Reducing medium for the cultivation of Porphyromonas gingivalis.

Porphyromonas gingivalis, a micro-organism frequently associated with human periodontal diseases, is commonly cultured under a reducing atmosphere in enclosed cabinets or glove boxes. This paper reports a modified Wilkins-Chalgren (MWC) medium for the culture of P. gingivalis under atmospheric conditions at 37 degrees C. On the basis of preliminary tests, WC broth was supplemented as follows: 500 mg/l cysteine hydrochloride; 250 mg/l sodium thioglycolate; and 1,000 mg/l sodium bicarbonate. Three P. gingivalis isolates (JKG-I, 33277 and A7436) showed very similar growth over 24 and 48 h periods when cultured both in WC medium in an anaerobic chamber and in the MWC medium. Culture of these isolates both in the anaerobic chamber and in the MWC medium yielded very similar data with respect to trypsin-like activity, total protease activity, and reactivity to monoclonal antibodies specific for P. gingivalis. Growth in the MWC medium varied over a 3- to 4-fold range for seven additional isolates (JKG-7, D86B6, D13B11, D84D2, JKG9, D67D9, D82F5) over 24 and 48 h periods.

Salivary levels of alpha 2-macroglobulin, alpha 1-antitrypsin, C-reactive protein, cathepsin G and elastase in humans with or without destructive periodontal disease.

Five host-response indicators were measured by enzyme-linked immunosorbent assays on unstimulated whole saliva samples from 45 adults (19 male, 26 female). The participants were distributed among four dentate groups representing oral health (I), gingivitis (II), moderate periodontitis (III), and severe periodontitis (IV), and one group of edentulous volunteers (V). Levels of the host-response indicators varied widely, from zero, primarily with groups I and V, to relatively high values with groups II, III and IV. The levels ranged as follows: alpha 2-macroglobulin, 0-4941 ng/ml; alpha 1-antitrypsin, 2-2271 ng/ml; C-reactive protein, 0-472 pg/ml; cathepsin G, 0-6035 ng/ml; elastase, 0-164 ng/ml (free), 0-732 ng/ml (bound to alpha 1-antitrypsin), and 0-318 ng/ml (bound to alpha 2-macroglobulin). Statistical evaluation by planned contrasts showed that levels of host-response indicators for group I were significantly lower (except for alpha 1-antitrypsin) than for groups II, III, and IV. A trend analysis of groups I-IV showed that mean scores (again, except for alpha 1-antitrypsin) increased significantly in a positive, monotonic manner. Group V showed significantly lower values for elastase than in the other groups. The findings demonstrate that these factors can be detected in whole saliva and suggest that, except for alpha 1-antitrypsin, their levels are directly related to an individual's periodontal status.

Trypsin-like activity levels of Treponema denticola and Porphyromonas gingivalis in adults with periodontitis.

Treponema denticola (Td) and Porphyromonas gingivalis (Pg) are associated with human moderate and severe adult periodontal diseases. This study quantifies these two anaerobes and their trypsin-like (TL) activities in subgingival plaque collected from both clinically healthy and periodontally diseased sites of human periodontitis patients. Antigen levels of the microorganisms were determined by monoclonal antibodies and TL activities were measured by the fluorescent substrate Z-gly-gly-arg-AFC in a disc format. Significant positive correlations were observed between the antigen levels and the TL activities when the data were subjected to statistical analyses both on a site-specific and on a patient basis. Anaerobe synergism was found between Td and Pg in a continental US population, and positive correlations were found between anaerobe levels (individually and total) and clinical indicators of adult periodontitis.

Measurements of interleukin-6 in gingival crevicular fluid from adults with destructive periodontal disease.

This paper describes a study of whether or not the amounts of interleukin-6 (IL-6) in gingival crevicular fluid (GCF) are correlated with periodontal clinical measures. A sensitive ELISA was developed to measure IL-6 in GCF. Two male and 3 female adult subjects with periodontal disease were examined at their first appointments, after 3 months, and after 6 months. Data were obtained on plaque index (PI), bleeding index (BI), probing depth (PD), and on the IL-6 content of GCF samples from 16 sites per subject for a total of 240 measurements. Significant correlations were found between BI and IL-6 (P < 0.005) and between PD and IL-6 (P < 0.05), but not between PI and IL-6. Only 6 out of the 80 sites (in 3 of the 5 subjects) showed PD increases of at least 2 mm. However, for each of these 3 subjects, the amounts of IL-6 in the GCF samples from these sites were markedly higher than the mean amounts of IL-6 in the GCF samples from the remaining sites. These findings suggest that IL-6 may be a useful indicator of periodontal disease, although more extensive longitudinal studies are needed to determine the real clinical value of this GCF component.

