Root-surface caries in rats and humans: inhibition by a non-antimicrobial property of tetracyclines

The incidence of root caries has been found to increase as the population ages and as edentulism becomes less prevalent due to improved dental awareness and care, and as exposure of roots due to gingival recession has also increased in the elderly. The mechanism of root caries is thought to be mediated by both bacterial and mammalian proteases produced by plaque and the periodontal tissues, respectively. In the current study, a rat model of periodontal disease was used in which gnotobiotic rats were infected intra-orally with a periodontal pathogen (P. gingivalis). Infecting the rats with P. gingivalis increased the collagenase activity in the gingival tissue in association with severe alveolar bone loss. Treating P. gingivalis-infected rats with doxycycline or CMT-1 prevented the destruction of the periodontium by MMPs, thus preventing exposure of roots to subgingival bacterial plaque and host tissue collagenases and the subsequent development of root caries. In addition, a low-dose doxycycline (LDD, 20 mg bid, non-antimicrobial dose) for 3 months was used in humans predisposed to increased root caries as the result of heavy use of smokeless (chewing) tobacco, causing gingival recession, subgingival plaque accumulation with Gram-negative bacteria, increased gingival crevicular fluid flow (GCF), and elevated GCF collagenase. Daily administration of LDD in smokeless tobacco patients reduced the GCF collagenase and prevented the further development of root caries.     

High levels of soluble intercellular adhesion molecule-1 (ICAM-1) in crevicular fluid of periodontitis patients with plaque

The intercellular adhesion molecule-1 (ICAM-1) is a membrane-bound molecule involved in cell-cell adhesive interactions which is upregulated on inflammatory epithelial cells. The levels of soluble ICAM-1 (sICAM-1) shed into the gingival crevicular fluid (GCF) were studied in healthy patients and patients with gingivitis, adult periodontitis or rapidly progressive periodontitis, using an ELISA technique. Clinical parameters including plaque index, gingival index, probing depth, and bleeding on probing were recorded following careful sampling of GCF with standardised filter strips. In GCF, sICAM-1 levels were higher for patients with plaque (p=0.04) and for patients with inflammation (p=0.02), but did not correlate with disease classifications. These results suggest that elevated GCF sICAM-1 levels may represent increased shedding of this molecule in the interstitial fluid as a result of membrane-bound ICAM-1 upregulation on ICAM-1 gingival-bearing cells in relation with plaque accumulation and inflammation.     

PC-assisted network for quality control in film development

The purpose of the present investigation was to evaluate the influence on sampling repetition of aspartate aminotransferase (AST) level with 10-minute intervals. Tests based on the composition of gingival crevicular fluid (GCF) for detection of active periodontitis and GCF require the repetition of sampling. Two 30-second samples of GCF were harvested with 10-minute intervals from 123 sites in 10 healthy subjects and 20 periodontitis patients. AST activity of the first samples in periodontitis subjects were approximately 7.8% greater than that of the second samples. The difference were not significant (P > 0.05). But in healthy subjects the difference were significant (P < 0.05). AST activity correlation positively with bleeding index (BI) and probing pocket depth (PD).     

Interleukin-1 beta concentration of gingival crevicular fluid

The concentration of interleukin-1 beta is elevated in inflamed gingival tissue. Therefore a method for the measurement of interleukin-1 beta (Il-1 beta) in gingival crevicular fluid (GCF) using a commercially available Il-1 beta ELISA was developed. GCF was collected with periopaper strips and 4 protocols of sampling using filter paper strips were tested; the method with a recovery rate of 111.9% (SD: +/- 14.5%) was chosen for subsequent analysis of all samples. Il-1 beta concentration in GCF of periodontitis patients and a healthy control group was determined. Patients (n = 19, mean age: 29.3 years) had not been treated. The healthy control group (n = 14, mean age: 22.8 years) showed, after a hygiene regimen of 2 weeks, no clinical signs of gingival/periodontal inflammation. Probing depth, clinical attachment level, bleeding upon probing, and a modified plaque index were recorded. Il-1 beta could be detected in all GCF samples. The concentration ranged between 22.8 ng/ml and 150 ng/ml in the healthy control group and between 85.8 ng/ml and 882.2 ng/ml in the periodontitis patients. No sex-related differences were noted. According to our present results the determination of GCF Il-1 beta concentration is possible using commercially available test kits if the principle of sample preparation is adapted to the specific requirements of GCF analysis.     

