidase activity and very strong arginine and lysine cysteine protease activities. Prevotella spp. had inhibitor-resistant dipeptidyl peptidase activity and arginine cysteine protease activity that was much weaker but biochemically similar to P. gingivalis. Treponema denticola possessed a strong trypsin-like serine protease activity as well as very weak dipeptidyl peptidase and chymotrypsin-like activities that were sensitive to some cysteine protease reagents. Actinobacillus actinomycetemcomitans showed a novel alanine- and lysine-specific activity, but its nature was unclear.

A comparison of cysteine and serine proteinases in human gingival crevicular fluid with tissue, saliva and bacterial enzymes by analytical isoelectric focusing.

Gingival crevicular fluid (GCF) contains several different proteinase activities and the study sought to clarify their sources. Gingival tissue and GCF were collected from chronic periodontitis patients. Gel-filtration chromatography of crude tissue extracts yielded cathepsin B and tryptase fractions sensitive to cysteine and serine proteinase inhibitors, respectively. Cell sonicates of suspected periodontal pathogens were prepared from broth cultures of reference strains. Of these, Porphyromonas gingivalis showed much the strongest activity and this had an effector response consistent with the metal-dependent cysteine proteinase described by others. Banding patterns in GCF, tissue and bacterial samples were compared on substrate-impregnated overlay membranes applied to isoelectric focusing gels. On Z-Val-Lys-Lys-Arg-AFC overlays, GCF had bands corresponding to tissue cathepsin B and the enzyme from P. gingivalis, though a contribution from Treponema denticola could not be ruled out. Use of D-Val-Leu-Arg-AFC overlays showed GCF activity similar to tissue tryptase. In GCF there were additional bands that did not correspond to any tissue or bacterial samples and on Z-Ala-Ala-Lys-AFC overlays these closely resembled activity in parotid saliva. The results confirmed that GCF contains tissue cathepsin B and tryptase, while the apparent presence of enzymes from P. gingivalis and possibly T. denticola is consistent with previous reports linking activity to these organisms. The saliva bands demonstrated that contamination of GCF may occur despite rigorous collection procedures.

Comparison of host tissue and bacterial dipeptidyl peptidases in human gingival crevicular fluid by analytical isoelectric focusing.

Earlier work has shown that gingival crevicular fluid (GCF) contains dipeptidyl peptidase (DPP) activities that resemble those in host tissue. Here, further comparisons were made with enzymes from suspected periodontal pathogens. Gingival tissue and GCF were collected from patients with chronic periodontitis. DPP II and DPP IV fractions with acid and alkaline pH optima, respectively, were separated from crude tissue extracts by gel-filtration chromatography. Bacterial cell sonicates were prepared from broth cultures of reference strains. There was moderate to strong DPP activity with Capnocytophaga spp., Porphyromonas gingivalis and Prevotella spp., very weak activity with Treponema denticola and no detectable activity with Actinobacillus actinomycetemcomitans or Fusobacterium nucleatum. Banding patterns in GCF, tissue and bacterial samples were compared on substrate-impregnated overlay membranes applied to isoelectric focusing gels. In gels washed with acid buffer, GCF had bands corresponding to tissue DPP II. Use of an alkaline washing buffer showed GCF activity which closely matched tissue DPP IV that had been pretreated with neuraminidase, an enzyme found by others in the gingival crevice. P. Gingivalis gave multiple bands and several of these had counterparts in GCF. The apparent presence in GCF of the DPP from P. gingivalis is consistent with the association of this organism with destructive periodontitis.

The effects of Acacia arabica gum on the in vitro growth and protease activities of periodontopathic bacteria.

The antibacterial activity of acacia gum was assessed using fresh isolates and reference strains of Actinobacillus actinomycetemcomitans, Capnocytophaga spp., Porphyromonas gingivalis, Prevotella intermedia and Treponema denticola. A fine aqueous suspension of gum was produced by sonication and then a soluble fraction isolated by centrifugation and membrane filtration. These preparations were incorporated into columbia agar at doubling concentrations. Growth of P. gingivalis and P. intermedia cultures on the agar was inhibited by whole gum sonicate at concentrations of 0.5-1.0% w/v. Both species showed reduced susceptibility when horse blood was present in the agar. The gum soluble fraction did not inhibit growth of any bacterial culture. The effect of acacia on bacterial proteases was examined with cell sonicates from log phase broth cultures. Enzyme activities were determined by fluorimetric assay with various synthetic peptide substrates. Most protease activities reduced in the presence of 0.5% w/v gum sonicate, with the trypsin-like activities of P. gingivalis and P. intermedia proving most sensitive. The gum soluble fraction was nearly always less inhibitory than the sonicate. The action of acacia gum against suspected periodontal pathogens and their enzymes suggests that it may be of clinical value.

Dipeptidyl peptidase II- and IV-like activities in gingival tissue and crevicular fluid from human periodontitis lesions.