A sensitive method for the detection of immune complexes in human gingival crevicular fluid.

An assay for immune complexes [ICs] in gingival crevicular fluid [GCF] could be potentially useful to ascertain the risk of periodontal disease activity at specific oral sites. This paper describes and ELISA-based method sufficiently sensitive to measure ICs in GCF samples at levels below 1 microgram/ml. Static GCF samples taken from 5 adults showed IC levels ranging from 5 to 166 micrograms/ml. Additional tests of 10 GCF samples from 1 adult were conducted with complement components and indicated that either C1q or rabbit anti-human C3d was suitable as a capture agent in the assay procedure. Further application of this assay may help to assess the usefulness of IC levels in GCF samples as possible diagnostic indicators of periodontal disease activity.

Detection of microbial trypsin-like enzymes by use of an agar gel.

Certain oral micro-organisms associated with periodontal diseases elaborate trypsin-like enzymes. This study describes a single assay for these enzymes, based on the incorporation of the synthetic substrate N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA) into ion-agar gel. The system can provide quantitative or qualitative data within 24 h and is suitable for use in the dental clinic.

Fibronectin levels of unstimulated saliva from naval recruits with and without chronic inflammatory periodontal disease.

Past studies have suggested that gingival crevicular fluid is produced more readily in persons with severe periodontal diseases than in persons with healthy gingivae. In this study, salivary fibronectin was selected as an index of total gingival crevicular fluid flow. Our purpose was to determine whether a relationship could be found between salivary fibronectin level and periodontal disease status. Unstimulated saliva was collected from 20 healthy and 20 periodontally-diseased naval recruits. The periodontally-diseased subjects included 10 with localized juvenile periodontitis and 10 with moderate to severe periodontitis. Mean subject ages and salivary flow rates were similar for the 2 groups. Although 2 of the periodontally-diseased subjects showed unusually high fibronectin levels, the mean level for the remaining 18 subjects did not differ significantly from the mean of the healthy group, and no association of periodontal disease status with salivary fibronectin content was seen. Consequently, it was not evident from salivary fibronectin levels that the content of gingival crevicular fluid in unstimulated whole saliva differed significantly for persons with or without severe periodontal disease, except possibly for extreme cases of disease.

Susceptibility of fibronectin to degradation by various gram-negative and gram-positive oral micro-organisms.

The degradative effects on tritiated human plasma fibronectin (FN) of proteases associated with twenty five Gram-negative (GN) and fifteen Gram-positive (GP) oral microbial isolates were examined. Ninety-two per cent of the GN and 20% of the GP isolates degraded the FN. In the GN group, the degradative effects were greatest for Bacteroides gingivalis and Treponema denticola, while in the GP group Streptococcus faecalis produced appreciable, and the two Lactobacillus casei isolates moderate and low, levels of FN degradation.

The effects of basic and acidic synthetic polypeptides on the adherence of the oral bacteria, Streptococcus mutans and Streptococcus sanguis, to hydroxyapatite.

Two basic and two acidic synthetic polypeptides that bind strongly to hydroxyapatite at neutral pH were tested to determine their influence on adsorption of two Streptococcus mutans and two Streptococcus sanguis strains to hydroxyapatite. The adsorption of the strains was significantly enhanced or reduced by the basic and acidic agents, respectively. Study of acidic polypeptides provided evidence of competition between the polypeptides and the bacterial cells for hydroxyapatite adsorption sites.

Salivary pH-rise activities in caries-free and caries-active naval recruits.