A relationship between proteinase activity and clinical parameters in the treatment of periodontal disease

The objective of this research was to determine the effectiveness of a biochemical assay which measures proteolytic enzyme activity in gingival crevicular fluid (GCF) and to relate this enzyme activity to clinical parameters traditionally utilized for periodontitis detection. A clinical trial was conducted on 8 periodontitis subjects with > or =4 sites exhibiting a loss of attachment of > or =5 mm and probing depths of > or =5 mm with bleeding on probing. On each subject, a plaque index was performed, followed by GCF sampling at those sites which exhibited a loss of attachment and probing depths. GCF was analyzed for activity against benzoyl-L-arginine-p-nitroanilide in the presence (BAPNA w/gly-gly) and the absence (BAPNA w/o gly-gly) of glycyl-glycine and against MeOSuc-Ala-Ala-Pro-Val-pNA and Suc-Ala-Ala-Pro-Phe-pNA for neutrophil serine proteinases activity (elastase and cathepsin G, respectively). Subsequently, a gingival index was performed, attachment levels and probing depths were recorded using a constant force probe with bleeding on probing being noted. A split-mouth design was employed and half mouths were randomly assigned to the following treatment groups: group A, half of the mouth received scaling/root planing and polishing: group B, half of the mouth received no treatment (control). Subjects were treated, then instructed on toothbrushing and interdental cleaning. After 4 weeks, subjects returned to receive a plaque index; GCF sampling, gingival index, attachment levels, probing depths and bleeding on probing as described above. Using a paired Student t-test, the findings suggest that BAPNA w/gly-gly was significantly less in treatment sites than in non-treated control sites (p=0.05). No such correlation was found for other activities, including neutrophil serine proteinases which were shown to occur in GCF in free, proteolytically active forms. In addition, significant treatment effects were detected for probing depths (p= 0.03) which reduced by 1.3 mm and attachment levels (p=0.02) which gained 0.7 mm. The reduction of P. gingivalis from treated periodontitis sites as detected by a significant decrease in BAPNA w/ gly-gly may prove to be a valuable marker for periodontal disease activity.     

Differential expression of CR3, Fc epsilon RII and Fc gamma RIII on polymorphonuclear leukocytes in gingival crevicular fluid

Polymorphonuclear leukocytes (PMNLs) are the most numerous cell population among the cellular infiltrates in gingival crevicular fluid (GCF) and play important roles in the host-defensive system in the gingival crevices. We determined the percentage of neutrophils, eosinophils and basophils in total PMNLs by light microscopic observation using Randolph-methylene blue staining, then assessed flow cytometric differences in the expression of CR3, Fc gamma RIII, Fc epsilon RII, LFA-1 alpha, and LFA-1 beta on PMNL in GCF and peripheral blood (PB) from 21 patients with adult periodontitis (AP) and 13 healthy donors. Percentages of basophils and eosinophils were higher in GCF than in PB. In both AP patients and healthy subjects, expression of CR3 and Fc epsilon RII was higher while Fc gamma RIII was lower in GCF than in PB. The statistical analysis showed that the expressions of Fc gamma RIII and Fc epsilon RII on GCF PMNLs were lower in AP patients than in healthy subjects. Expressions of LFA-1 alpha and beta on GCF were similar to those on PB PMNLs. PB PMNLs stimulated in vitro with Porphyromonas gingivalis culture supernatant and fMLP displayed an expression pattern of CR3, Fc gamma RIII and Fc epsilon RII on GCF PMNLs. However, C5a and IL-1 failed to induce changes in Fc gamma RIII and Fc epsilon RII. The results indicate that GCF neutrophils are activated, present enhanced adhesion and a decreased IgG-binding ability which would reflect that they are at the terminal stage of activation, and that GCF contains a larger eosinophil fraction than in PB. Moreover, these GCF eosinophils appear to be activated.     

Subgingival temperature: relation to gingival crevicular fluid enzymes, cytokines, and subgingival plaque micro-organisms