Gingival tissue and gingival crevicular fluid were collected from patients with chronic periodontitis. Gel filtration chromatography of crude tissue extracts yielded separate fractions active against Lys-Ala-7-amino-4-trifluoromethyl-coumarylamide (AFC) at acid pH and Gly-Pro-AFC at alkaline pH. The molecular weights, pH optima and inhibitor responses of these activities were consistent with those of dipeptidyl peptidases (DPP) II and IV, respectively. When tested with the same substrates, crevicular fluid was also found to contain DPP II- and IV-like activities with very similar pH profiles and inhibitor responses to those in tissue. The close resemblance suggested that the crevicular fluid enzymes were derived mainly from inflamed gingival tissues. Slight differences in the DPP II-like activities might be explained by the additional presence in crevicular fluid of enzymes from subgingival bacteria. With use of appropriate buffers, a third substrate, Ala-Pro-AFC, gave selective detection of both DPP II- and IV-like activities in tissue and crevicular fluid. Assays with Ala-Pro-AFC had the advantage of greater sensitivity, especially with DPP II-like activity. Raised levels of this enzyme have previously been found in the gingiva of periodontitis patients and thus DPP II-like activity in crevicular fluid might prove of value in monitoring disease activity.

The immediate- and medium-term effects of Meswak on the composition of mixed saliva.

Chewing sticks or Meswaks are used for teeth cleaning in many parts of the world. They contain substances that may reduce caries and periodontal disease. The present study consisted of 2 parts. In a short-term experiment, volunteers chewed on an inert eliciting agent (pyrogen-free rubber) and then a piece of Meswak, each for 5 min. For the medium-term experiment, volunteers brushed with either Meswak or a conventional toothbrush 5 x a day for 2 weeks. Saliva produced immediately after chewing Meswak showed statistically significant increases in calcium and chloride, but decreases in phosphate and pH as compared with controls. In the medium-term experiment, saliva samples collected 4 h after the last use of Meswak or toothbrush showed no significant differences in any of the components examined (calcium, magnesium, chloride, phosphate, IgA, IgG, lactate dehydrogenase and aspartate transaminase). Gingival and plaque indices, however, were significantly lower after brushing with Meswak. Salivary calcium promotes mineralization of tooth enamel and chloride inhibits calculus formation. Our results thus indicate that Meswak releases substances into saliva that could improve oral health. Calcium and chloride values were similar to those of controls after 4 h and thus frequent use of Meswak may be necessary to maintain a favorable salivary environment.

The finding of antiplaque features in Acacia Arabica type of chewing gum.

The gum of Acacia Arabica is described in the British pharmacopoeia as a source of useful medicaments. It is believed to be of value for treating gingivitis and for reducing plaque. 2 blind crossover trials were carried out to evaluate the antiplaque potential of Acacia gum compared with sugar free gum. In trial 1, the mean gingival and plaque scores were lower after 7 days of using Acacia compared with sugar-free gum but the differences were insignificant. In trial 2, daily photographic assessment of erythrocine-stained plaque showed lower scores after Acacia gum compared with sugar-free gum. The total difference in scores for each day from each individual between the 2 treatments was highly significant (p less than 0.05). This implies the presence of substances in Acacia gum which, compared with ordinary gum, primarily inhibit the early deposition of plaque.

Photographic assessment of the antiplaque properties of sanguinarine and chlorhexidine.

The purpose of this study was to assess the effect of rinsing and topical application of sanguinarine on labial surface plaque accumulation, compared with topically applied water, and compared with rinsing with chlorhexidine, while refraining from all oral hygiene measures for 5 days. Color photographs of the disclosed plaque were taken at the end of each phase of treatment and blindly traced using an Apple II micro-computer graphic tablet digitizer. The plaque score for each tooth was calculated by dividing the computer reading of the labial plaque surface area by the total labial surface area of the tooth. Topically applied sanguinarine showed better plaque reduction than mouthrinsing with sanguinarine. There was a significant reduction in plaque accumulation after rinsing with chlorhexidine compared with topically applied sanguinarine, water, and mouthrinsing with sanguinarine. The results of this study indicate that chlorhexidine is a more superior antiplaque agent than sanguinarine. The use of an Apple II micro-computer graphic tablet digitizer provided a valuable method for accurate plaque assessments.

Unusual pigmentation of the gingiva. Report of two different types.

Two unusual types of oral mucosal pigmentation are reported. The first type is due to the use of an oral hygiene aid that is obtained from the bark of a plant, Juglans regia, that is known as Derum or Dendasa. The second type is that of a self-induced tattoo of the buccal maxillary gingiva created with soot obtained from burned wood in clay ovens.

A method for producing monoclonal antibodies to human oral epithelial cells by the hybridoma technique.

Female BALB/c mice 6-8 weeks old were immunized intravenously (IV) with two injections of 30,000 human oral epithelial cells two weeks apart. The sera of these mice, and none of the controls, were positive by indirect immunofluorescence to cell suspensions of human epithelial cells of gingivae, tonsils and skin, but were negative to sections of human spleen, kidney and liver. The fluorescence was apparent only in the prickle cell layer, not at the basement membrane region nor in the connective tissue. The spleen cells from one of the selected immunized mice were fused to mouse myeloma cells, using polyethylene glycol (1500 MW), three days after a third IV booster injection of 30,000 epithelial cells. Three of the monoclonal cell lines produced have shown different staining patterns with various sizes of epithelial cells in suspension, results that differ from the staining pattern obtained with epithelial cells using polyclonal sera. These monoclonal antibodies were directed against antigens that differ from blood group A, B and HLA antigens on the surface of epithelial cells.