Kleinberg (1967) (Archs oral Biol. 12, 1457-1473) has observed that the incubation of a salivary sediment/supernatant mixture with a low concentration of glucose produces a pH drop followed by a pH rise. As this pH-rise effect could elevate oral pH in vivo, the degree of such activity could be directly related to caries resistance. The pH-rise of paraffin-stimulated whole saliva samples from 54 caries-free and 54 caries-active male naval recruits was compared in two assays. The first, conducted with sediment/supernatant/2.8 mM glucose mixtures, showed no significant differences for mean pH versus time profiles for 26 caries-free and 26 caries-active subjects. Concomitant assays, in which 3.33 mM arginine and water replaced the supernatants in the assay mixtures, showed slightly higher mean pH/time profiles, with higher minima for the caries-free subjects. These profile differences were statistically significant for the arginine-containing mixtures, a result attributed to different microbial distributions for the caries-free and caries-active sediments. The second assays were conducted on saliva supernatants from 28 caries-free and 28 caries-active subjects, employing a strain of Lactobacillus casei in place of salivary sediment in the assay system. No statistically significant differences were evident between the mean pH/time profiles for the two groups of subjects, although a significant positive correlation was observed between pH minimum and bicarbonate content of the samples. Thus no relationship of salivary pH-rise activity and caries experience were found.

Purification and properties of endo-1, 3-alpha-D-glucanase from Pseudomonas.

An endo-1, 3-alpha-D-glucanase (EC 3.2.1.59) was purified from cell-free culture supernatants of Pseudomonas NRRL-B-12324. The enzyme was purified 8.7-fold to a specific activity of 78.1 U/mg of protein. The enzyme was inducible and had an isoelectric point of 4.6 and a Km of 80.0 mM in terms of anhydroglucose units. Two distinct peaks of activity were resolved by gel filtration with two different supporting media, whereas only one peak of activity was resolved by isoelectricfocusing. The two peaks were assigned molecular weight values of 67,400 and 279,000. The pH optimum was near 5.0 and the temperature optimum was near 56 degrees C. Additional gel filtration data indicated that the enzyme functions as an endohydrolase.

Salivary protein polymorphisms in caries-free and caries-active adults.

Several salivary proteins are known to exhibit genetic polymorphism, and this study was undertaken to determine whether there was an association of caries experience with specific parotid salivary protein phenotypes. Parotid saliva collected from 46 caries-free and 47 caries-active naval recruits was subjected to SDS, anionic, and cationic polyacrylamide slab gel electrophoresis. Distributions of the acidic proline-rich (Pr), parotid acidic (Pa), double band (Db), parotid size variant (Ps), parotid middle band (Pm), and parotid basic (Pb) protein phenotypes were similar in both caries-free and caries-active subjects. Further, overall protein patterns on SDS, anionic, and cationic slab gels did not differ between the two groups.

Dextranase-producing organisms in dental plaque from caries-free and caries-active naval recruits.

Dental plaque samples from caries-free and caries-active naval recruits were assayed for the prevalence of dextranase-producing organisms. These organisms were found in the plaque of all of the subjects. Mean percentages of dextranase-producing organisms with respect to total colony count for the 2 groups of subjects were not significantly different.

Determination, by carbon-13 nuclear magnetic resonance spectroscopy, of the composition of glucans synthesized by enzymes of the cariogenic organism Streptococcus mutans.

The 13C-n.m.r. spectra of water-soluble and -insoluble glucans synthesized by enzymes isolated from six strains of Streptococcus mutans are interpreted. The glucans are shown to be composed primarily of alpha-(1 linked to 3)- and alpha-(1 linked to 6)-linked glucosyl residues, and the relative abundance of each linkage is estimated from peak areas. Treatment of water-insoluble glucans with dextranase is found to result in water-soluble and -insoluble products, the former enriched in alpha-(1 linked to 6)-linkages and the latter in alpha-(1 linked to 3)-linkages. The structural conclusions arrived at by 13C-n.m.r. spectroscopy are consistent with data from methylation analysis and 1H-n.m.r. spectroscopy.

The hypermineralization of diseased root surfaces.

Root surfaces which have lost their attachment due to chronic inflammatory periodontal disease present significant increases in calcium, magnesium, phosphorus, and fluoride. The clinical significance of this effect is not known in regards to wound healing, but it may be helpful in control of cervical sensitivity.

Permeability of cellophane membranes to parotid during dialysis.

Pooled paratoid saliva was dialyzed in cellophane membranes against water for periods of up to 1 week and loss of proteins was monitored by acrylamide gel-electrophoresis. A gradual loss of cationic proteins was observed whereas anionic proteins were not appreciably affected. Loss of the cationic proteins could be greatly reduced by performing dialyses against dilute electrolyte solutions rather than water. These effects were attributed primarily to electrostatic changes associated with the dialysis membranes.