There have been no reports on the relationship of subgingival temperature to specific gingival crevicular fluid (GCF) components. Therefore, the purpose of this cross-sectional study was to determine whether there was any relationship between subgingival temperature and GCF levels of neutrophil elastase (NE), myeloperoxidase (MPO), beta-glucuronidase (BG), interleukin-1 alpha (IL-1), and interferon alpha (IFN). Furthermore, another objective was to confirm an association of subgingival temperature with clinical parameters and specific subgingival plaque micro-organisms as has been reported earlier. 27 human subjects each having healthy (n = 50), gingivitis (n = 59) and periodontitis (n = 53) sites were evaluated. The plaque index (PI), subgingival temperature, probing depth, attachment loss, bleeding index and gingival index were measured. GCF was sampled following the measurement of the PI and removal of the supragingival plaque. GCF samples were assayed for the enzymes NE, BG, MPO and the cytokines IFN-alpha and IL-1 alpha. A sterile Gracey curette was utilized at each sampled site to collect subgingival plaque. The plaque samples were evaluated using an immunoassay. Subgingival temperature was found to directly correlate with all clinical parameters (p < 0.001). Significant, albeit not large, correlations were found between subgingival temperature and NE (r = 0.35, p < 0.001), MPO (r = 0.26, p < 0.001) and BG (r = 0.23, p < 0.01). Temperature was found to correlate positively with E. corrodens (r = 0.33, p < 0.02) and F. nucleatum (r = 0.25, p < 0.05) but not with P. intermedia (r = 0.02, p = 0.9), P. gingivalis (r = 0.20, p = 0.1) and A. actinomycetemcomitans (r = 0.01, p > 0.9). In conclusion, subgingival temperature is correlated with the GCF enzymes, NE, MPO and BG as well as the clinical parameters and specific plaque micro-organisms associated with periodontal disease.     

Polymorphonuclear neutrophil chemotaxis in Chinese patients with post-juvenile periodontitis and rapidly progressive periodontitis

The purpose of this study was to investigate polymorphonuclear neutrophil (PMN) chemotaxis in Chinese patients with post-juvenile periodontitis (post-JP) and rapidly progressive periodontitis (RPP). Peripheral blood PMNs were isolated from nine post-JP and 13 RPP patients. Eight patients with adult periodontitis (AP) and seven clinically healthy subjects were used for comparison. Clinical data, including the plaque index and gingival index, of each subject were recorded. The "modified Boyden chamber technique" was used for PMN chemotaxis assay. Our results showed that the PMN chemotaxis index in patients with post-JP and RPP were significantly depressed compared to that in healthy subjects or patients with AP. There was no significant difference in PMN chemotaxis between post-JP and RPP, or between AP and healthy subjects. There was also no significant correlation between the plaque index, or gingival index, and PMN chemotaxis in any group. These results suggest that there is a PMN chemotaxis defect in most Chinese patients affected by post-JP or RPP, and that the PMN chemotaxis defect is not associated with clinical parameters such as the plaque index or gingival index.     

Thoracic diagnosis with electron-beam computed tomography

Posterior interproximal alveolar bone in 59 women, within 5 years after menopause, was assessed at baseline and after 2 years of supportive periodontal therapy (history of moderate/advanced periodontitis) using digitized image analysis. Baseline lumbar spine bone mineral density, smoking status, and yearly serum estradiol (E2) levels also were obtained to group subjects. An additional 16 non-periodontitis postmenopausal women were followed 2 years for clinical and estrogen status. 2-min GCF IL-1beta levels averaged from 2 baseline periodontal pockets (in periodontitis subjects) and 2 non-periodontitis sites (in non-periodontitis and periodontitis subjects) were determined with an enzyme immunoassay. A progressive and stable site were also monitored every 6 months for GCF IL-1beta in 15 patients. Results after 2 years indicated that 17 subjects had no posterior interproximal sites losing > or =0.4 mm of alveolar crest bone height, while 13 subjects had > or =3 such sites. Using analysis of variance, none of the above clinical groupings resulted in a significant difference in mean baseline or longitudinal GCF IL-1beta levels. However, when subjects who lost alveolar crest bone height were considered, E2-sufficient subjects had significantly depressed baseline GCF IL-1beta (in past-periodontitis sites) compared to E2-deficient patients (9.1+/-2.1 versus 31.7+/-10.2 pg/2-min sample, p<0.05), suggesting E2 influences gingival IL-1beta production in progressive periodontitis patients.     

The OSPAR Commission and Ministerial Meeting, 20-24 July 1998, Sintra Lisbon

Our aim was to study protease activity in GCF from inflamed sites with or without tissue destruction. 19 patients with both periodontitis and gingivitis sites and 12 patients having gingivitis alone participated in the study. GCF samples were collected by an intracrevicular washing method. The protease activity was measured as degradation of FITC-conjugated casein. To obtain a semiquantitative estimate of the harvested GCF volume, we measured the transferrin concentration in the wash-fluid. The protease activity was significantly higher in the deep pockets in periodontitis patients than in shallow pockets in the same patients. This difference was still higher when the ratio of protease activity to the amount of transferrin in the sample was plotted. Although protease activity was lower in samples from gingivitis patients than in the deep pockets in periodontitis patients, the difference was not significant. About 90% of the activity could be inhibited by the addition of an excess amount of alpha-1-antitrypsin (A1AT). This study shows that protease activity is higher in inflamed sites with tissue destruction than in inflamed sites without. Most of this activity could be inhibited by A1AT, which suggests that the activity is due to an imbalance between protease and antiprotease rather than to proteases insensitive to A1AT.     

Analysis of gingival crevicular fluid intracytoplasmic enzyme activity in patients with adult periodontitis and rapidly progressive periodontitis. A longitudinal study model with periodontal treatment

In the present study, the activity of 3 functionally related enzymes, creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST) levels in the rest and flow gingival crevicular fluid (rGCF, fGCF) from patients with rapidly progressive periodontitis (RPP) and adult periodontitis (AP) were determined before and after periodontal treatment, including maintenance. When rGCF and fGCF mean enzyme levels were compared, rGCF was found to contain approximately twice as much enzyme levels than fGCF throughout the study. The findings of the present study revealed that both the rGCF and fGCF samples also contained higher CK, LDH, and AST levels than serum samples. Baseline clinical parameters and GCF enzyme levels presented a significant decline throughout the non-surgical and surgical treatment phases in both patient groups, with surgical treatment being more effective. Despite clinical stability, in the AP group levels of LDH and AST showed a tendency to increase in the third month, while enzyme levels still continued to decrease in the RPP group, who received additional antibiotics during the surgical phase. These findings suggest that GCF intracytoplasmic enzyme profile is related with periodontal status and successful periodontal treatment, in addition to clinical improvement, has a significant effect on this profile. Analysis of biochemical events, more specifically intracytoplasmic enzyme levels in GCF, are likely to offer a sensitive measure of periodontal pathology which may help in overcoming the existing limitations of clinical parameters. For this purpose, analysis of rGCF intracytoplasmic enzymes seems to be more beneficial.     

Immunohistochemical study of neutrophil- and fibroblast-type collagenases and stromelysin-1 in adult periodontitis

Eight adult periodontitis (AP) patients were studied immunohistochemically to determine the presence of matrix metalloproteinases (MMPs) MMP-1, MMP-3, and MMP-8 in the marginal gingival and gingival granulation tissue specimens obtained from periodontal flap surgery after scaling and root planing. Clinically healthy gingival tissue specimens obtained from impacted third-molar extraction operations served as controls. MMP-type-specific antisera were applied by the avidin-biotin-peroxidase complex staining method. Moderate immunoreactivity for neutrophil collagenase (MMP-8) was found both in the AP patients' marginal gingival connective tissue and in gingival granulation tissue specimens. Immunoreactivity for fibroblast-type collagenase (MMP-1) and stromelysin-1 (MMP-3) was detected only in the AP patients' gingival granulation tissue specimens. In the control specimens, no immunoreactivity for the MMPs could be detected. For the first time, this finding demonstrates immunohistochemically the presence of MMP-8 in human inflamed gingiva in situ, and further highlights the importance of MMP-8 in periodontal tissue destruction, evidently during the acute phase(s) of the disease. However, our results confirm and extend previous studies indicating that other types of MMPs from resident gingival cell sources also seem to participate in the chronic and destructive course of periodontal inflammation.     

Crevicular fluid level of beta-glucuronidase in relation to clinical periodontal parameters and putative periodontal pathogens in early-onset periodontitis

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.     

Long-term sub-antimicrobial doxycycline (Periostat) as adjunctive management in adult periodontitis: effects on subgingival bacterial population dynamics

Previous trials had indicated that various schedules of sub-antimicrobial doxycycline significantly reduced gingival crevicular fluid (GCF) collagenase activity in adult patients with periodontitis with no evidence of emergent tetracycline-resistant (Tcr) marker oral flora. The purpose of this nine-month study was to expand these observations, emphasizing newer microbial diagnostic methods. Subgingival paper point samples were obtained at baseline (BL), 3, 6, and 9 months. Four subject treatment groups in a double-blind design were evaluated by mechanical scaling and root planing (SRP) and/or 20 mg doxycycline BID (Periostat). Thirty-eight patients entered the study at baseline (BL). Dark-field microscopy on 260 samples showed that morphotype distribution was independent of treatment schedule. Culture analysis of the 3 most prevalent isolates recovered showed that Streptococcus and Prevotella species accounted for approximately 85% of the 724 cultures. There did not appear to be any overgrowth or replacement by opportunistic oral flora. Of 658 susceptibility patterns evaluated by Etest, the MIC50/90 and mode MIC showed stable patterns, independent of treatment group. Our findings were different from those of previously published reports, but may be partly explained by the lack of universally standardized methods in oral microbiology and interpretive criteria for susceptibility testing.     

Oxidative inactivation of alpha 1-proteinase inhibitor by alveolar epithelial type II cells

The aim of this work was to evaluate the ability of guinea pig alveolar epithelial type II cells to generate significant amounts of reactive oxygen species to inactivate alpha 1-proteinase inhibitor (alpha 1-PI). Inactivation of alpha 1-PI was evaluated by its inhibitory activity against porcine pancreatic elastase and was expressed as a percentage. The same experiments were performed in parallel with alveolar macrophages (AM) obtained from the same animals and with MRC-5 fibroblasts. Both type II cells and AM released significant amounts of hydrogen peroxide and superoxide, whereas the fibroblasts did not. Unstimulated type II cells (0.5 +/- 2%), AM (1.2 +/- 1.5%), and fibroblasts (0.5 +/- 0.5%) were unable to inactivate alpha 1-PI. Addition of phorbol myristate acetate did not increase their ability to inactivate alpha 1-PI. In contrast, type II cells (79.7 +/- 7%) and AM (80.1 +/- 8%) dramatically inactivated alpha 1-PI in the presence of myeloperoxidase (25 mU/ml), whereas fibroblasts did not. Addition of catalase to the reaction significantly prevented the inactivation of alpha 1-PI. Western blot analysis of alpha 1-PI did not reveal a significant proteolysis of alpha 1-PI, which supports the hypothesis that, in the presence of neutrophil-derived myeloperoxidase, type II cells may oxidatively inactivate alpha 1-PI.     

Tricuspid valve endocarditis in a non-drug addict associated with peliosis hepatis

The aims of the present study were to investigate whether the tachykinins substance P and neurokinin A were present in gingival crevicular fluid in both periodontal health and disease and to study the relationship with periodontal inflammation. Gingival crevicular fluid (GCF) was collected from a healthy, a gingivitis and a periodontitis site in 20 subjects with periodontitis and from a healthy site in 20 subjects without periodontitis. The volume of GCF was measured and each sample subsequently analysed for substance P and neurokinin A by radioimmunoassay. There were significantly increased levels of substance P-like immunoreactivity (SP-LI) and neurokinin A-like immunoreactivity (NKA-LI) in gingivitis and periodontitis sites compared with healthy sites. Both tachykinins were significantly elevated in periodontitis affected subjects, with significantly more tachykinin-like immunoreactivity at healthy sites in periodontitis affected compared with periodontally-healthy subjects. Despite the considerable individual variation in the levels of SP-LI and NKA-LI, both tachykinins were present at levels at which they could have biological activity. It is concluded that substance P and neurokinin A may have a r?le in the pathogenesis of periodontal disease and that further investigations could prove useful in clarifying the mechanisms through which neuropeptides could modulate periodontal health and disease.     

Correlation of interleukin-1 beta, interleukin-6, and periodontitis

In order to understand the role of IL-1 beta and IL-6 in the periodontal tissue destruction coincident to periodontitis, we assessed the levels of these two mediators in both the gingival tissue and the serum of patients with periodontal disease and of periodontally healthy subjects. In addition, production of IL-6 by six healthy human gingival fibroblast (HGF) strains in response to IL-1 beta was also investigated. The levels of IL-1 beta and IL-6 in gingival tissues and in serum were examined by ELISA. Both mediators were observed to increase in diseased tissues of patients with adult periodontitis, and there was a positively significant relationship between both mediators and clinical assessments of periodontal destruction. Moreover, a significant correlation was also noted between levels of IL-1 beta and IL-6 in gingival tissues of periodontitis patients (r = 0.4334, p < 0.01). However, there was no significant difference in the serum levels of IL-1 beta and IL-6 between periodontitis patients and periodontally healthy controls. In fibroblast cultures, confluent monolayers of HGF were incubated with recombinant human IL-1 beta for 48 h at 37 degrees C in 5% CO2 and air. At the end of the culture period, supernatants were collected and assayed for IL-6 activity by inducing proliferation in the IL-6-dependent hybridoma cell line 7TD1. A dose-dependent stimulatory effect of IL-1 beta on IL-6 production by HGF was noted, wherein 3 strains exhibited higher IL-6 activity than the other 3. These data indicate that the levels of IL-1 beta and IL-6 in gingival tissues are closely related to the severity of periodontal disease and that the IL-1 beta and IL-6 produced in gingival tissues may not reflect these two mediators levels in serum. Moreover, IL-1 beta responsiveness of HGF in IL-6 production depends on both the concentration of IL-1 beta and cells of individual subjects. Since HGF are present in periodontal lesion, it is possible that IL-6 secretion stimulated by exposure to inflammatory cell products such as IL-1 beta may participate in the destruction of periodontal tissue in periodontitis.